This work was supported by The VENOMICS project, European project grant N&#176; 278346 through the Seventh Framework Program (FP7 HEALTH 2011-2015). The VENOMICS project is a collaboration between several research institutions and companies in Europe:
AFMB, Aix-Marseille Universit&#233; (France), CEA Saclay (France), NZYTech (Portugal), SistemasGenomicos (Spain), University de Liege (Belgium), VenomeTech (France), Vitamib (France), Zealand Pharma (Denmark)
This work was supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01.
The authors would also like to thank Mr. Jeremy Turchetto for assisting with preparations for the shooting of the video.

<ol>
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The authors declare that they have no competing financial interests.

There is no single universal protocol for the expression of soluble, folded, functional proteins. To be cost- and time-efficient, most laboratories or protein core facilities working with multiple targets therefore use high throughput protein expression screening to find the best &#8216;generic&#8217; combination of variables to obtain a soluble active protein for the majority of targets. We have identified DsbC as being a generally applicable fusion partner for the soluble expression of disulfide-rich peptides and proteins11. Using DsbC fusions and high throughput methods, within a week the soluble expression of multiple targets can be observed11 and then additional variables, such as those discussed in the introduction, can be screened in subsequent rounds on those targets that require further optimization. The protocols described herein are aimed at the expression of disulfide-rich proteins and peptides. However, for users wishing to express non-reticulated proteins in a high throughput manner, the corresponding protocols have been published previously and can be found elsewhere22,24.
The high throughput setup is ideal for a number of applications, including the screening of a large number of different proteins for soluble expression or the screening of a large number of expression constructs (including various fusion tags) for several target genes at the same time (or multiple expression constructs for a single target) in order to improve success rates. The platform can also be used for the benchmarking and validation of new protocols on a large number of targets. Other applications include the screening of variants for a single difficult target, e.g., all orthologs or members of the same family, or to test the success of production of a panel of mutants of a single target in one experiment. This protocol has also been used in combination with co-expression vectors (with one tagged protein only) to allow the pull down and preliminary characterization of protein-protein complexes followed by more thorough biophysical analysis to confirm the correct complex formation and stoichiometry33. The amount of protein purified is sometimes suitable for micro-assays (functional tests, protein-DNA34 or protein-protein interaction assays). There are several advantages to the high throughput expression screening strategy: (i) the ability to test a large number of targets or a large number of variables in a single experiment, (ii) limited batch-to-batch variation, (iii) the simplicity and ease of working at a smaller-scale using deep-wells, (iv) scalability and reproducibility at larger scale, (v) the potential for automation, and(vi) simplicity of tracking and handling (no labeling of individual tubes, less mistakes introduced when using the plate format than with the handling of individual tubes in the mixing or exchanging of clones).
Although not discussed in the protocol section, there are several important considerations for the preparation of the experiment that will be briefly discussed below. For a more thorough discussion please see our previous publication24.For maximum efficiency, it is beneficial to have a suitable system for high throughput cloning, to simplify the sub-cloning of large numbers of targets. For the initial phase of the VENOMICS project, we utilize the versatile Gateway recombination system25 that allows subcloning in any destination vector at a pace of hundreds of clones per week. Protocols for Gateway recombination cloning can be found on the Invitrogen website. Other alternatives for high throughput cloning include ligation-independent cloning (LIC)35,36 and restriction-free (RF) cloning37.There are multiple ways to obtain the target genes for expression, including by PCR from template DNA, from entry clone collections or as synthetic genes, which is the strategy chosen for the VENOMICS project. Synthetic genes can be ordered with recombination sites on each end of the gene and gene synthesis allows easy codon optimization of the target gene sequence (to exclude rare E. coli codons). This is recommended but not essential. For targets without codon optimization that contain a high number of rare codons, Rosetta 2 (DE3) pLysS (which carries tRNAs for rare codons that are not highly expressed in E. coli) may be better suited than BL21 (DE3) pLysS. While there are strains available that limit the reduction of disulfide bonds in the normally reducing environment of the cytoplasm, in our hands they have not been as successful as regular E. coli strains11.
