This work was supported by a Wellcome Project Grant (078043/Z/05/Z) to R.O.J.W.

PART A: High-throughput growth of bacterial cultures.
1. Grow Bacteria Overnight in 2 ml of Autoinduction Medium Using 24-well Plates
<ol>
 <li>Sterilise the 24-well plates by microwaving.</li>
 <li>Inoculate 1.5 ml of autoinduction medium (Overnight Express Medium) with freshly grown bacterial colonies or frozen glycerol stocks. We normally inoculate three wells per mutant with three individual cloned colonies. Reserve six wells for positive and negative controls. For the positive controls we grow up three wildtype clones and for the negative controls we grow up 2 clones which have been transformed with a non-expressing plasmid. Check for sufficient sterilization of the plate by leaving one well blank for the medium-only control.</li>
 <li>Grow the cells for 18 hr at 37 &deg;C and shaking at 250 rpm. We use lac-inducible BL21 (DE3) Rosetta 2 cells. Autoinduction medium contains a mixture of glucose and lactose. The bacteria initially feed on glucose and then begin to use lactose, which also induces the expression of the recombinant proteins.</li>
 <li>Remove the lid and place the plate on the robotic platform.</li>
</ol>
PART B: Robotic purification of recombinant proteins.
2. Prepare the Robotic Platform
<ol>
 <li>
Make up the wash buffer consisting of 20 mM imidazole, 0.1% Triton X-100, 0.5 M NaCl, 20 mM Tris-acetate, pH 7.9, 10 mM MgOAc2, 0.7 mM ZnOAc2, 10% glycerol, and the elution buffer consisting of 0.5 M imidazole, 0.1 % Triton X-100, 0.5 M NaCl, 20 mM Tris-acetate, pH 7.9, 10 mM MgOAc2, 0.7 mM ZnOAc2, 10% glycerol. These buffers will be used to wash the beads after the tagged proteins have been bound to them, or to elute the proteins from the beads, respectively.</li>
 <li>Make up the bacterial diluent (100 ml distilled water with 15 &mu;l antifoam reagent). This solution will be used to make dilutions of the bacterial overnight cultures for optical density (A600) measurements. The presence of antifoam in the diluent prevents the formation of air bubbles that would interfere with the plate reader measurements.</li>
 <li>Make up the lysis solution consisting of 10x FastBreak reagent, 2 &mu;l lysonase per sample and 15 mM MgOAc2. FastBreak contains a mix of detergents and salts that break the bacterial cell walls and facilitate the release of intracellular proteins. Lysonase is a proprietary mix of lysozyme and a nuclease. Lysozyme assists in disrupting the cell wall and the nuclease digests the released bacterial nucleic acids.</li>
 <li>Optional: Make up a 6 M guanidine hydrochloride solution. Some recombinant proteins tend to stick to the Teflon-coated pipetting needles. The guanidine hydrochloride solution denatures proteins and washes them off more effectively than water that is routinely used to rinse the washable robotic pipetting tips after each step.</li>
 <li>Prepare the BCA (bicinchoninic acid) protein quantitation reagent by mixing bicinchoninic acid solution (reagent A) and the copper sulphate solution (reagent B): Peptide bonds reduce Cu2+ from the CuSO4 component present in the BCA reagent to Cu1+ whereby the amount of Cu1+ is proportional to the number of peptide bonds present in the solution. In a second step, two molecules of bicinchoninic acid chelate Cu1+ resulting in an absorbance shift to 562 nm, resulting in a purple colour. The colour reaction is time-dependent and usually needs several hours to be definitive. After that the colour is stable for several hours.</li>
 <li>Fill troughs or plates of the right size and place them in their pre-allocated positions on the robotic platform.</li>
 <li>Place one 24-well plate for the guanidine hydrochloride waste, two clear 96-well plates for OD600 and absorbance measurements and one blue 96-well plate for the purified proteins on their positions on the platform. Place one 96-deepwell plate on the magnetic stand.</li>
 <li>Dilute MagneHis Ni-particles which bind proteins by forming chelates with their His-tags 5-fold in distilled water and fill them into the bead-stirring unit. Switch the stirrer on to keep the beads in suspension.</li>
 <li>Switch on the robotic platform and flush the pipetting needles for several minutes to remove air bubbles which would otherwise interfere with the pipetting accuracy. The re-usable pipetting needles are flushed in between individual pipetting steps. Alternatively, disposable tips could be used. All subsequent steps are carried out robotically. The robotic protocol is available on request.</li>
</ol>
3. Cell Growth is Checked by Measuring the OD600
<ol>
 <li>10 &mu;l of overnight culture is diluted in 90 &mu;l of diluent solution.</li>
 <li>Measure the OD600 to ensure that the bacteria have grown to similar densities (Figure 1).</li>
</ol>
4. The Cells are Broken up to Release the Proteins and to Allow Bead-binding
<ol>
 <li>100 &mu;l of magnetic Ni-bead suspension is distributed into each well of a second 24-well plate.</li>
