The authors would like to acknowledge Dr. Evette Radisky and Alexandra Hockla at the Mayo Clinic in Jacksonville, Florida, for providing the pET-3a-pro-MMP-3cd plasmid as the template for cloning the Hisx6pro-MMP-3cd gene, and their comments, along with Dr. Paul Hartley from the Nevada Genomics Center at the University of Nevada, Reno, for DNA sequencing. The authors would also like to thank Cassandra Hergenrader for helping with part of protein expression. M.R.-S. would like to thank the NIH-P20 GM103650-COBRE Integrative Neuroscience grant and the UNR R&#38;D mICRO SEED Grant Award.

1. MMP expression
<ol>
	<li>Cloning and transformation of pET-3a-Hisx6-pro-MMP-3cd into R2DP cells
	<ol>
		<li>Digest the pET-3a plasmid (see the Table of Materials) with NdeI and BamHI restriction enzymes in Digest Buffer (see the Table of Materials). In a total reaction volume of 40 &#181;L, add 4 &#181;L of Digest Buffer, 33 &#181;L of 100 ng/&#181;L plasmid, and 1.5 &#181;L of each restriction enzyme and allow the reaction to proceed for ~2 h until completion at 37 &#176;C.</li>
		<li>Perform a PCR reaction on the MMP-3cd sequence to insert an N-terminal His-tag. Use 25 &#181;L of PCR Mix (see the Table of Materials), 2.5 &#181;L of 10 &#181;M primers (Supplemental Figure S1), and 1.25 &#181;L of the 100 ng/&#181;L insert sequence. Add sterile water to a final reaction volume of 50 &#181;L.</li>
		<li>Run the PCR product and digested vector on a 1% agarose gel. Purify the gel bands using a Gel Recovery Kit (see the Table of Materials) per the manufacturer&#39;s protocol.</li>
		<li>Clone the amplified PCR product into the digested vector between the NdeI and BamHI restriction sites using DNA Assembly Mix (see the Table of Materials). Use online tools to determine the required volume of the insert and cut vector for a total reaction volume of 15 &#181;L.</li>
		<li>Thaw a 50 &#181;L aliquot of high-transformation efficiency cells (see the Table of Materials) on ice until thawed. Prewarm SOC Growth Medium (see the Table of Materials) to 37 &#176;C and LB-ampicillin (LB Amp) plates (see the Table of Materials).</li>
		<li>Add 1-2 &#181;L of the pET-3a-Hisx6-pro-MMP-3cd assembly reaction to the 50 &#181;L aliquot. Incubate on ice for 30 min.</li>
		<li>Heat-shock the cells by incubating at 42 &#176;C for 30 s. Incubate on ice for 2 min.</li>
		<li>Add 950 &#181;L of SOC growth medium to each transformant mixture. Shake for 1 h at 250 rpm and 37 &#176;C.</li>
		<li>Plate 100 &#181;L of the transformants on LB Amp plates and incubate overnight at 37 &#176;C.</li>
		<li>Inoculate each isolated colony in 10 mL of LB Amp medium. Shake overnight at 250 rpm and 37 &#176;C.</li>
		<li>Extract plasmid DNA per manufacturer&#39;s protocol for the miniprep kit (see the Table of Materials). Confirm the sequence of the construct using T7 forward and reverse primers (Supplemental Figure S1). 
		NOTE: The pET-3a-Hisx6-pro-MMP-3cd construct DNA can be stored at -20 &#176;C. When ready, proceed with transformation into R2DP cells.</li>
		<li>Thaw one 20 &#181;L aliquot of R2DP cells (see the Table of Materials) on ice for 2-5 min. Prewarm SOC growth medium to room temperature and LB Amp CamR plates to 37 &#176;C (see the Table of Materials).</li>
		<li>Add 1 &#181;L of 100 ng/&#181;L sequence-confirmed pET-3a-Hisx6-pro-MMP-3cd to the 50 &#181;L aliquot. Stir gently to mix and return the tube to ice.</li>
		<li>Incubate the tube on ice for 5 min.</li>
		<li>Heat-shock the cells by incubating at 42 &#176;C for exactly 30 s. Do not shake.</li>
		<li>Place the cells on ice for 2 min.</li>
		<li>Add 80 &#181;L of room-temperature SOC medium to the transformant mixture. Shake for 1 h at 250 rpm and 37 &#176;C.</li>
		<li>Plate the transformants on LB Amp CamR plates and incubate overnight at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Growth and induction
	<ol>
		<li>Inoculate a single, isolated colony of R2DP pET-3a-Hisx6-pro-MMP-3cd transformant from an LB Amp CamR plate in 10 mL of LB Amp CamR media at 37 &#176;C. Shake at 250 rpm overnight (~16 h). Save aliquots from each culture and prepare 40% (v/v) glycerol (see the Table of Materials) stocks if desired.</li>
		<li>Per overnight culture, inoculate a 1 L flask containing 500 mL of LB Amp CamR medium to an optical density at 600 nm (OD600) of 0.05-0.1. 
		NOTE: This should return the cells to logarithmic growth.</li>
		<li>Measure the OD600 at several time points, typically for 3-4 h, until it falls between 0.4 and 0.6.</li>
		<li>Before induction, aliquot a fraction of culture into a 1.5 mL microfuge tube (see the Table of Materials) and label it Un-induced Fraction. Store it at -80 &#176;C for gel analysis. If not running an SDS-PAGE gel, skip this step and proceed to step 1.2.5.</li>
		<li>Induce the cultures to a final concentration of 1 mM using 1 M isopropyl-&#223;-D-thiogalactopyranoside (IPTG) stock (see the Table of Materials). Continue to incubate in the 37 &#176;C shaker for an additional 3-4 h. 
		NOTE: During expression, the reader should determine the optimal OD600 at the time of induction and the IPTG concentration. If the yield drops substantially after purification, the imidazole concentration in purification buffers may require adjustment, or the cell pellet may need to be sonicated further.</li>
		<li>Before centrifuging the cultures, aliquot a fraction of culture into a second 1.5 mL microfuge tube and label it Induced Fraction. Store it at -80 &#176;C for gel analysis. If not running an SDS-PAGE gel, skip this step and proceed to step 1.2.7.</li>
		<li>Centrifuge the cell culture in 250 mL conical bottles (see the Table of Materials) at maximum speed and 4 &#176;C for 10 min.</li>
		<li>Repeat step 1.2.7 until the cultures are completely pelleted. 
		NOTE: PAUSE: Cell pellets can be frozen at -80 &#176;C and thawed later for further processing. Otherwise, skip this step and proceed to step 1.3.1.</li>
	</ol>
	</li>
	<li>Inclusion body extraction and solubilization 
	NOTE: Prepare fresh 10 M urea, preferably no earlier than one day in advance, stirring thoroughly until dissolved completely. Do not heat or autoclave urea; store it at room temperature.
	<ol>
		<li>Resuspend the pellet (from step 1.2.8) in lysis buffer (see the Table of Materials). Per gram of pellet, add 3 mL of lysis buffer and resuspend by vortexing or pipetting. Shake overnight at 4 &#176;C.</li>