Analytical scale affinity purification is performed from the test expression cultures in order to recover the soluble fusion proteins and quantify yields. Quantitative data can be obtained on the soluble yields of fusion proteins expressed within a range of 0.1 &#8211; 100 mg/L of culture. If a target is not soluble, alternative expression and induction temperatures, strains or media can be tested before different fusion partners are pursued. For soluble expression of disulfide-rich proteins, we previously ranked the effect of fusion partners as DsbC &#62; DsbA &#62; GST &#62; MBP &#62; TRX &#62; HIS-tag for cytoplasmic expression11. Periplasmic expression is another possibility that may aid the successful folding of disulfide-rich targets. The periplasm is a less reducing environment than the cytoplasm and contains useful redox chaperones to assist disulfide bonding. DsbC, DsbA, and MBP proteins are normally localized to the periplasm by their periplasmic signal sequences. This provides the opportunity to exploit these tags to direct the disulfide-rich targets to the periplasmic space in order to assist folding. For intractable targets, the next step would be to purify the insoluble HIS-tagged target from inclusion bodies, solubilize and refold (this is out of the scope of this protocol and will not be discussed here39). This can be fairly simple for targets with only one or two disulfide bonds, but becomes increasingly more difficult as the number of disulfide bonds increases. Alternatively, and particularly for proteins and peptides with four or more disulfide bonds, it may be beneficial to try more complex production systems such as yeast, insect or mammalian expression.
With advances in miniaturization and automation, HTP electrophysiology lab-on-a-chip technologies28 will undoubtedly be the way of the future for functional analyses. We envisage that for most purposes (perhaps with the exception of structural studies) this will negate the need for large scale cultures. Small scale cultures will not only be useful for screening expression conditions, but also be able to provide sufficient amounts of sample for these miniaturized functional assays. The ability to produce multiple targets in parallel in sufficient quantities for functional characterization will lower the costs of culturing and using these kind of platforms, expression and characterization of recombinant proteins will become more cost and time-effective.
The protocols herein have been applied to the expression of disulfide-rich peptides and proteins in the initial phase of the FP7 European VENOMICS project. Venoms are an excellent source of bioactive peptides that often have interesting pharmacological potential. However, their production is challenging due to their complex disulfide-bonding patterns and small size. Using high-throughput platforms like the one described herein, the VENOMICS project aims to generate a library of 10,000 novel venom peptides to reproduce the diversity observed in nature. This library will be exploited for the characterization of disulfide-rich peptides with potential pharmacological or therapeutic applications with the aim of developing new drugs. The platform is currently being used for benchmarking and validating new protocols for use in the VENOMICS project.

Motivated by the advancement of genomics and accelerated rate of discovery of new proteins, high throughput pipelines have been developed to parallelize traditional approaches for the screening and identification of optimal protein production strategies. Potential variables to be optimized include, but are not limited to, varying expression strains1,2, temperature3,4, media2,3, target variants5, fusion partners6-13, co-expression with chaperones14,15, cytoplasmic or periplasmic expression16-18, and purification buffer components3. By implementing high throughput approaches, many variables or many targets can be tested in parallel with a high level of efficiency, while limiting batch-to-batch variation. In our experience, the strategy also gives good reproducibility upon scale-up using the same culture (temperature, media, aeration, etc.) and purification conditions (same resin, buffers, etc.). Several high throughput platforms have been used in the past decade to identify conditions for soluble protein expression, namely through varying parameters such as fusion partners, expression strains or temperature19-23.
