We thank Tomer Zohar for work on scFv screening and characterization assays. A portion of this work was supported by award number F32CA150622 from the National Cancer Institute (to AJK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

<ol>
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J., 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=Engineering+antibody+fitness+and+function+using+membrane-anchored+display+of+correctly+folded+proteins.">Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins.</a> J. Molec. Biol. 416 (1), 94-107 (2012).</li><li>DeLisa, M. P., Tullman, D., Georgiou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+quality+control+in+the+export+of+proteins+by+the+bacterial+twin-arginine+translocation+pathway.">Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway.</a> Proc Natl Acad Sci U S A. 100 (10), 6115-6120 (2003).</li><li>Fisher, A. C., Kim, W., DeLisa, M. 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The authors declare that they have no competing financial interests.

Engineering antibodies for cytoplasmic activity is a difficult task due to the reducing milieu of the cytoplasm, which impedes the formation of stabilizing disulfide bonds6,7. This causes most antibodies to be cytoplasmically inactive unless they are engineered for stability and solubility in the cytoplasm, in addition to being engineered for binding affinity. The existing methods of phage display, bacterial surface display, and yeast surface display methods all use the secretory pathway14-16 for the display of engineered antibodies, but these methods have no means to engineer intracellular folding. Antibodies engineered using inner-membrane display have improved cytoplasmic stability and solubility because the folding quality control of the Tat pathway prevents translocation of antibodies that are poorly folded and unstable in the cytoplasm. This method simplifies the iterative process of engineering intracellular antibodies for affinity and solubility, as the two properties are engineered in one step. Although this method was designed for engineering antibodies with solubility in the reducing intracellular environment, it could also be applied to engineering antibodies to function in non-reducing conditions, since the proteins engineered using this method maintain their folding in the oxidizing environment of the periplasm.
Although this technique simplifies the process of engineering antibodies with high affinity and high cytoplasmic solubility, several limitations are important to consider when using this protocol. When analyzing the secondary screen ELISA signals to identify promising scFv variants, the threshold for discerning between potentially interesting variants and those that may not exhibit adequate antigen-binding is not likely to be apparent until after several clones have been further characterized. It is important to look for improved binding over the parent antibody; however, an abnormally high signal could be indicative of avidity29 or aggregation effects30, a challenge which is not unique to the inner-membrane display screening approach. A key limitation to remember when using this protocol is the inability to recover spheroplasts after panning, as they are non-viable (unpublished data). This necessitates the DNA amplification and transformation steps to recover the antibody-encoding plasmids.
Several critical steps of the protocol enable the simultaneous engineering of folding and binding of antibodies. For screening to be successful, the scFv library being screened must be expressed as a fusion to the ssTorA signal peptide. Without this sequence, antibodies will not be directed to the Tat pathway and thus will not be translocated to the periplasm19. Additionally, it is imperative that a C-terminal epitope tag is fused to the antibodies to allow detection of the displayed antibodies in the binding assays. Clearly, the E. coli strain used to express the scFvs must also have the necessary Tat pathway machinery, but this is true of the commonly used E. coli strains.
Modifications to this protocol are possible to improve its potential to isolate antibodies with the desired characteristics. A subtractive panning step may be completed prior to panning against the target antigen to deplete the scFv library of non-desired constituents. The library spheroplasts can be incubated with magnetic beads coated with BCCP alone or coated with a non-desired protein, and the spheroplasts that bind to those beads can be discarded before screening the remaining unbound spheroplasts for binding to the desired target. As mentioned in the Representative Results, a method to improve the affinity of an isolated scFv is to include a soluble competitor in the panning reaction to compete with the scFvs displayed on the spheroplasts. Because the soluble competitor is a purified protein, no DNA is amplified from it, so only sequences of the scFvs displayed on the spheroplasts will be recovered in the PCR reaction. Additionally, this method could be extended to engineering other types of antibodies or to non-antibody binding proteins.
E. coli inner-membrane display is a powerful platform for engineering antibodies with high affinity and high levels of intracellular solubility. This method is particularly suited for efficient engineering of antibodies designed to function in the intracellular environment. These intracellular antibodies are already being explored as potential therapeutics in a number of fields, including neurodegenerative diseases, cancer, and viral infections31. This technique will enable more widespread use of intracellular antibodies as tools for research and medicine in these fields and any other field where studying a protein target in situ is desired.