		<li>Add 1.25 mL of 10% (w/v) sodium deoxycholate (see the Table of Materials) per 1 L of culture. Shake at room temperature for 30 min at 150 rpm.</li>
		<li>Add 10 &#181;L of DNase I (see the Table of Materials) per 1 L of culture. Shake at room temperature for 30 min at 150 rpm.</li>
		<li>Centrifuge for 10 min at 13,000 &#215; g and 4 &#176;C.</li>
		<li>Set aside a fraction of Lysed MMP for gel analysis. Store it at -80 &#176;C. If not performing gel analysis, proceed to step 1.3.6. 
		NOTE: After centrifugation, the pellet may be stringy and not compactly packed, making it risky to discard the supernatant. If this is the case, then skip step 1.3.6 and proceed to step 1.3.7.</li>
		<li>Discard the supernatant from the centrifuged samples. 
		NOTE: The protocol can be paused at this point and the cell pellets frozen at -80 &#176;C and thawed later. Otherwise, skip this step and proceed to step 1.3.7.</li>
		<li>Resuspend the pellet in 100 mL/L culture of Inclusion Body Buffer (see the Table of Materials) by pipetting up and down.</li>
		<li>During sonication, keep the samples on ice to prevent overheating. Sonicate each sample for 6 cycles of 15 s, output 5, and 50% pulse. Allow 15 s rest periods for cooling between cycles. 
		NOTE: If necessary, transfer the samples into 50 mL conical tubes for further centrifugation (see the Table of Materials). Centrifuge for 10 min at 13,000 &#215; g and 4 &#176;C.</li>
		<li>Set aside a fraction of Sonicated MMP for gel analysis. Store it at -80 &#176;C. If not performing gel analysis, proceed to step 1.3.11.</li>
		<li>Check the pellet. If stringy, repeat steps 1.3.8-1.3.10. If the pellet is compact, discard the supernatant and proceed to step 1.3.12. 
		NOTE: Sonication in Inclusion Body Buffer can be repeated to recover more protein from the lysed cell debris. However, too much sonication can cause shearing, which harms MMP yield. The protocol can be paused at this stage, and the cell pellets can be frozen at -80 &#176;C and thawed later.</li>
		<li>Resuspend each pellet from a 1 L culture in 5 mL of Solubilization Buffer (see the Table of Materials) by pipetting. Incubate for at least 30 min on ice to allow the proteins to solubilize.</li>
		<li>Set aside a fraction of Solubilized MMP for gel analysis. Store it at -80 &#176;C. If not performing gel analysis, proceed to step 1.3.14.</li>
		<li>Centrifuge the cells for 10 min at 13,000 &#215; g and 4 &#176;C. DO NOT DISCARD THE SUPERNATANT.</li>
		<li>If a pellet forms/remains after centrifugation, pour the supernatant into a separate 50 mL conical tube. Resuspend the pellet in another 5 mL of Solubilization Buffer (per 1 L of culture) by pipetting up and down.</li>
		<li>Centrifuge for 10 min at 13,000 &#215; g and 4 &#176;C. DO NOT DISCARD THE SUPERNATANT.</li>
		<li>Repeat steps 1.3.13 and 1.3.14 until a little to no pellet forms after centrifugation or only gray precipitate remains. Pool the supernatants. Discard or store the pellet at -80 &#176;C for additional sonication.</li>
	</ol>
	</li>
</ol>
2. MMP purification and refolding
<ol>
	<li>His-tag (HT) affinity purification
	<ol>
		<li>Per the manufacturer&#39;s protocol, fill a gravity-flow column (see the Table of Materials) with well-mixed Ni-NTA resin (see the Table of Materials). Allow the resin to settle and separate from the storage buffer such that a distinct line forms between the two layers. 
		NOTE: Never allow the resin to dry, as air will penetrate the resin and harm protein yield. In between uses, perform the resin regeneration procedure described in section 2.2.</li>
		<li>Allow the storage buffer to drain. Fill the column with two resin-bed volumes of HT Equilibration Buffer.</li>
		<li>Drain the HT Equilibration Buffer and discard. As the column is draining, centrifuge the protein extract at 13,000 &#215; g for 1 min and filter-sterilize using a 0.22 &#181;m filter (see the Table of Materials).</li>
		<li>Swap the waste container for a 50 mL conical tube labeled HT Flowthrough. Add the prepared protein extract to the column.</li>
		<li>Reapply the flowthrough to maximize binding.</li>
		<li>Set aside a fraction of Flowthrough Fraction for gel analysis. Store it at -80 &#176;C. If not performing gel analysis, proceed to step 2.1.7.</li>
		<li>Immediately wash the resin with 15 mL of HT Wash Buffer (see the Table of Materials). Collect the flowthrough in 15 mL conical tubes (see the Table of Materials) labeled HT Wash. 
		NOTE: Absorbance values at 280 nm (A280) were obtained via spectrophotometry and used along with molecular weight and extinction coefficient, &#949;, to estimate protein concentrations. For denatured Hisx6-pro-MMP-3cd, the molecular weight is 29.86 kDa, and &#949; is 34.38 M-1 cm-1.</li>
		<li>Blanking against HT Wash Buffer, measure and record the A280. Repeat steps 2.1.7 and 2.1.8 with additional wash fractions. Once the A280 approaches baseline and impurities have been minimized, proceed to step 2.1.9.</li>
		<li>Set aside a fraction of Wash Fraction for gel analysis. Repeat for multiple wash fractions. Store the fractions at -80 &#176;C. If not performing gel analysis, proceed to step 2.1.10.</li>
		<li>Immediately elute His-tagged proteins by adding 5 mL of HT Elution Buffer (see the Table of Materials). Collect the flowthrough as 0.5-1 mL fractions in microfuge tubes labeled HT Elution.</li>
		<li>Set aside a fraction of Elution Fraction for gel analysis. Repeat for multiple elution fractions. Store the fractions at -80 &#176;C. If not performing gel analysis, proceed to step 2.1.12.</li>
		<li>If the A280 is &#62;0.3 mg/mL, dilute the fraction with HT Equilibration Buffer (see the Table of Materials). 
		NOTE: The eluted fraction must be diluted to an A280 of 0.3 mg/mL or less to prevent precipitation during dialysis. The protocol can be paused here and the pooled fractions frozen at -80 &#176;C and thawed later. Otherwise, skip this step and proceed to step 2.2.1.</li>
	</ol>
	</li>
	<li>Resin regeneration
	<ol>
		<li>Wash the resin with ten resin-bed volumes of HT Regeneration Buffer (see the Table of Materials) and ten resin-bed volumes of sterile water.</li>
		<li>Store the resin as a 50% slurry in 20% (v/v) ethanol in water.</li>
	</ol>
	</li>
</ol>
3. Protein refolding
NOTE: For smaller volumes, dialysis cassettes can be used at a lower risk of sample loss. Dialysis tubing is required if larger volumes are used (see the Table of Materials).
<ol>
	<li>Dialysis 
