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Article

High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria

by
José Rodrigues
1,†,
Vanessa T. Almeida
1,†,
Ana L. Rosário
1,
Yong Zi Tan
2,3,‡,
Brian Kloss
4,
Filippo Mancia
2,* and
Margarida Archer
1,*
1
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal
2
Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY 10032, USA
3
National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA
4
Center on Membrane Protein Production and Analysis, New York Structural Biology Center, New York, NY 10027, USA
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this publication.
‡
Present address: Molecular Medicine Program, The Hospital for Sick Children, Toronto, ON, Canada.
Processes 2021, 9(4), 629; https://doi.org/10.3390/pr9040629
Submission received: 4 March 2021 / Revised: 24 March 2021 / Accepted: 26 March 2021 / Published: 2 April 2021
(This article belongs to the Special Issue Applications of Structural Biology Techniques in Enzymology)
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Abstract

:
Studies on membrane proteins can help to develop new drug targets and treatments for a variety of diseases. However, membrane proteins continue to be among the most challenging targets in structural biology. This uphill endeavor can be even harder for membrane proteins from Mycobacterium species, which are notoriously difficult to express in heterologous systems. Arabinofuranosyltransferases are involved in mycobacterial cell wall synthesis and thus potential targets for antituberculosis drugs. A set of 96 mycobacterial genes coding for Arabinofuranosyltransferases was selected, of which 17 were successfully expressed in E. coli and purified by metal-affinity chromatography. We herein present an efficient high-throughput strategy to screen in microplates a large number of targets from Mycobacteria and select the best conditions for large-scale protein production to pursue functional and structural studies. This methodology can be applied to other targets, is cost and time effective and can be implemented in common laboratories.

1. Introduction

Membrane proteins represent 20 to 30% of open-reading frames of all genomes sequenced [1,2] and perform essential functions in cells, such as transportation, signal transduction and energy production [3]. They also play important roles in several diseases and, as a result, are attractive therapeutic targets, estimated to represent more than 30% of all marketed drugs [4,5,6]. However, biochemical and structural characterization of membrane proteins have several bottlenecks, namely toxicity by excess of mRNA levels of the target protein [7], toxicity caused by heterologous expression [8], membrane lipid composition [9,10], detergent extraction and solubility [11,12], which ultimately results in low amounts of membrane protein produced.
Many efforts have been devoted on the development of protocols to efficiently produce membrane proteins in Escherichia coli. An elegant approach to accelerate this process involves the fusion of green fluorescent protein (GFP) to monitor the expression and purification processes [13,14]. A commonly used strategy consists of varying different parameters simultaneously, such as expression vectors with different tags and promoters, host strains, homologues or solubilizing detergents [15,16]. High-throughput (HTP) protein production platforms have also been developed by Structural Genomics Consortia, such as JCSG [17], Northeast SGC [18] or the New York Consortium on Membrane Protein Structure (NYCOMPS, New York, NY, USA) [19,20].
Based on the HTP strategy to express prokaryotic membrane proteins previously developed at NYCOMPS, we have devised an accessible protocol to screen mycobacterial membrane proteins, which are difficult to express in heterologous systems [21]. An initial set of 96 target genes was assembled from the genomic sequences coding for Arabinofuranosyltransferases (AraTs) from 14 different Mycobacterium species. Even though Mycobacteria (Mb) proteins display low yield of production in E. coli, it remains one of preferred hosts for heterologous expression of Mb proteins and is compatible with HTP strategies.
The AraT targets are integral membrane enzymes that play a pivotal role in the synthesis of arabinan, an important component of Mycobacteria cell envelope [22,23]. AraTs transfer arabinose in the furanose conformation (Araf) from a single donor decaprenylphosphoryl-β-d-arabinose (DPA) to the arabinan domain of arabinogalactan (AG) of mycobacterial cell wall [24]. The cell envelope is crucial for growth and virulence of pathogenic Mycobacteria [25] and is a major contributor to resistance against common antibiotics [26]. AraT family comprises 7 sub-families: Embs (EmbA, EmbB and EmbC), which are inhibited by the first-line antitubercular drug Ethambutol (EMB), and Afts (AftA, AftB, AftC and AftD), which are potential novel therapeutic targets against tuberculosis.
In this study, we present a simple and cost-effective methodology to screen AraTs from different Mycobacterium species and chose the most promising targets to proceed biochemical and structural studies.