We recently used our high throughput screening approach for the expression of soluble disulfide-rich proteins11. The proteins selected were not only from venomous sources, but also included disulfide-rich enzyme inhibitors from a wide range of species including plants, pigs, cows and humans. The experiment compared the effects of 12 different fusion partners and three different expression strains on the solubility and folding of 28 disulfide-reticulated proteins. We demonstrated that using DsbC as a fusion partner for production in the strain BL21 (DE3) pLysS vastly outproduced (in both yield and number of soluble proteins obtained) any other combination of strain and fusion tested11. The results of this experiment formed the basis for adapting our original general high throughput pipeline (which has been used for the expression screening of a wide range of proteins)22,24 into one more suited for the expression of disulfide-rich targets. Disulfide-rich proteins from animal venoms are of particular interest. Venoms are a complex mixture of bioactive peptides and proteins, with potential value pharmacologically and therapeutically. However, expression of disulfide bond-containing proteins is not trivial. These proteins generally contain between one to seven disulfide bonds, and must be oxidized with the correct disulfide-bonding patterns in order to be active. Currently, the platform is being used for screening the expression of a large number of disulfide-rich animal venom proteins as part of the FP7 European VENOMICS Project (www.venomics.eu) and benchmarking novel protocols for the high throughput expression of thousands of targets. Here, an automated method is provided for high throughput small-scale expression screening and purification (see Figure 1) applied to disulfide-rich animal venom proteins. The strategy for disulfide rich peptides and proteins utilizes a HIS-tag for purification and the redox-active fusion partner, DsbC, creating cleavable HIS-DsbC fusions to the target proteins (see Figure 2).
While the focus of the protocols herein is automation using a liquid handling robot and HTP electrophoresis, these methods are also suitable for a high throughput manual approach, meaning that even laboratories with a basic setup can take advantage of the protocols without any prerequisite for expensive equipment. Manual protocols for the transformation to purification and analysis (not specific to disulfide-rich proteins) have been published elsewhere24 and will not be repeated here. The throughput of the manual procedure (from expression clone, produced by recombinational cloning25, to analysis of soluble protein levels) is 96 (using SDS-PAGE detection) or 384 (4 x 96; using dot blot and SDS-PAGE26) cultures per week (see Figure 1). This can be increased if performed in a semi-automated way (using a liquid handling robot and dot blot26 or HTP electrophoresis, such as with a Caliper GXII LabChip system22 for analysis of results) to up to 1,152 (12 x 96) cultures in parallel over one week, as described herein. Culturing is performed in deep well 24 (DW24) format so that regular shaking incubators can be used in contrast to cultures grown in deep well 96 (DW96) format, which necessitate the use of short orbital high-speed shaking incubators for sufficient aeration (shaking at 800 rpm). The use of auto-induction media27 also simplifies expression, eliminating the manual induction step. Even where laboratories already use pre-defined expression and purification conditions, these can be transferred directly into this HTP system simply to improve efficiency. A detailed schematic of the high throughput screening pipeline for disulfide-rich proteins is provided in Figure 3. The parameters in the pipeline were selected based on extensive screening experiments11,22, which allowed us to choose the most useful conditions for protein production.
Characterization can be performed on tagged proteins purified directly from small-scale expressions in pilot studies where tens of micrograms of sample is sufficient, or for sensitive functional assays and binding assays (for example, low volume HTP patch clamp systems28). The same can even be performed on the untagged targets after cleavage, provided the tag and protease are removed (for example, by reverse phase HPLC). Quality control can also be performed by mass spectrometry (to confirm the expected size and oxidation state) or chromatographic methods (to confirm purity or heterogeneity)29. Sometimes tag cleavage is unnecessary or even undesirable (particularly for poorly soluble proteins30,31), so in this protocol cleavage is optional. Regardless, in all constructs there is a TEV protease cleavage site (ENLYFQ/[G]32) directly preceding the target gene to produce native protein after cleavage (see Figure 2 and Discussion). If cleavage of the fusion tag is desired, cleavage can be tested (on the elution fraction or &#8216;on column&#8217;) at the small scale to analyze efficiency, optimize conditions if required and obtain reliable estimates of yields for subsequent scale-up experiments.
There are two options for the volume of beads used during the affinity purification, depending on the aims and expectations of the experiment. To be able to capture as much protein as possible (to purify for pilot assays or MS, or to extrapolate for scale-up yields) a final volume of 200 &#181;l of resin should be used, allowing detection of soluble protein in the range of 1-100 mg/L culture before saturation of the system (see protocol (A) in Section 8.1). However, if the aim of the experiment is the detection of low amounts of soluble proteins then a final volume of 50 &#181;l of resin is suitable, allowing detection of soluble protein in the range of 0.1-25 mg/L culture (see protocol (B) in Section 8.2).