	NOTE: The optimum protein concentration for dialysis is ~0.3 mg/mL. If significant precipitation occurs during dialysis, reduce the urea concentration gradient between each dialysis using a stepwise dialysis method and add more intermediate steps (e.g., from 6 M to 5 M, and then 5 M to 4 M, rather than skipping the 5 M stage). As freeze-thaw cycles damage the cellular and protein structure, it is vital to minimize pauses in the protocol.
	<ol>
		<li>Per the manufacturer&#39;s protocol, use the appropriate amount of dialysis tubing according to the volume of Elution Fraction samples.</li>
		<li>Submerge the eluted MMP fractions in dialysis tubing in 1 L of Dialysis Buffer 1 (see the Table of Materials). Stir the tubing and its contents on a magnetic stirrer for no less than 8 h at 4 &#176;C.</li>
		<li>Transfer to 1 L of Dialysis Buffer 2 (see the Table of Materials). Stir the tubing and its contents on a magnetic stirrer for no less than 8 h at 4 &#176;C.</li>
		<li>Transfer to 1 L of Dialysis Buffer 3 (see the Table of Materials). Stir the tubing and its contents on a magnetic stirrer for no less than 8 h at 4 &#176;C.</li>
		<li>Transfer the sample into new 50 mL conical tubes and label them as Dialyzed MMP.</li>
		<li>Examine the tube for any precipitate. If precipitate has formed, centrifuge the sample for 1 min at 13,000 &#215; g and 4 &#176;C.</li>
		<li>Transfer the supernatant into new 15 mL conical tubes and label them as Refolded MMP.</li>
		<li>Set aside a fraction for gel analysis and label it as Refolded MMP. Store it at -80 &#176;C. If not performing gel analysis, proceed to step 3.1.9. 
		NOTE: If yields are low, the precipitate can be dissolved in HT Equilibration Buffer and steps in section 3.1 repeated with dialysis tubing. If gel analysis is not to be performed or if the protocol must be paused here, freeze the samples at -80 &#176;C and thaw them later. If yields are in the desired range, proceed to step 3.2.1.</li>
	</ol>
	</li>
	<li>Reconcentration 
	NOTE: The extinction coefficients for refolded and denatured Hisx6-pro-MMP-3cd are expected to be the same; hence, A280 calculations are not affected.
	<ol>
		<li>Reconcentrate the sample up to 0.5 mg/mL. Use a 400 mL stirred cell (see the Table of Materials) to concentrate the sample to 15 mL. To prevent foaming, use a 50 mL reconcentration tube to concentrate further if needed. 
		NOTE: If a precipitate forms, it can be pelleted and dissolved in HT Equilibration Buffer. Then, repeat sections 3.1 and step 3.2.1. Otherwise, continue to step 3.2.2.</li>
		<li>Set aside a fraction for gel analysis and label it as Concentrated MMP. 
		&#8203;NOTE: The protocol can be paused here and the samples frozen at -80 &#176;C and thawed later.</li>
	</ol>
	</li>
</ol>
4. Activation
<ol>
	<li>4-Aminophenylmercuric acetate (APMA) activation 
	NOTE: APMA is highly toxic. Make a fresh stock solution of 20 mM APMA before activation, and always work under a fume hood when using APMA. Discard the APMA waste into its container.
	<ol>
		<li>Per 1 mL aliquot of MMP (1 mg/mL), add 50 &#181;L of 20 mM APMA (see the Table of Materials) to reach a final APMA concentration of 1 mM. Incubate overnight at 37 &#176;C.</li>
		<li>If a precipitate forms, centrifuge it at maximum speed for 10 min at 4 &#176;C. Store the supernatant in a 1.5 mL microfuge tube labeled Activated MMP. Discard the precipitate into a container marked for APMA waste.</li>
		<li>Set aside a fraction for gel analysis and label it Activated MMP. 
		NOTE: The protocol can be paused here and the samples frozen at -80 &#176;C and thawed later. If not performing gel analysis, proceed to step 4.2.1. After activation, the molecular weight and extinction coefficient of MMP-3cd are 19.40 kDa and 28.42 M-1 cm-1, respectively.</li>
	</ol>
	</li>
	<li>Desalting
	<ol>
		<li>Remove APMA from the activated MMP-3cd sample with a 2 mL desalting column (see the Table of Materials), following the manufacturer&#39;s protocol.</li>
		<li>Set aside a fraction for gel analysis and label it Desalted MMP. Store it at -80 &#176;C. If not performing gel analysis, proceed to section 4.3 with the remaining samples. 
		NOTE: The protocol can be paused here and the samples frozen at -80 &#176;C and thawed later.</li>
	</ol>
	</li>
	<li>Running the SDS-PAGE gels
	<ol>
		<li>Run all protein fractions on SDS-PAGE gels: Un-induced Fraction, Induced Fraction, Lysed MMP, Sonicated MMP, Solubilized MMP, Flowthrough Fraction, Wash Fraction, Elution Fraction, Refolded MMP, Concentrated MMP, Activated MMP, and Desalted MMP.</li>
	</ol>
	</li>
	<li>Long-term storage of MMP-3
	<ol>
		<li>Add 0.05% (v/v) nonionic surfactant (see the Table of Materials) to the desalted MMP-3cd samples and store them at -80 &#176;C.</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioredoxin+fusion+construct+enables+high-yield+production+of+soluble,+active+matrix+metalloproteinase-8+(MMP-8)+in+Escherichia+coli.">Thioredoxin fusion construct enables high-yield production of soluble, active matrix metalloproteinase-8 (MMP-8) in Escherichia coli.</a> Protein Expression and Purification. 122, 64-71 (2016).</li><li>Maity, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GST-His+purification:+A+two-step+affinity+purification+protocol+yielding+full-length+purified+proteins.">GST-His purification: A two-step affinity purification protocol yielding full-length purified proteins.</a> Journal of Visualized Experiments: JoVE. (80), e50320 (2013).</li><li>Stefan, A., Ceccarelli, A., Conte, E., Mont&#243;n Silva, A., Hochkoeppler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifaceted+benefits+of+protein+co-expression+in+Escherichia+coli.">The multifaceted benefits of protein co-expression in Escherichia coli.</a> Journal of Visualized Experiments: JoVE. (96), e52431 (2015).</li><li>Yadavalli, R., Sam-Yellowe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HeLa+based+cell+free+expression+systems+for+expression+of+Plasmodium+rhoptry+proteins.">HeLa based cell free expression systems for expression of Plasmodium rhoptry proteins.</a> Journal of Visualized Experiments: JoVE. (100), e52772 (2015).</li><li>Zeytuni, N., Zarivach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+M.+magneticum+strain+AMB-1+magnetosome+associated+protein+MamA&#916;41.">Purification of the M. magneticum strain AMB-1 magnetosome associated protein MamA&#916;41.</a> Journal of Visualized Experiments: JoVE. (37), e1844 (2010).</li></ol>