 <li>900 &mu;l of each small-scale bacterial culture is transferred into the 24-well plate containing the beads. Add 100 &mu;l of the 10x FastBreak/lysonase mix.</li>
 <li>The 24-well plate is transferred to the shaking platform for rapid shaking (800 rpm) for 30 min at room temperature. Bacterial cell walls are disrupted by a combination of mechanical forces and chemical action and the recombinantly produced proteins are released into solution. With their affinity tags they immediately bind the paramagnetic chelating nickel beads.</li>
</ol>
5. The Beads are Washed
<ol>
 <li>The cell-lysates containing the resuspended magnetic beads are transferred to a 96-deepwell plate which is positioned on a stand with magnetic rods that slide between the wells. The 96-well plates have got a square cone-shaped bottom which allows easier removal of the supernatant. The wells can hold volumes of up to 2.1 ml but are filled with much smaller volumes. This enables us to perform vigorous shaking steps without sample cross-contamination by splashing.</li>
 <li>The pipette tips are washed between individual pipetting steps with 6 M guanidine-HCl. This step is crucial for the purification of TFIIB and other "sticky" proteins to avoid cross-contamination. With other proteins it may be sufficient to rinse the needles extensively with water between the pipetting steps.</li>
 <li>The magnetic rods of the magnetic stand attract the paramagnetic beads, pulling them away from the centres of the wells and allow the pipetting needles free access to the supernatant. The supernatant is discarded.</li>
 <li>500 &mu;l of wash buffer is first added to the 24-well plate and subsequently transferred to the 96-well plate to ensure the complete transfer of the beads. The 24-well plate is removed from the shaker.</li>
 <li>Another 500 &mu;l of wash buffer is added directly to the 96-well plate, the plate is transferred to the shaker and vigorously shaken for 1 min. The plate is moved back to the magnetic stand and the wash buffer discarded.</li>
 <li>This step is repeated twice and the wash procedure is finished by removing any buffer remnants from the plate.</li>
</ol>
6. Elute the Proteins in Elution Buffer
<ol>
 <li>100 &mu;l of elution buffer is added to the beads, the plate is moved to the shaker and vigorously shaken for 30 min at room temperature.</li>
 <li>The plate is moved back to the shaker and the eluate, containing the purified recombinant protein, is transferred to a new plate.</li>
</ol>
7. Measure the Concentrations of the Purified Proteins
<ol>
 <li>190 &mu;l of BCA reagent mix is transferred to a clear 96-well plate and 10 &mu;l of the protein solution added.</li>
 <li>After several hours of incubation (typically 5-6 or overnight), measure the absorbance and compare it to BSA standards to determine the concentrations of each of the purified protein preparations (Figure 2).</li>
 <li>The purified proteins can now be used for downstream applications at the appropriate concentrations (Figure 3).</li>
</ol>
8. Representative Results
The purification protocol offers two quality control stages, examples of which are shown. We are able to identify and document potential problems at the bacterial growth stage (Figure 1) and later on upon assessing the yields of the purified proteins (Figure 2). We typically purify proteins and test them in triplicate. This, in combination with the two quality control steps, gives us confidence that any variation we observed in our functional assays are due to the mutant phenotype (Figure 3) and not caused by experimental variations or failed purifications. The yields obtained typically range from 50-200 &mu;g and are more than sufficient for various functional assays.
<img alt="Figure 1" src="/files/ftp_upload/4110/4110fig1.jpg" /> 
 Figure 1. Histogram of the OD measurements of a 24 well plate with overnight cultures. Three clones of six TFIIB mutant variants as well as of the wildtype TFIIB have been grown. Two clones carrying a non-expressing plasmid and a well with medium only serve as negative controls. The OD measurements show that there are small variations in the growth rates between individual cultures.
<img alt="Figure 2" src="/files/ftp_upload/4110/4110fig2.jpg" /> 
 Figure 2. Protein yields obtained from these cultures as determined by a BCA assay and confirmed by SDS PAGE. One of the variants is not expressed at high levels. In combination with Figure 1, we can conclude that this was not due to differential cell growth but due to protein expression not being induced properly.
<img alt="Figure 3" src="/files/ftp_upload/4110/4110fig3.jpg" /> 
 Figure 3. Representative result of a transcription assay. We measured the stimulation activity of TFIIB variants on the production of small abortive transcripts by RNAP. Here, the stimulation effects of a full library of single amino acid substitutions of TFIIB residue K87 are shown. The high degree of reproducibility is confirmed by small error rates. A sample gel showing the performance of three mutants as compared to wildtype (wt), negative controls (NC) and elution buffer only controls is depicted underneath.