Antibodies capable of folding and functioning in the intracellular environment are promising tools for both research and therapeutic applications. They have the ability to modulate protein activity by binding to a target protein inside cells to prevent protein-protein interactions, disrupt protein-nucleic acid interactions, or prevent substrate access to enzymes1-5.
Although antibodies have much potential for intracellular applications, engineering them for proper folding and solubility in the intracellular environment while maintaining the ability to bind to a target antigen is challenging. The reducing cytoplasmic environment prevents the formation of the disulfide bonds normally required for the stable folding of full-length antibodies and antibody fragments, including single-chain variable fragment (scFv) antibodies6,7. A number of directed evolution approaches have been employed to engineer antibodies with high affinities for target antigens8-10. These approaches commonly use phage display, yeast surface display, or bacterial surface display to screen large libraries of antibodies11-13. These methods are powerful and effective for identifying antibodies that bind to targets, yet they depend on the secretory pathway to transport proteins that will be displayed14-16. The secretory pathway translocates unfolded proteins from the reducing cytoplasm into the endoplasmic reticulum lumen in yeast or into the periplasm in bacteria. The proteins then fold under oxidizing conditions and are displayed on the cell surface or packaged into phage particles to screen for binding affinity17,18. As a result, antibodies isolated using these techniques will not necessarily fold well in the cytoplasm, and intracellular solubility must often be engineered separately if the antibodies will be used in intracellular applications.
To improve the efficiency of engineering antibodies that are well folded in the cytoplasm, we previously reported the success of MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins), a method for screening an scFv antibody library using Escherichia coli inner-membrane display19. Bacterial inner-membrane display relies on the twin-arginine translocation (Tat) pathway for transporting displayed antibodies, in contrast to other common display methods that use the secretory pathway. The Tat pathway contains a quality control mechanism that only allows soluble, correctly folded proteins to be transported from the E. coli cytoplasm, across the inner membrane, and into the periplasm20,21. Overexpressed Tat substrates (i.e., proteins targeted to the Tat pathway with an N-terminal fusion to the Tat signal peptide ssTorA) that are well folded in the cytoplasm form a long-lived translocation intermediate with the N-terminus in the cytoplasm and the C-terminus in the periplasm19. This allows display of correctly folded Tat substrates, including antibody fragments, on the periplasmic face of the E. coli inner membrane. After removing the outer membrane by enzymatic digestion to generate spheroplasts, antibodies are exposed to the extracellular space (Figure 1). This allows Tat substrates displayed on the inner membrane to be screened for binding to a specific target. Importantly, harnessing the Tat pathway for cell-surface display ensures that only the antibodies in the library that are well folded in the cytoplasm will be interrogated for binding, allowing simultaneous engineering of binding affinity and intracellular folding. In this protocol, we describe how to display an scFv library on the E. coli inner membrane, pan the library against a target antigen, and perform a secondary screen to identify the most promising constituents of the library. While we focus the protocol on scFvs, the method could be applied to engineering any protein whose application requires binding and intracellular folding.
<img alt="Figure 1" src="/files/ftp_upload/54583/54583fig1.jpg" /> 
Figure 1. Tat inner-membrane display. In E. coli, scFv antibodies that are expressed as a fusion to the ssTorA signal sequence and correctly folded in the cytoplasm are transported across the inner membrane. A translocation intermediate forms, where the scFvs are anchored in the inner membrane with the N-terminus in the cytoplasm and the C-terminus in the periplasm. The E. coli outer membrane is enzymatically digested to form spheroplasts, thereby exposing the anchored antibodies to the extracellular space and making them available for detection by using an antibody that binds to the C-terminally fused epitope tag on the displayed antibody. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54583/54583fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1824">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">scFv library</td>
			<td style="width:205px;">Varies</td>
			<td style="width:135px;">N/A</td>
			<td style="width:859px;">A suitable scFv library should be obtained from a commercial or academic source.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MC4100 E. coli cells</td>
			<td>Coli Genetic Stock Center</td>
			<td>6152</td>
			<td>Cells need to be chemically competent or electrocompetent, depending on the selected transformation method.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Difco dehydrated culture media LB Broth, Miller (Luria-Bertani)</td>
			<td style="width:205px;">BD</td>
			<td style="width:135px;">244610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Chloramphenicol (Cm)</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:135px;">BP904-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium phosphate, dibasic (Na2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Potassium phosphate, monobasic (KH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Bovine serum albumin (BSA)</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:135px;">BP9706-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Fisher Scientific</td>