The authors declare that they have no competing financial interests.

The large-scale production of soluble, human, recombinant MMPs remains a challenging task. Mammalian cells can express functional MMPs at high costs and long wait times, whereas E. coli rapidly produce high quantities of MMP inclusion bodies that must be purified and refolded11,16. R2DP cells significantly increase the yield of MMP inclusion bodies, enabling a more cost-effective and productive MMP refolding process. However, E. coli lack the posttranslational machinery needed to fold MMPs, and though engineered strains show greatly improved expression levels, only some intermediates are properly folded upon denaturant removal4. Consequently, occasional precipitation of MMPs is still expected during refolding and activation. These results show that a significant portion of purified Hisx6-pro-MMP-3cd yield is lost after refolding, activation, and desalting. 
These stages can be further optimized by adding more dialysis stages along with testing concentrations of MMP-3cd and APMA. However, per liter of culture, the yield of functional MMP-3cd is 49-fold higher in R2DP cells than BL21(DE3) cells. Additionally, the proportion of functional, desalted MMP-3cd recovered from purified Hisx6-pro-MMP-3cd rose from 3.7% for BL21(DE3) cells to 14% for R2DP cells. Therefore, R2DP cells offer a viable alternative to current MMP production options, such as expression in mammalian cells, offering more competitive yields per liter of culture.
This protocol describes a detailed method to express and purify Hisx6-pro-MMP-3cd in R2DP E. coli cells, along with the activation and refolding of MMP-3cd. As the protocol takes several days to complete, careful planning is critical to minimize the loss of functional MMPs due to multiple freeze-thaw cycles. As R2DP cells are the key improvement in this method, it is paramount to optimize expression yield, as large quantities can be lost through refolding and activation. During expression, the operator should determine the optimal OD600 and IPTG concentration prior to induction. After His-tag purification, if yield drops substantially, then the resin may need to be regenerated or replaced, or the cell pellet may need to be sonicated further. 
If significant precipitation occurs during dialysis, reduce the changes in urea concentration between stages in stepwise dialysis by adding more stages (e.g., from 6 M to 5M, and then 5 M to 4 M, rather than skipping the 5 M stage). Once refolded, and particularly after activation, MMPs are substantially more prone to precipitation or degradation through autoproteolysis17. After pH and salt concentrations of all buffers have been optimized and desired tests have been performed, all steps following dialysis should be completed with urgency. 
Growing attention toward MMPs as potential targets for therapeutics has been met with rapid innovations in protein engineering for improving binding, inhibition, and selectivity of MMP therapeutics9. Consequently, once-ambitious prospects in the field of MMP therapeutics are steadily becoming more attainable6. The need for fast, reliable methods to recover soluble, active MMPs undoubtedly will become more imperative with time.