<ol>
	<li>Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleotide+addition+cycle+of+RNA+polymerase+is+controlled+by+two+molecular+hinges+in+the+Bridge+Helix+domain.">The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain.</a> BMC Biol. 8, 1741-7007 (2010).</li><li>Nottebaum, S., Tan, L., Trzaska, D., Carney, H. C., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+polymerase+factory:+a+robotic+in+vitro+assembly+platform+for+high-throughput+production+of+recombinant+protein+complexes.">The RNA polymerase factory: a robotic in vitro assembly platform for high-throughput production of recombinant protein complexes.</a> Nucleic Acids Res. 36, 245-252 (2008).</li><li>Tan, L., Wiesler, S., Trzaska, D., Carney, H. C., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bridge+helix+and+trigger+loop+perturbations+generate+superactive+RNA+polymerases.">Bridge helix and trigger loop perturbations generate superactive RNA polymerases.</a> J. Biol. 7, 40 (2008).</li><li>Wiesler, S. C., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+linker+domain+of+basal+transcription+factor+TFIIB+controls+distinct+recruitment+and+transcription+stimulation+functions.">The linker domain of basal transcription factor TFIIB controls distinct recruitment and transcription stimulation functions.</a> Nucleic Acids Res. 39, 464-474 (2011).</li><li>Weinzierl, R. O., Wiesler, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+Function+of+TFIIB.">Revealing the Function of TFIIB.</a> Transcription. , (2011).</li><li>Bell, S. D., Magill, C. P., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+and+regulated+transcription+in+Archaea.">Basal and regulated transcription in Archaea.</a> Biochemical Society transactions. 29, 392-395 (2001).</li><li>Parvin, J. D., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+topology+and+a+minimal+set+of+basal+factors+for+transcription+by+RNA+polymerase+II.">DNA topology and a minimal set of basal factors for transcription by RNA polymerase II.</a> Cell. 73, 533-540 (1993).</li><li>Tyree, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+minimal+set+of+proteins+that+is+sufficient+for+accurate+initiation+of+transcription+by+RNA+polymerase+II.">Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II.</a> Genes. 7, 1254-1265 (1993).</li><li>Batada, N. N., Westover, K. D., Bushnell, D. A., Levitt, M., Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+nucleoside+triphosphates+and+role+of+the+entry+site+to+the+RNA+polymerase+II+active+center.">Diffusion of nucleoside triphosphates and role of the entry site to the RNA polymerase II active center.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 17361-17364 (2004).</li><li>Liu, X., Bushnell, D. A., Wang, D., Calero, G., Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+an+RNA+Polymerase+II-TFIIB+Complex+and+the+Transcription+Initiation+Mechanism.">Structure of an RNA Polymerase II-TFIIB Complex and the Transcription Initiation Mechanism.</a> Science (New York, N.Y). 327, 206-209 (2010).</li><li>Kostrewa, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+II-TFIIB+structure+and+mechanism+of+transcription+initiation.">RNA polymerase II-TFIIB structure and mechanism of transcription initiation.</a> Nature. 462, 323-330 (2010).</li><li>Werner, F., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+modulation+of+RNA+polymerase+core+functions+by+basal+transcription+factors.">Direct modulation of RNA polymerase core functions by basal transcription factors.</a> Molecular and cellular biology. 25, 8344-8355 (2005).</li></ol>

No conflicts of interest declared.