Production can be scaled up, if required, to obtain milligram quantities of purified targets for further structural and functional studies using the conditions identified for soluble expression. The details of scale-up protocols used at AFMB have been discussed elsewhere22,24.
Further details relevant to the experimental setup, critical steps within the protocol, modifications and trouble-shooting and limitations of the technique are provided in the discussion. Please read the discussion before commencing the experiments.
Throughout the protocols we expect a success rate of 90% at each step (for example, at least 90% of the cultures must grow at any given step). If the success rate of any step in the experiment falls below 90% the samples are discarded and the experiment is repeated for the full collection of constructs. However, this success rate is not applicable to the number of constructs that express as soluble proteins or the proportion of constructs that cleave with 100% efficiency, as this will be highly dependent on the proteins tested.
The specific details for the set-up of the robot worktable are provided for each protocol (also see Figure 4), however they can be adapted as required for alternative worktable set-ups. The robot hardware (Tecan) consists of a 96-multichannel arm (MCA96), robotic manipulator (RoMa) and the 8-channel liquid handling head (LiHa). All steps utilizing the MCA96 can also be performed using the LiHa if an MCA96 is not available, however they will take longer because the LiHa will need to be washed between steps. While the robot is technically not a sterile environment, the inclusion of antibiotics generally ensures that there are not problems with contamination or sterility.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tecan Freedom EVO 200 liquid handling robot</td>
			<td>Tecan Group Ltd.</td>
			<td></td>
			<td>Protocols can be adapted to any liquid handling robot with a vacuum manifold for plates. 
			http://www.tecan.com/platform/apps/product/index.asp?MenuID=2694&#38;ID=5270&#38;Menu=1&#38;Item=21.1.8</td>
		</tr>
		<tr>
			<td>96-well PCR (PCR96) plates</td>
			<td>Greiner Bio-One</td>
			<td>652270</td>
			<td> 
			http://us.gbo.com/bioscience/detail/2504/</td>
		</tr>
		<tr>
			<td>LB medium</td>
			<td></td>
			<td></td>
			<td>Autoclaved for sterility.</td>
		</tr>
		<tr>
			<td>Antibiotic stocks</td>
			<td></td>
			<td></td>
			<td>Kanamycin (50 mg/ml), Ampicillin (100 mg/ml), Chloramphenicol (34 mg/ml in ethanol), store stocks at -20 &#176;C. Use a 1 in 1000 dilution.</td>
		</tr>
		<tr>
			<td>Deep-well 96 (DW96) plate with 2.42 ml volume capacity</td>
			<td>Greiner Bio-One&#160;</td>
			<td>780270</td>
			<td>Autoclaved for sterility. 
			http://us.gbo.com/bioscience/detail/2462/</td>
		</tr>
		<tr>
			<td>Expression vectors</td>
			<td></td>
			<td></td>
			<td>For the expression of target constructs. Store at &#8722;20 &#176;C.</td>
		</tr>
		<tr>
			<td>BL21 (DE3) pLysS competent cells</td>
			<td></td>
			<td></td>
			<td>Or alternative E. coli expression strain of the user&#39;s choice. Store at &#8722;80 &#176;C.</td>
		</tr>
		<tr>
			<td>Breathseal breathable adhesive film</td>
			<td>Greiner Bio-One</td>
			<td>676050</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR machine with 96-well plate block</td>
			<td></td>
			<td></td>
			<td>For the transformation heat shock and boiling of SDS-PAGE/Caliper samples.</td>
		</tr>
		<tr>
			<td>24-well sterile tissue culture plates</td>
			<td>Greiner Bio-One</td>
			<td>662165</td>
			<td> 
			http://us.gbo.com/bioscience/detail/2220/</td>
		</tr>
		<tr>
			<td>LB-agar</td>
			<td></td>
			<td></td>
			<td>Autoclaved for sterility.</td>
		</tr>
		<tr>
			<td>200 &#956;l sterile disposable tips</td>
			<td>Tecan Group Ltd.</td>
			<td>30 038 617</td>
			<td> 
			http://www.tecan.com/platform/apps/product/index.asp?MenuID=2283&#38;ID=4118&#38;Menu=1&#38;Item=21.4.1.2</td>
		</tr>
		<tr>
			<td>Multitron Shaking Incubator, with 3 mm throw</td>
			<td>Infors</td>
			<td>AJ103</td>
			<td>Not essential, a regular shaking incubator can also be used. 