Most complex eukaryotic proteins undergo elaborate posttranslational modifications after expression, requiring highly assisted protein folding and co-factors to be functional1. Producing large amounts of soluble human protein in a bacterial host remains a significant challenge due to high costs and the lack of robust expression and purification methods, even for smaller-scale laboratory experiments2,3. MMPs, human endopeptidases with large molecular weight, are usually expressed as insoluble inclusion bodies when expressed in E. coli. Extraction of soluble human MMPs often leads to a laborious, time-consuming solubilization and refolding process4.
MMPs have critical roles in both physiological and pathogenic processes. Human MMPs are a family of 23 zinc endopeptidases, categorized by structure and substrate specificity, and differentially expressed in spite of a highly conserved catalytic domain5,6. MMPs are secreted as inactive zymogens, regulated via posttranslational activation and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs)7,8,9,10. Though initially recognized for their role in ECM turnover, MMPs have also been implicated in development, morphogenesis, tissue repair, and remodeling8. Dysregulation of MMPs has been notably linked to cancer along with neurodegenerative, cardiovascular, and fibrotic diseases, among other illnesses5,7.
The development of robust large-scale MMP production methods is critical to ensure the success of future studies of MMP mechanisms through biochemical and cell-based assays. Various MMPs have been previously expressed in bacteria11, including Hisx6-tagged MMPs, without altering MMP activity12,13,14,15. However, these methods include tedious, long steps that might be difficult to replicate.
Mammalian cells can also be used to express many different human proteins while ensuring the proper posttranslational modifications16. Although the mammalian expression system is an ideal choice to produce recombinant human proteins with proper post-translational modifications, the main disadvantages of this method are initial low yields, costly growth media and reagents, long timelines to reach stable expression lines, and risk of contamination with other species such as fungi or bacteria2,11. Moreover, MMP production in mammalian cell lines yields impurities from associated cellular proteins such as TIMPs or fibronectins11. Unlike the slow cell growth observed in mammalian cells, the bacterial expression system offers large-scale protein production in a short period along with simpler media and growth requirements. However, due to the lack of other associated cellular proteins (i.e., TIMPs) in bacterial expression systems, active MMPs at higher concentrations are subject to degradation through autoproteolysis, resulting in poor MMP yield17.
This paper describes a detailed method for bacterial expression, purification, and activation of recombinant Hisx6-pro-MMP-3cd using E. coli as an expression host due to its affordability, simplicity, and success in producing higher yields of MMPs2,3,18. Since E. coli lacks the protein folding machinery and posttranslational processing required for recombinant MMPs and other complex proteins, many E. coli strains have been engineered to overcome these limitations, making E. coli a more suitable host for expression of recombinant human MMP-3cd,19,20. For instance, the R2DP strain used in this study enhances eukaryotic expression by supplying a chloramphenicol-resistant plasmid containing codons rarely used in E. coli.
As described in this protocol, after overexpression of relatively pure inclusion bodies from the pET-3a vector (Figure 1) in R2DP cells, Hisx6-pro-MMP-3 catalytic domain (MMP-3cd) proteins are extracted and denatured4. Hisx6-pro-MMP-3cd3,19 was purified using&#160;affinity tag chromatography. Upon refolding and dialysis, the pro-MMP-3cd (zymogen) was activated by 4-aminophenylmercuric acetate (APMA), and SDS-PAGE analysis is used to evaluate yields and the need for further purification5,21. This protocol describes expression, purification, and activation of soluble MMP-3cd as an example. However, it may be also used as a guide for expression of other MMPs and human proteases with similar expression, and activation mechanisms (Figure 2). For other proteins other than MMP-3cd, the reader is advised to determine optimal buffer compositions and methods for their target protein before attempting this protocol.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63263/63263fig01.jpg" /> 
Figure 1: Plasmid map of the pET-3a-Hisx6-pro-MMP-3cd plasmid. The pET-3a vector includes an ampicillin resistance gene. An N-terminal Hisx6-tag sequence is cloned into the pET-3a-based vector, including pro-MMP-3cd, to yield the pET-3a-Hisx6-pro-MMP-3cd construct under control of T7 promoter between BamHI and NdeI restriction sites. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63263/63263fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63263/63263fig02.jpg" /> 