			<td>BP220-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP1521</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminetetraacetic acid (EDTA), 0.5 M</td>
			<td>Fisher Scientific</td>
			<td>BP2482-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Magnesium chloride (MgCl2)</td>
			<td>Fisher Scientific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Lysozyme</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:135px;">L3790-10X1ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex mixer</td>
			<td>VWR</td>
			<td>97043-564</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bicine</td>
			<td>Fisher Scientific</td>
			<td>BP2646100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">D-Biotin</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:135px;">BP232-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Fisher Scientific</td>
			<td>BP1755-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BugBuster Master Mix (cell lysis detergent)</td>
			<td>EMD Millipore</td>
			<td>71456</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vivaspin 2 MWCO, 3000 daltons</td>
			<td style="width:205px;">GE Healthcare Sciences</td>
			<td>28932240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Target antigen</td>
			<td>Varies</td>
			<td>N/A</td>
			<td>Purified target antigen may be purchased or produced/purified.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Dynabeads MyOne Streptavidin T1</td>
			<td style="width:205px;">Invitrogen</td>
			<td style="width:135px;">65601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Dynamag-2 magnet</td>
			<td style="width:205px;">Invitrogen</td>
			<td style="width:135px;">12321D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube rotator</td>
			<td>VWR</td>
			<td>13916-822</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">PCR primers</td>
			<td style="width:205px;">IDT</td>
			<td style="width:135px;">N/A</td>
			<td>Primer sequences are as described in the protocol.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">10X T4 DNA ligase reaction buffer</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">B0202S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">T4 Polynucelotide kinase (PNK)</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">M0201S</td>
			<td>Make sure the T4 ligase buffer used in the primer phosphorylation reaction contains 1 mM ATP.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">5X Phusion HF buffer pack</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">B0518S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Deoxynucleotide (dNTP) solution mix, 10 mM each dNTP</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">N0447L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Phusion DNA polymerase</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">M0530S</td>
			<td>Other high-fidelity polymerases may be used as an alternative, but the annealing temperature in Table 3 must be adjusted.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">C1000 Touch thermal cycler with dual 48/48 fast reaction module</td>
			<td>Bio-Rad</td>
			<td>185-1148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>Promega</td>
			<td>V3121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">SYBR Safe DNA gel stain</td>
			<td style="width:205px;">Invitrogen</td>
			<td style="width:135px;">S33102</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Wizard SV gel and PCR clean-up system</td>
			<td>Promega</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">T4 DNA ligase</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:627px;">Microdialysis membrane filter</td>
			<td style="width:205px;">EMD Millipore</td>
			<td style="width:135px;">VSWP04700</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>BD</td>
			<td>214030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">96-well polystyrene round-bottom cell culture plates</td>
			<td style="width:205px;">VWR</td>
			<td style="width:135px;">10062-902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Costar general polystyrene assay plate lids</td>
			<td style="width:205px;">Corning</td>
			<td style="width:135px;">3931</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microtitre plate shaker</td>
			<td style="width:205px;">VWR</td>
			<td>12620-926</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Costar 96 well EIA/RIA Easy Wash clear flat bottom polystyrene high bind microplate</td>
			<td style="width:205px;">Corning</td>
			<td style="width:135px;">3369</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Bel-blotter polycarbonate 96-well replicating tool</td>
			<td style="width:205px;">Bel-Art Products</td>
			<td>378760002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Instant nonfat dry milk</td>
			<td>Quality Biological</td>
			<td>A614-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tween 20 (polysorbate 20)</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">PopCulture reagent (concentrated cell lysis detergent)</td>
			<td style="width:205px;">EMD Millipore</td>
			<td>71092-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Monoclonal ANTI-FLAG M2-Peroxidase(HRP) antibody produced in mouse</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td>A8592</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">SigmaFast OPD</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:135px;">P9187-50SET</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sulfuric acid (H2SO4), 10N solution</td>
			<td>Fisher Scientific</td>
			<td>SA200-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reynolds Wrap aluminum foil</td>
			<td>VWR</td>
			<td>89079-075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BioTek Epoch microplate spectrophotometer</td>
			<td>Fisher Scientific</td>
			<td>11120570</td>
			<td></td>
		</tr>
	</tbody>
</table>