			http://www.infors-ht.com/index.php/en/</td>
		</tr>
		<tr>
			<td>Plate incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtiter plate</td>
			<td></td>
			<td></td>
			<td>For glycerol stocks and elution using purification protocol B.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td></td>
			<td></td>
			<td>For the preparation of glycerol stocks.</td>
		</tr>
		<tr>
			<td>200 &#956;l disposable tips</td>
			<td>Tecan Group Ltd.</td>
			<td>30 038 616</td>
			<td> 
			http://www.tecan.com/platform/apps/product/index.asp?MenuID=2283&#38;ID=4118&#38;Menu=1&#38;Item=21.4.1.2</td>
		</tr>
		<tr>
			<td>Adhesive tape pads&#160;</td>
			<td>Qiagen</td>
			<td>19570</td>
			<td> 
			http://www.qiagen.com/Products/Catalog/Lab-Essentials-and-Accessories/Tape-Pads</td>
		</tr>
		<tr>
			<td>Bactinyl</td>
			<td>Orapi Group</td>
			<td></td>
			<td>Or equivalent microbial disinfectant.</td>
		</tr>
		<tr>
			<td>ZYP-5052 medium</td>
			<td></td>
			<td></td>
			<td>See Table 1 for recipes of components.</td>
		</tr>
		<tr>
			<td>Deep well 24 (DW24) plates,10 ml capacity&#160;</td>
			<td>Whatman</td>
			<td>7701-5102</td>
			<td>Autoclaved for sterility. 
			http://www.whatman.com/UNIPLATECollectionandAnalysisMicroplates.aspx#7701-5102</td>
		</tr>
		<tr>
			<td>Flat-bottomed, clear microtiter plate</td>
			<td>Greiner Bio-One</td>
			<td>655101</td>
			<td>For absorbance readings. 
			http://us.gbo.com/bioscience/detail/2029/</td>
		</tr>
		<tr>
			<td>Plate reading spectrophotometer</td>
			<td></td>
			<td></td>
			<td>Optional. For measuring OD600nm of cultures.</td>
		</tr>
		<tr>
			<td>Centrifuge with rotor for deep well plates&#160;</td>
			<td></td>
			<td></td>
			<td>Suitable for 3800 x g.</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td></td>
			<td></td>
			<td>50 mg/ml in water. Store at &#8722;20 &#176;C.</td>
		</tr>
		<tr>
			<td>Imidazole ACS grade</td>
			<td>Merck</td>
			<td>IX0005-1</td>
			<td>A high quality grade of imidazole must be used so that it will not interfere with A280 readings for calculating protein yield.</td>
		</tr>
		<tr>
			<td>Lysis buffer</td>
			<td></td>
			<td></td>
			<td>50 mM Tris, 300 mM NaCl, 10 mM imidazole pH 8 containing 0.25 mg/ml lysozyme (or your preferred buffer). Add lyzozyme from stocks each time and do not store for extended periods.</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sonicator (Ultrasonic processor XL, adapted for deep well plates)</td>
			<td>Misonix Inc.</td>
			<td></td>
			<td>Not essential. Lysozyme alone is normally sufficient for nearly complete lysis.</td>
		</tr>
		<tr>
			<td>DNase&#160;</td>
			<td></td>
			<td></td>
			<td>2 mg/ml stock in water, filter sterilized. Store aliquots at &#8722;20 &#176;C.</td>
		</tr>
		<tr>
			<td>Magnesium sulphate (MgSO4)</td>
			<td></td>
			<td></td>
			<td>2M stock in water, autoclaved.</td>
		</tr>
		<tr>
			<td>SDS-PAGE or Caliper sample buffer&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Binding buffer</td>
			<td></td>
			<td></td>
			<td>50 mM Tris, 300 mM NaCl, 10 mM imidazole pH 8 (or your preferred buffer). Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td></td>
			<td></td>
			<td>50 mM Tris, 300 mM NaCl, 50 mM imidazole pH 8 (or your preferred buffer). Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Elution buffer</td>
			<td></td>
			<td></td>
			<td>50 mM Tris, 300 mM NaCl, 250 mM imidazole pH 8 (or your preferred buffer). Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>96-well Receiver/Filter Plate 20 &#181;m, 1.8 ml capacity</td>
			<td>Macherey-Nagel</td>
			<td>740686.4</td>
			<td> 
			http://www.mn-net.com/ProductsBioanalysis/Accessories/tabid/10909/language/en-US/Default.aspx</td>
		</tr>
		<tr>
			<td>200 &#956;l disposable wide bore tips</td>
			<td>Tecan Group Ltd.</td>
			<td>30 050 348</td>
			<td> 
			http://www.tecan.com/platform/apps/product/index.asp?MenuID=2283&#38;ID=4118&#38;Menu=1&#38;Item=21.4.1.2</td>
		</tr>
		<tr>
			<td>Ni Sepharose 6 Fast Flow resin</td>
			<td>GE Healthcare</td>
			<td>17-5318-02</td>
			<td>For purification of target fusion proteins. Store at 4 &#176;C. Wash and equilibrate before use. 