Figure 2: Bacterial expression of pro-MMP-3cd, purification, refolding, and activation. 1.1: pET-3a-Hisx6-pro-MMP-3cd plasmid was transformed into BL21(DE3) or R2DP Cells. 1.2: Pro-MMP-3cd protein expression was induced using IPTG. 1.3: Chemical lysis and sonication are used to extract Hisx6-pro-MMP-3cd proteins that are mainly insoluble and found in the inclusion bodies. Urea was used to denature and solubilize protein from inclusion bodies. 2.1. Denatured Hisx6-pro-MMP-3cd protein was purified via affinity chromatography purification. 3. The eluted Hisx6-pro-MMP-3cd was slowly refolded during dialysis through gradual removal of urea from the buffer. 4. Finally, refolded MMP-3cd protein was activated using APMA by removing the N-terminal pro-peptide domain. APMA is later removed from the solution through desalting. The numbers correspond to protocol sections describing these steps. Abbreviations: MMP-3cd = Matrix metalloproteinase-3 catalytic domain; APMA = 4-aminophenylmercuric acetate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63263/63263fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>0.22 &amp;micro;m sterile filter</td><td>Sigma Aldrich</td><td>SLGP033RS</td><td>Used to remove some contaminants from the protein extract before purification, and prevent the Ni-NTA column from clogging</td></tr><tr><td>1 L Erlenmeyer flasks</td><td>Thermo Fisher Scientific</td><td>S76106F</td><td>n/a</td></tr><tr><td>1 L glass bottles</td><td>Thermo Fisher Scientific</td><td>06-414-1D</td><td>n/a</td></tr><tr><td>1.5 mL microfuge tubes</td><td>Thermo Fisher Scientific</td><td>02-682-002</td><td>n/a</td></tr><tr><td>15 mL conical tubes</td><td>Thermo Fisher Scientific</td><td>339650</td><td>n/a</td></tr><tr><td>18 G, 1-in. beveled needle</td><td>Amazon</td><td>B07S7VBHM2</td><td>Used in combination with the dialysis casette</td></tr><tr><td>2 mL desalting column</td><td>Thermo Fisher Scientific</td><td>89890</td><td>Removes APMA following activation</td></tr><tr><td>2-(N-Morpholino)ethanesulfonic acid (MES)</td><td>Thermo Fisher Scientific</td><td>AAA1610422</td><td>n/a</td></tr><tr><td>250 mL conical bottle cushions</td><td>Thermo Fisher Scientific</td><td>05-538-53A</td><td>Stabilize conical bottles during large-volume centrifugation</td></tr><tr><td>250 mL conical bottles</td><td>Thermo Fisher Scientific</td><td>05-538-53</td><td>n/a</td></tr><tr><td>400 mL stirred cell</td><td>Sigma Aldrich</td><td>UFSC40001</td><td>Re-concentrates a much larger volume than the centrifugal filter unit. Rosetta2(DE3)pLysS cells produce high volumes of protein that may exceed the 15 mL limit of the centrifugal filter unit</td></tr><tr><td>4-aminophenylmercuric acetate (APMA)</td><td>Sigma Aldrich</td><td>A9563-5G</td><td>Activates MMP-3 by cleaving the propeptide</td></tr><tr><td>5 mL syringe</td><td>Thermo Fisher Scientific</td><td>NC0829167</td><td>Used in combination with the dialysis casette</td></tr><tr><td>50 mL conical tubes</td><td>Thermo Fisher Scientific</td><td>339650</td><td>Used for storage in many purification steps</td></tr><tr><td>50 mL re-concentration tube</td><td>Sigma Aldrich</td><td>UFC901024D</td><td>Used for re-concentrating protein samples after dialysis or removing contaminants</td></tr><tr><td>Agar</td><td>Thermo Fisher Scientific</td><td>BP1423-500</td><td>Buffer ingredient that solidifies autoclaved LB media upon cooling</td></tr><tr><td>Ampicillin</td><td>Thermo Fisher Scientific</td><td>BP1760-25</td><td>Antibiotic used with pET3a vector; used at 100 &amp;micro;g/mL in LB media</td></tr><tr><td>BamHI</td><td>NEB</td><td>R3136S</td><td>Restriction enzyme to be used with the pET3a vector</td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Thermo Fisher Scientific</td><td>600-30-23</td><td>The calcium ion stabilizes MMP structure</td></tr><tr><td>Cell spreaders</td><td>Thermo Fisher Scientific</td><td>50-189-7544</td><td>Can be used to spread cells across a petri dish after transformation</td></tr><tr><td>Chloramphenicol</td><td>Thermo Fisher Scientific</td><td>22-055-125GM</td><td>Antibiotic used with pET3a vector; used at 34 &amp;micro;g/mL in LB media</td></tr><tr><td>Dialysis Buffer 1</td><td>n/a</td><td>n/a</td><td>20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 &amp;micro;M ZnCl&lt;sub&gt;2&lt;/sub&gt;, 4 M Urea.</td></tr><tr><td>Dialysis Buffer 2</td><td>n/a</td><td>n/a</td><td>20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 &amp;micro;M ZnCl&lt;sub&gt;2&lt;/sub&gt;, 2 M Urea.</td></tr><tr><td>Dialysis Buffer 3</td><td>n/a</td><td>n/a</td><td>20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM CaCl&lt;sub&gt;2&lt;/sub&gt; , 1 &amp;micro;M ZnCl&lt;sub&gt;2&lt;/sub&gt;.</td></tr><tr><td>Dialysis clips</td><td>Thermo Fisher Scientific</td><td>68011</td><td>Used in combination with snakeskin dialysis tubing</td></tr><tr><td>Dialysis tubing</td><td>Thermo Fisher Scientific</td><td>88243</td><td>Alternative dialysis method that holds much larger sample volumes, but with higher risk of sample loss</td></tr><tr><td>Digest buffer</td><td>NEB</td><td>B7204S</td><td>Buffer used in digesting the pET3a vector</td></tr><tr><td>Disposable cuvettes</td><td>Thermo Fisher Scientific</td><td>21-200-257</td><td>Used to measure the bacterial culture OD during growth and expression</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Thermo Fisher Scientific</td><td>D107125G</td><td>Assists with protein denaturation by reducing any disulfide bonds</td></tr><tr><td>DNA assembly mix</td><td>NEB</td><td>E2621S</td><td>Used to ligate the Hisx6-pro-MMP-3cd PCR product and digested pET3a vector</td></tr><tr><td>DNase I</td><td>NEB</td><td>M0303S</td><td>Endonuclease for degrading unfavorable DNA contaminants that could later affect protein purification</td></tr><tr><td>Ethanol</td><td>Thermo Fisher Scientific</td><td>A995-4</td><td>n/a</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Thermo Fisher Scientific</td><td>J15694-AE</td><td>Used in denaturation. Prevents oxidation and subsequent formation of disulfide bonds</td></tr><tr><td>Gel recovery kit</td><td>Promega</td><td>A9281</td><td>Isolates and purifies DNA from agarose gels</td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific</td><td>G33-500</td><td>Used