The automated recombinant protein purification method described here allows the production and purification of a large number of mutant proteins in a small-scale format under highly reproducible conditions with minimal human intervention. Figures 1 and 2 show results of systematic quality-controls and examples of the purified proteins. Figure 3 shows that the purified transcription factors used in this example perform in a highly reproducible manner in functional assays.
Even though the procedure was developed for the purification of archaeal TFIIB, it is widely applicable for the purification of affinity-tagged proteins. The use of such automated purification protocols will thus significantly facilitate the biochemical analysis of recombinant proteins and will thus further our understanding of protein-protein interactions on a scale that is difficult to achieve manually.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Overnight Express Instant TB Medium</td>
 <td>Merck Chemicals Ltd </td>
 <td>71491-4 </td>
 <td></td>
 </tr>
 <tr>
 <td>FastBreak</td>
 <td>Promega Ltd.</td>
 <td>V8573 </td>
 <td></td>
 </tr>
 <tr>
 <td>Lysonase Bioprocessing Reagent/ 1 ml </td>
 <td>Merck Chemicals Ltd </td>
 <td>71230 </td>
 <td></td>
 </tr>
 <tr>
 <td>Antifoam 204</td>
 <td>Sigma-Aldrich Company Ltd</td>
 <td>A6426</td>
 <td></td>
 </tr>
 <tr>
 <td>MagneHis Ni-Particles</td>
 <td>Promega</td>
 <td>V8565 </td>
 <td></td>
 </tr>
 <tr>
 <td>Imidazole</td>
 <td>Sigma-Aldrich Company Ltd</td>
 <td>56750 </td>
 <td></td>
 </tr>
 <tr>
 <td>Trizma base</td>
 <td>Sigma-Aldrich Company Ltd.</td>
 <td>93362</td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>VWR</td>
 <td>27810.295 </td>
 <td></td>
 </tr>
 <tr>
 <td>Bicinchoninic Acid protein determination </td>
 <td>Sigma-Aldrich Company Ltd </td>
 <td>BCA1-1KT </td>
 <td></td>
 </tr>
 <tr>
 <td>Deep Well Plate 2.2 ml Square Wells PP pk10 </td>
 <td>Anachem Ltd </td>
 <td>1810-00 </td>
 <td></td>
 </tr>
 <tr>
 <td>Microplate MicroWell 96 well flat bottom polystyrene not treated 12 sleeves of 5 plates clear 0.4 ml well volume 128 mm x 86 mm Thermo Scientific Nunc </td>
 <td>Fisher Scientific Ltd.</td>
 <td>DIS-984-090M </td>
 <td></td>
 </tr>
 <tr>
 <td>Microplate Blue</td>
 <td>VWR</td>
 <td>NUNC367001 </td>
 <td></td>
 </tr>
 <tr>
 <td>24-Well Blocks RB (24) </td>
 <td>Qiagen</td>
 <td>19583 </td>
 <td></td>
 </tr>
 <tr>
 <td>Guanidine hydrochloride</td>
 <td>VWR</td>
 <td>ALFAA13543.0B </td>
 <td></td>
 </tr>
 <tr>
 <td>Washable needle for TheOnyx</td>
 <td>Aviso GmbH</td>
 <td>8152-317001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Reagent Rack for Magnetic Beads </td>
 <td>Aviso GmbH</td>
 <td>8152-035003 </td>
 <td></td>
 </tr>
 <tr>
 <td>Plate Reader Synergy HT</td>
 <td>BioTek</td>
 <td>4200-000043</td>
 <td></td>
 </tr>
 <tr>
 <td>Robotic Platform TheOnyx 44OH/150/100</td>
 <td>Aviso GmbH</td>
 <td>8145-050046</td>
 <td></td>
 </tr>
 <tr>
 <td>Microplate Shaker Variomag Teleshaker</td>
 <td>Inheco</td>
 <td>3800047 </td>
 <td></td>
 </tr>
 <tr>
 <td>96-well Magnet Type A</td>
 <td>Qiagen</td>
 <td>36915 </td>
 <td></td>
 </tr>
 </tbody>
</table>

high-throughput purification of affinity-tagged recombinant proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

Watch this Scientific Journal Video about High-throughput Purification of Affinity-tagged Recombinant Proteins at JoVE.com

High-throughput Purification of Affinity-tagged Recombinant Proteins

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

high-throughput-purification-of-affinity-tagged-recombinant-proteins

Nanyang Technological University

Research

JoVE Journal

Biology

14.1K Views.  Imperial College London. We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

Video: High-throughput Purification of Affinity-tagged Recombinant Proteins

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...