			http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/17531802</td>
		</tr>
		<tr>
			<td>Tobacco Etch Virus (TEV) protease</td>
			<td></td>
			<td></td>
			<td>Optional, for cleavage. 2 mg/ml, without reducing agents in storage buffer. Store at &#8722;80 &#176;C.</td>
		</tr>
		<tr>
			<td>96-well 0.22 &#181;m filter plate</td>
			<td>Millipore</td>
			<td>MSGV N22 10</td>
			<td>Optional. To filter the soluble fraction after cleavage. 
			http://www.millipore.com/catalogue/item/msgvn2210</td>
		</tr>
		<tr>
			<td>SDS-PAGE/dot blot equipment or Caliper Labchip GXII equipment</td>
			<td></td>
			<td></td>
			<td>Electrophoresis apparatus and choice of gel type is at the user&#8217;s discretion.</td>
		</tr>
		<tr>
			<td>Spectrophotometer and cuvettes</td>
			<td></td>
			<td></td>
			<td>For measuring absorbance at 280 nm (A280) to calculate yield of soluble proteins. Not required if the analysis is done on the Caliper.</td>
		</tr>
		<tr>
			<td>ZipTip pipette tips</td>
			<td>Millipore</td>
			<td></td>
			<td>Optional. The type of ZipTip is at the user&#39;s discretion. C4 resin should be used for larger proteins, while for peptides C18 may be better. Alternative methods for desalting of samples may also be used to clean up samples before further quality control. 
			http://www.millipore.com/catalogue/module/c5737#0</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials are listed in the order in which they are required in the Protocol section. Reagents can be stored at room temperature unless noted otherwise. Reference numbers for the author&#8217;s preferred choice of materials are provided, however equivalent products may also be suitable. For reagents where the brand will not influence the outcome of the experiment, the company details have been omitted.</td>
		</tr>
	</tbody>
</table>

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.

Representative results are shown in Figure 6 for the expression screening of 96 disulfide-rich proteins from the VENOMICS pipeline. The proteins are arranged by increasing number of disulfide bonds then increasing number of residues. The peptides were expressed in the cytoplasm with a HIS-tag and DsbC fusion partner. When using the recommended culture conditions, an OD600&#160;of 12 is normally achieved. The peptides were purified using protocol 8.2-B with a final volume of 50 &#181;l of nickel affinity resin, so a maximum of ~25 mg/L fusion protein could be detected in this experiment.