for making glycerol stocks, which are frozen at -80 &amp;deg;C</td></tr><tr><td>Gravity flow column</td><td>BioRad</td><td>7321010</td><td>Used for Ni-NTA purification of recombinantly His-tagged proteins</td></tr><tr><td>High-transformation efficiency cells</td><td>NEB</td><td>C2987</td><td>High-transformation efficiency cells with greater chance of success for cloning the N-terminal His-tag into the pET3a-pro-MMP-3cd construct</td></tr><tr><td>HT Elution Buffer</td><td>n/a</td><td>n/a</td><td>20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 6 M urea, 250 mM imidazole. Adjust pH to 7.4</td></tr><tr><td>HT Equilibration Buffer</td><td>n/a</td><td>n/a</td><td>20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 6 M urea. Adjust pH to 7.4</td></tr><tr><td>HT Regeneration Buffer</td><td>n/a</td><td>n/a</td><td>20 mM MES, 0.1 M NaCl. Adjust pH to 5.0</td></tr><tr><td>HT Wash Buffer</td><td>n/a</td><td>n/a</td><td>20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 6 M urea, 25 mM imidazole. Adjust pH to 7.4</td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Thermo Fisher Scientific</td><td>A144C-212</td><td>Used to pH buffers</td></tr><tr><td>Imidazole</td><td>Thermo Fisher Scientific</td><td>AAA1022122</td><td>Mimics the histidine side group. Used to separate non-specifically binding proteins from the his-tagged target protein</td></tr><tr><td>Inclusion Body Buffer</td><td>n/a</td><td>n/a</td><td>20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl, 5 mM DTT, 2% v/v Triton X 100, 0.5 M Urea. Adjust pH to 8.0</td></tr><tr><td>Isopropyl-&amp;szlig;-D-thiogalactopyranoside (IPTG)</td><td>Thermo Fisher Scientific</td><td>FERR0392</td><td>A reagent that induces target gene expression in pET3a. Make 0.5 mL 1 M aliquots, filter sterilize and store in -20 &amp;deg;C</td></tr><tr><td>LB Amp Cam&lt;sup&gt;R&lt;/sup&gt; media</td><td>n/a</td><td>n/a</td><td>To be poured into a sterible 1 L bottle or 1 L flask. For 1 L, add 25 g LB Broth. Sterilize by autoclaving. Once cooled to below 50 &amp;deg;C, add ampicillin to 100 &amp;micro;g/mL and chloramphenicol to 34 &amp;micro;g/mL</td></tr><tr><td>LB Amp Cam&lt;sup&gt;R&lt;/sup&gt; plates</td><td>n/a</td><td>n/a</td><td>To be poured into sterile petri dishes. Pour until the petri dish lid is completely covered. 1 L of media yields 40-60 plates. For 1 L: 25 g LB Broth, 16 g Agar. Sterilize by autoclaving. Once cooled to below 50 &amp;deg;C, add ampicillin to 100 &amp;micro;g/mL and chloramphenicol to 34 &amp;micro;g/mL</td></tr><tr><td>LB Broth</td><td>Thermo Fisher Scientific</td><td>BP1426-2</td><td>Pre-mixed with tryptone, yeast extract, and sodium chloride</td></tr><tr><td>Lysis Buffer</td><td>n/a</td><td>n/a</td><td>50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl, 0.133 g/mL lysozyme, 0.49% v/v Triton X-100. Adjust pH to 8.0</td></tr><tr><td>Lysozyme</td><td>MP Biomedicals</td><td>195303</td><td>Used in protein extraction. Enzyme that lyses bacterial cell walls</td></tr><tr><td>Miniprep kit</td><td>Promega</td><td>A1330</td><td>If successful, extracts the pET3a-pro-MMP-3cd construct from transformants</td></tr><tr><td>NdeI</td><td>NEB</td><td>R0111S</td><td>Restriction enzyme to be used with the pET3a vector</td></tr><tr><td>Ni-NTA resin</td><td>Thermo Fisher Scientific</td><td>PI88221</td><td>Used to bind recombinant his-tagged proteins. This strong interaction can be displaced with higher concentrations of imidazole</td></tr><tr><td>Nonionic surfactant</td><td>Thermo Fisher Scientific</td><td>PI28316</td><td>Storage detergent for preventing MMP aggregation. Minimizes interactions between hydrophobic residues on the MMP surface and water molecules, without disrupting catalytic activity.</td></tr><tr><td>PCR mix</td><td>NEB</td><td>M0492S</td><td>A PCR reagent for inserting an N-terminal his-tag into the pET3a-pro-MMP-3cd vector</td></tr><tr><td>pET plasmid</td><td>Addgene</td><td>n/a</td><td>The pET3a vector offers ampicillin resistance, inducible expression of a target gene, and sequencing with T7 primers</td></tr><tr><td>Petri dishes</td><td>VWR</td><td>25384-342</td><td>Used for plating transformants on LB agar media</td></tr><tr><td>R2DP cells</td><td>Novagen</td><td>714033</td><td>BL21 derivatives with enhanced expression of eukaryotic proteins. Contain tRNAs of codons found to be rare in e. coli</td></tr><tr><td>SOC growth media</td><td>NEB</td><td>B9020S</td><td>Non-selective growth media for rapid growth during transformation</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Thermo Fisher Scientific</td><td>BP358-1</td><td>Used in buffers and helps with protein stability</td></tr><tr><td>Sodium deoxycholate</td><td>Thermo Fisher Scientific</td><td>PI89905</td><td>Detergent used in protein extraction. Lyses cell walls</td></tr><tr><td>Solubilization Buffer</td><td>n/a</td><td>n/a</td><td>20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM DTT, 6 M Urea. Adjust pH to 8.0</td></tr><tr><td>Tris base</td><td>Thermo Fisher Scientific</td><td>BP152-1</td><td>Common buffer used in the physiological pH range. Temperature-sensitive</td></tr><tr><td>Triton X-100</td><td>Thermo Fisher Scientific</td><td>M1122980101</td><td>Detergent used for cell lysis</td></tr><tr><td>Urea</td><td>Thermo Fisher Scientific</td><td>AAJ75826A7</td><td>First chaotropic agent for disrupting protein secondary structure</td></tr><tr><td>Zinc chloride (ZnCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Thermo Fisher Scientific</td><td>AAA162810E</td><td>Stabilizes MMP structure. The zinc ion is found in the catalytic site of MMP-3</td></tr></tbody></table>