bacterial inner-membrane display for screening a library of antibody fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

The intracellular protein folding quality control mechanism of the Tat pathway in E. coli limits transport across the inner cell membrane to proteins that are well folded in the reducing cytoplasmic environment. By overexpressing a fusion of an scFv to the ssTorA signal sequence (the signal sequence from the TorA protein, which is naturally transported by the Tat pathway20), translocation is stalled, resulting in display of scFvs on the inner membrane19. After enzymatic disruption of the outer membrane, the displayed antibodies are made available for screening for antigen-binding activity. The ability to take advantage of the Tat pathway for scFv display was shown by Karlsson et al.19 (Figure 6). The scFv antibodies scFv13 and scFv13.R4 were fused to either the native ssTorA sequence or a modified ssTorA that lacks the arginine-arginine residue pair recognized by the Tat pathway. scFv13.R4 was engineered by Martineau et al. from scFv13 through four rounds of directed evolution and is known to fold well in the cytoplasm9. This scFv was displayed on the inner membrane, but only when expressed as a fusion to the native ssTorA signal sequence (Figure 6). Contrarily, scFv13 is not well folded cytoplasmically9, so it is not displayed well on the inner membrane, regardless of the signal sequence to which it is fused. Additionally, if the scFvs were expressed in cells that lacked the TatC protein, a vital component of the Tat machinery20,28, display was not observed, showing the important link between inner-membrane display and the Tat pathway. These results demonstrate that only proteins that contain the Tat signal peptide and that are correctly folded in the cytoplasm are displayed on the inner membrane, allowing transport through the Tat pathway to function as a screen for intracellular folding.
<img alt="Figure 6" src="/files/ftp_upload/54583/54583fig6.jpg" /> 
Figure 6. Detection of displayed scFvs on the inner membrane. Flow cytometry analysis was performed to detect the display of poorly folded scFv13 and well-folded scFv13.R4 on the inner membrane. scFvs were fused to native ssTorA or ssTorA(KK), where the Arg-Arg pair in the ssTorA sequence was modified to Lys-Lys. The C-terminal FLAG epitope tags on the scFvs were detected with a fluorescein isothiocyanate (FITC)-conjugated anti-FLAG antibody. Cells without the TatC protein (&#916;tatC) and ssTorA-scFv13 without the FLAG tag were tested as controls. M indicates the median fluorescence value. Reprinted from reference 19 with permission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54583/54583fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Inner-membrane display can successfully isolate scFv antibodies with high levels of affinity for a target protein and high levels of cytoplasmic solubility. Additionally, subsequent rounds of directed evolution using inner-membrane display improve antibody characteristics19. To demonstrate this, an error-prone PCR library based on scFv13, which has a low level of binding affinity for &#946;-galactosidase, was panned against the target antigen &#946;-galactosidase using the display and panning method described in the protocol. scFv 1-4 was isolated after one round of mutagenesis and panning, and exhibited higher binding affinity to &#946;-galactosidase than scFv13 (Figure 7A) and a higher level of cytoplasmic solubility (Figure 7B).
A new library, based on scFv 1-4, was made using error-prone PCR, and panning of this second-generation library against &#946;-galactosidase was done using a modification of the described protocol. The panning against &#946;-galactosidase for the second round of evolution was done in the presence of purified, soluble scFv 14 as a competitor to improve the likelihood of isolating clones with higher affinity than scFv 1-4. After this second round of mutagenesis and panning, scFv 2-1 and scFv 2-3 were isolated using the ELISA-based secondary screening. These scFvs not only exhibited higher binding affinity for &#946;-galactosidase than scFv13, but also exhibited better binding than the first-round clone scFv 1-4. scFv 2-1 exhibited &#946;-galactosidase binding comparable to that of scFv13.R4 (Figure 7A). scFv 2-3 also shows a further increase in cytoplasmic solubility compared to scFv 14, highlighting the simultaneous engineering of solubility and antigen-binding. Since affinity and soluble expression of the scFvs are screened for simultaneously, it is possible that a selected scFv has moderate solubility but high binding or vice versa. For example, scFv 2-1 has lower soluble expression than scFv 2-3, but it exhibits higher binding affinity to &#946;-galactosidase.
<img alt="Figure 7" src="/files/ftp_upload/54583/54583fig7.jpg" /> 
Figure 7. Target-binding and cytoplasmic expression of scFv variants isolated using inner-membrane display. (A) scFvs were expressed in the cytoplasm of E. coli cells (e.g., without the ssTorA signal sequence) with a hexahistidine (6&#215;-His) tag and purified using nickel-nitrilotriacetic acid spin-columns. The binding of the purified scFvs to &#946;-galactosidase was measured with an ELISA. Purified scFvs were loaded onto &#946;-galactosidase-coated ELISA plates, and the bound scFvs were detected with an anti-6&#215;-His antibody. The data are an average of six replicates, and the error bar shows standard error of the mean. (B) The soluble and insoluble fractions of the cell lysates from cells expressing the scFvs cytoplasmically were analyzed by a Western blot probed with an anti-6&#215;-His antibody. Total protein concentration was used to normalize the loading of the samples. Reprinted (A) and adapted (B) from reference 19 with permission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54583/54583fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments at JoVE.com

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

bacterial-inner-membrane-display-for-screening-library-antibody

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.4K Views.  University of Maryland. We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Video: Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results.
Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY&#160; II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input.
Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.

Crystallographic fragment screening at the Helmholtz-Zentrum Berlin is performed using a workflow with dedicated compound libraries, crystal handling tools, fast data collection facilities&#160;and largely automated data analysis. The presented protocol intends to maximize the output of such experiments to provide promising starting points for downstream structure-based ligand design.

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...