Figure 6A shows the electrophoresis result from the Caliper LabChip system (showing the fusion before cleavage) and the scoring system based on extrapolation to yield in mg/L culture of the fusion protein (in levels of 0.1 to 2 mg/L culture, 2 to 10 mg/L culture, 10 to 25 mg/L culture and not detected.) Note that the cleaved DsbC tag normally runs at around 32 kDa, rather than 27 kDa as expected. Similarly, the DsbC fusions also run around 5 kDa higher than expected. For a lot of the targets we not only see the intact fusion (upper band), but also the fusion partner alone (lower band). For some of these targets optimizing the culture conditions can improve the ratio of the intact fusion (upper band) compared to DsbC alone allowing an increase of the final yield. The scoring is based only on the level of intact fusion. Only 16 out of 96 proteins could not be detected at the fusion level. This corresponds to an overall success level of 83%. Of the 80 proteins that could be detected, 45 of these were detected at levels greater than 2 mg/L culture (56%). Depending on target and fusion, protein yields are usually in the range of 2-100 mg/L culture (although in this example using the protocol 8.2 B a maximum of ~25 mg/L fusion protein can be detected).
An analysis of the success by number of disulfide bonds present (shown in Figure 6B) shows reasonable success for all numbers of disulfide bonds tested (between 1 and 7), with the lowest success level being 66% for targets containing 6 disulfide bonds. An analysis of the distribution of expression success based on isoelectric point and number of residues&#160; (shown in Figure 6C) shows no particular bias for the technique, with both successfully expressed targets and targets that were not detected scattered throughout the plot.
Figure 6D shows an example of the mass spectrometry results obtained from electrospray ionization mass spectrometry (ESI-MS) for a single target, before and after reduction of the sample with DTT. Normally, such an exhaustive mass spectrometry analysis would not need to be performed (a reduced sample would not need to be analyzed), however for the purposes of thorough demonstration we have shown both results. The target shown is a 5.7 kDa disulfide-rich venom protein with 4 disulfide bonds. The spectrum on the left shows the results for the protein prior to reduction with DTT, as it was after cleavage and desalting without further intervention. The spectrum on the right hand side shows the protein after reduction with DTT followed by desalting to remove any excess DTT. The ions corresponding to the experimental masses are marked with arrows on the spectrum and the designation for each ion is shown in green. The experimental parent masses calculated for these ions (for the protein prior to reduction (-DTT) and after reduction (+DTT)) are shown in the table. The masses (5709.6 Da for the sample prior to reduction and 5717.6 Da for the reduced sample) exhibit a mass difference of 8 Da. A mass difference of 2 Da corresponds to the presence of 1 oxidized disulfide bond, therefore a mass difference of 8 Da indicates the presence of 4 disulfide bonds (as expected) in the non-reduced sample.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig1highres.jpg" width="500" /> 
Figure 1.&#160;Schematic representation of the high throughput expression screening protocol. Using this protocol, 96 to 384 conditions can be tested by a single person in one week using manual methods, or up to 1,152 conditions with the described semi-automated equipment. Expression plasmids are constructed using a recombination cloning technology so that numerous targets can be sub-cloned at one time. Once soluble expression conditions have been identified and cleavage performed, if desired, the protein can proceed to quality control to check oxidation and purity, microassays and/or large-scale production.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig2highres.jpg" width="500" /> 
Figure 2.&#160;Schematic for universal recombinational cloning and construct design. From the target entry clones, multiple expression vectors can be sub-cloned in a single experiment in a high throughput fashion (up to 6 x 96 entry clones in a week). The expressed protein encodes a HIS tag for nickel affinity purification. The DsbC (lacking its periplasmic signal sequence for cytoplasmic expression) fusion partner is used to increase solubility and/or aid folding and correct oxidation of the target protein. Note that the target coding sequence should contain an N-terminal TEV protease site (ENLYFQ) if tag cleavage is desired. The inset at the top left shows the production of the entry clones using a donor vector and the target sequences, which can be obtained by PCR or gene synthesis. Entry clones can also be obtained from commercial entry clone collections. Multiple recombinational cloning systems are available, however we utilize the Gateway system, as shown in the schematic.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig3highres.jpg" width="500" /> 