bacterial expression and purification of human matrix metalloproteinase-3 using affinity chromatography

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

When running samples on SDS-PAGE, because the protein is expressed in the form of insoluble inclusion bodies, the lysed and sonicated fractions should contain little to no Hisx6-pro-MMP-3cd extract, as the protein has not yet been resolubilized in urea. Figure 3 compares the His-tag purification elution fractions of Hisx6-pro-MMP-3cd from BL21(DE3) cells and R2DP cells. Elution fractions were pooled separately for both BL21(DE3) and R2DP cells before dialysis. Fractions from each step were run after the proteins were desalted (Figure 4). All Hisx6-pro-MMP-3cd samples display a band at approximately 30 kDa, and active MMP-3cd displays a band at approximately 20 kDa upon removal of the His-tag and pro-domain (Figure 4 and Supplemental Figure S1).
The total yield of protein in mg/L of E. coli culture was determined after purification and desalting of BL21(DE3) and R2DP cultures (Table 1). Using R2DP cells yields substantially higher levels of MMP expression. Whereas regular BL21(DE3) cells yielded only 3.5 mg of purified Hisx6-pro-MMP-3cd per liter of culture, R2DP cells produced 45 mg /L culture. Similarly, yields of functional, desalted MMP-3cd increased from 0.13 mg/L culture to 6.2 mg/L culture for BL21(DE3) and R2DP cells, respectively. Human pro-MMP-3cd overwhelms the cellular machinery of the standard BL21(DE3) strain because of its size (approximately 30 kDa) and the elaborate posttranslational modifications required that are exclusive to eukaryotes. The R2DP strains are BL21(DE3) derivatives, designed to enhance the expression of eukaryotic proteins. The R2DP strain carries tRNAs for AGA, AGG, AUA, CUA, GGA, CCC, and CGG, which are rarely used in E.coli but abundant in the pro-MMP-3cd DNA sequence. This is potentially a key factor in the increased levels of protein expression observed in R2DP cells.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63263/63263fig03.jpg" /> 
Figure 3: SDS-PAGE gel analysis of Hisx6-pro-MMP-3cd expression in BL21(DE3) and R2DP cells. (A) The first eight elution fractions of Hisx6-pro-MMP-3cd in BL21(DE3) cells. (B) The first eight elution fractions of Hisx6-pro-MMP-3cd in R2DP cells. Following extraction and solubilization of MMP inclusion bodies in urea, Hisx6-pro-MMP-3cd samples were purified through Ni-NTA chromatography column using batch-gravity flow. Gels are truncated to show only the elution fractions. Initially, due to high concentrations of protein, fractions 1-5 are 1 mL. Later fractions (6-8) are between 5 and 8 mL each. The Hisx6-pro-MMP-3cd band is observed at ~30 kDa. Abbreviations: MMP-3cd = Matrix metalloproteinase-3 catalytic domain; SDS-PAGE = sodium dodecylsulfate polyacrylamide gel electrophoresis; Ni-NTA = nickel-nitrilotriacetic acid; FT = flowthrough. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63263/63263fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63263/63263fig04.jpg" /> 
Figure 4: Proteolytic cleavage of His-tag and pro-domain upon activation of MMP-3cd. The induced, refolded, concentrated, activated, and desalted fractions of MMP-3cd in R2DP cells are shown. After dialysis, Hisx6-pro-MMP-3cd is concentrated and activated using APMA.&#160;Upon activation, the molecular weight of the activated MMP-3cd band is approximately 20 kDa, as opposed to the His-tagged zymogen, which remains at ~30 kDa. Impurities are removed in the activation and desalting stages. Abbreviations: MMP-3cd = Matrix metalloproteinase-3 catalytic domain; APMA = 4-aminophenylmercuric acetate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63263/63263fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Stages</td>
			<td colspan="3">BL21(DE3) cells</td>
			<td colspan="3">R2DP cells</td>
		</tr>
		<tr>
			<td>Volume (mL)</td>
			<td>Concentration (mg/mL)</td>
			<td>Yield (mg/L culture)</td>
			<td>Volume (mL)</td>
			<td>Concentration (mg/mL)</td>
			<td>Yield (mg/L culture)</td>
		</tr>
		<tr>
			<td>Purification</td>
			<td>23</td>
			<td>0.30</td>
			<td>3.5</td>
			<td>42</td>
			<td>2.1</td>
			<td>45</td>
		</tr>
		<tr>
			<td>Desalting</td>
			<td>1.5</td>
			<td>0.17</td>
			<td>0.13</td>
			<td>72</td>
			<td>0.17</td>
			<td>6.2</td>
		</tr>
	</tbody>
</table>
Table 1: Table of volumes and concentrations across stages of MMP-3cd purification. Hisx6-pro-MMP-3cd was expressed either in 2 L of a culture of BL21(DE3) or in 2 L of R2DP cells. Volume, concentration, and yield (mg per liter) culture are reported for BL21(DE3) and R2DP cells. Yield (mg per liter of culture) was obtained by dividing the total yield of protein (mg) obtained by volume of culture, which was 2 L for both the BL21(DE3) and R2DP cases. Protein amounts yields are reported for two stages: Hisx6-pro-MMP-3cd following Hisx6-tag purification and active MMP-3cd after desalting.
Supplemental Figure S1: Sequences of T7 primers and MMP-3cd protein before and after activation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63263/Supplemental Files.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography at JoVE.com

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...

bacterial-expression-purification-human-matrix-metalloproteinase-3

Nanyang Technological University

Research

JoVE Journal

Biochemistry

4.4K Views.  University of Nevada, Reno. His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

Video: Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become the most widely used protein purification method in the modern literature. But, the application of affinity chromatography to metal binding proteins, especially those with redox sensitive metals such as iron, is often limited to laboratories with access to a glove box - equipment that is not routinely available in the undergraduate laboratory. In this article, we demonstrate our benchtop methods for isolation, IMAC purification and metal-ion reconstitution of a poly-histidine tagged, redox-active, non-heme iron binding extradiol dioxygenase and the assay of the dioxygenase with varied substrate concentrations and saturating oxygen. These methods are executed by undergraduate students and implemented in the undergraduate teaching and research laboratory with instrumentation that is accessible and affordable at primarily undergraduate institutions.

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Biology

Recombinant Human Myosin-7a Production and Functional Characterization Protocol

MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common and severe form of deaf-blindness in humans. It is hypothesized that myosin-7a forms a transmembrane adhesion complex with other Usher proteins, essential for the structural-functional integrity of photoreceptor and cochlear hair cells. However, due to the challenges in obtaining pure, intact protein, the exact functional mechanisms of human myosin-7a remain elusive, with limited structural and biomechanical studies available. Recent studies have shown that mammalian myosin-7a is a multimeric motor complex consisting of a heavy chain and three types of light chains: regulatory light chain (RLC), calmodulin, and calmodulin-like protein 4 (CALML4). Unlike calmodulin, CALML4 does not bind to calcium ions. Both the calcium-sensitive, and insensitive calmodulins are critical for mammalian myosin-7a for proper fine-tuning of its mechanical properties. Here, we describe a detailed method to produce recombinant human myosin-7a holoenzyme using the MultiBac Baculovirus protein expression system. This yields milligram quantities of high-purity full-length protein, allowing for its biochemical and biophysical characterization. We further present a protocol for assessing its mechanical and motile properties using tailored in vitro motility assays and fluorescence microscopy. The availability of the intact human myosin-7a protein, along with the detailed functional characterization protocol described here, paves the way for further investigations into the molecular aspects of myosin-7a in vision and hearing.

Author Spotlight: Unraveling the Role of Myosin-7a and Usher Proteins in Hearing and Human Disease

This protocol details the procedures for recombinantly producing the human myosin-7a holoenzyme using the MultiBac Baculovirus system and for studying its motility using a tailored in vitro filament gliding assay.

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Bioengineering

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...