Figure 3.&#160;High throughput screening pipeline for multiple disulfide-rich targets. Targets are initially expressed as HIS-DsbC fusions in the cytoplasm of BL21 (DE3) pLysS E. coli at 37/17&#160;&#176;C using auto-induction medium (ZYP-5052). Purification is performed on nickel resin followed by detection of soluble constructs by HTP electrophoresis (or with dot blot/SDS-PAGE). If the first round of expression screening is unsuccessful, alternative culture conditions are tried. If constructs produce soluble proteins in high enough yields, microassays and quality control can be performed and, if required, large-scale production can be pursued. For targets where soluble yields are not high enough, expression screening can continue with alternative strains and temperatures then other fusion partners and periplasmic expression. Optional steps are indicated by dashed boxes.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig4highres.jpg" width="500" /> 
Figure 4.&#160;Robot Worktable setup for the HTP platform. The layout of our liquid handling robot worktable is shown, although alternative worktables can also be used provided there are equivalent sites available. The setup consists of a wash station (WS) for the (8-channel liquid handling head (LiHa)), two microplate carriers with 4 positions each (MP4, positions 1-4 and 5-8), a vacuum station with 2 positions (Te-VacS, positions 9 and 10), a microplate carrier with 3 positions (MP3, positions 11-13), two plate shakers with 2 positions each (Te-Shakers, positions 14-15 and 16-17) and a carrier for disposable tips with 3 positions (DiTi, positions 18-20). In addition there are two hotel carriers for deep well plates and one for microplates (not shown). The hardware installed on the liquid handling robot is a 96-multichannel arm (MCA96) for use with disposable tips, an 8-channel liquid handling head (LiHa) with fixed tips and a robotic manipulator (RoMa) that moves plates/equipment around on the worktable. The numbering of the positions is referred to throughout the protocol.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig5highres.jpg" width="500" /> 
Figure 5.&#160;Schematic for transferring from a single 96-well plate into four 24-well plates.
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig6highres.jpg" width="500" /> 
Figure 6.&#160;Representative results are shown for the expression screen of 96 disulfide-rich venom proteins. The proteins were expressed as HIS-DsbC fusions in the cytoplasm and purified using 50 &#181;l of Nickel resin (Protocol 8.2-B). A) Expression screening results, showing virtual gel and scoring for the expression yield. The contrast in the virtual gel has been adjusted lane-to-lane to compensate for very faint or intense bands. Note that for some targets two bands can be seen, the upper band corresponding to the intact fusion protein and the lower band corresponding to the fusion tag alone. B) The proportion of proteins expressed at each expression level compared to the number of disulfide bonds in the protein. The actual number of proteins in each group is overlayed on the graph. C) The distribution of expression levels based on isoelectric point (pI) and number of residues. D) An example of the mass spectrometry results for a 5.7 kDa disulfide-rich venom protein with 4 disulfide bonds. The spectrum on the left hand side shows the protein prior to reduction with DTT and the spectrum on the right hand side shows the protein reduced with DTT and then desalted. The ions corresponding to the experimental masses are marked with arrows and their assignments are shown in green. The experimental masses for the protein prior to reduction and after reduction are shown in the table and exhibit a mass difference of 8 Da, corresponding to the presence of oxidized protein before addition of reducing agent. <a href="https://www.jove.com/files/ftp_upload/51464/51464fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Recipe</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>ZY</td>
			<td>~928 ml 
			10 g tryptone 
			5 g yeast extract 
			925 ml water</td>
			<td>Mix and then autoclave to sterilize.</td>
		</tr>
		<tr>
			<td>2 M MgSO4</td>
			<td>100 ml 
			49.3 g MgSO4&#183;7H2O 
			~60 ml water</td>
			<td>Stir until dissolved then autoclave to sterilize.</td>
		</tr>
		<tr>
			<td>50x 5052</td>
			<td>1 L 
			250 g glycerol 
			730 ml water 
			25 g glucose 
			100 g &#945;-lactose</td>
			<td>Add in sequence, stir over heat until all dissolved then autoclave to sterilize.</td>
		</tr>
		<tr>
			<td>20x NPS</td>
			<td>1 L 
			900 ml water 
			66 g (NH4)2SO4 
			136 g KH2PO4 
			142 g Na2HPO4</td>
			<td>Add in sequence and stir until all dissolved then autoclave to sterilize.</td>
		</tr>
	</tbody>
</table>
Table 1. Recipe for components of ZYP-5052 medium.

Watch this Scientific Journal Video about High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli at JoVE.com

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.

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. ...

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23.9K Views.  Aix-Marseille Université. 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.