2. Materials and Methods

2.1. High-Throughput Cloning of Arabinofuranosyltransferase Genes

96 genes of AftA, AftB, AftC, AftD, EmbA, EmbB and EmbC were identified from 14 Mycobacterium genomes using a bioinformatics approach [19] (Table A1). Ligation Independent Cloning (LIC) was used to clone all selected targets, using the protocols previously described by Bruni and Kloss [27], with the following modifications: (1) target sequences were amplified by Polymerase Chain Reaction (PCR) using oligonucleotides that were compatible with LIC-adapted expression vectors (pNYCOMPS-N23 and pNYCOMPS-C23) containing appropriate overhangs with the start codon ATG, instead of the endogenous GTG start codon for some of the target genes; (2) XL10 E. coli was used for cloning purposes. All liquid handling was performed by hand, using multichannel pipettes. The resulting constructs were used to transform E. coli C41, C43 and BL21 (DE3) pLysS strains. Transformants were selected on LB [10 g/L Tryptone, 5 g/L Yeast Extract and 10 g/L NaCl] plates containing appropriate antibiotics: 100 µg/mL of ampicillin for E. coli C41 and C43 cells, 100 µg/mL of ampicillin and 34 µg/mL of chloramphenicol for E. coli BL21 (DE3) pLysS cells.

2.2. High-Throughput Expression Screening and Purification

Pre-cultures were grown overnight at 37 °C, 200 rpm in 600 µL of LB medium supplemented with the appropriate antibiotics. For this purpose, 96-well plates (VWR) were used. The overnight cultures were used to inoculate 2.5 mL of 2xYT medium [16 g/L Tryptone, 10 g/L Yeast Extract and 5 g/L NaCl], supplemented with antibiotics, in 24-well plates (UNIPLATE Collection and Analysis Microplate), at an initial optical density (OD600) of 0.03–0.08. Cells were grown at 37 °C, 200 rpm, until the cultures reached OD600 of 0.4–1.2 (2 to 2.5 h) (see Table A2), then cultures were cooled to 22 °C and gene expression was induced overnight (~16 h) with 0.25 mM isopropyl-β-d-thiogalactoside (IPTG). Cells were harvested the next day by centrifugation at 3200× g, for 20 min at 4 °C. OD600 measurements were done in TECAN Spark 10 M. Cell pellets were re-suspended in 300 µL of lysis buffer (BugBuster supplemented with 0.1 mg/mL Lysozyme, 3 U/mL Benzonase, 2 mM MgCl2 and 0.5 mM PMSF) through 10 min of vigorous shaking, using a plate shaker, at room temperature. Detergent n-Dodecyl β-d-maltoside (DDM) was added to the lysate at 1% (w/v) final concentration and the plate containing the samples was incubated for 2 h at 4 °C, with gentle agitation. For the separation of the insoluble cell debris, plates were centrifuged at 3200× g for 20 min, 4 °C and 250 µL of the supernatants were transferred to a 96-well filter plate containing a bed of 50 µL Ni-NTA agarose resin (HisPurTM Ni-NTA Spin plate (Thermo Scientific™, Waltham, MA, USA)), previously washed with double distilled water and equilibrated with buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 10 mM Imidazole, 0.1% DDM). Imidazole at 10 mM final concentration was added to each sample, to avoid unspecific binding of contaminants to the Ni-NTA resin. Plates were incubated for 15 min in a plate-shaker at 4 °C. The plates were then centrifuged, the flowthrough fractions collected and reloaded to the resin bed, repeating the 15 min incubation with the Ni-NTA resin bed. After the second incubation step, plates were centrifuged to remove unbound proteins. The resin was washed three times with 250 μL washing buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 60 mM Imidazole and 0.1% DDM) and finally eluted with 250 μL of elution buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 300 mM Imidazole and 0.05% DDM). Eluted samples were run on SDS-PAGE: 10% polyacrylamide gels were used for targets with molecular weights between 73–149 kDa (AftD, EmbA, EmbB and EmbC) and 12% polyacrylamide gels for 47–75 kDa targets (AftA, AftB and AftC).

2.3. Large Scale Protein Expression and Purification

50 mL cultures of each target in E. coli C41 cells were grown overnight in 250 mL flasks at 37 °C, 200 rpm, in LB medium supplemented with 100 µg/mL of ampicillin. The overnight cultures were used to inoculate 4 × 500 mL of 2xYT medium, supplemented with 100 µg/mL of ampicillin, in 2.5 L Thomson’s Ultra Yield™ Flasks (Oceanside, CA, USA), at an initial OD600 around 0.05. Cells were grown at 37 °C, 200 rpm, until the cultures reached OD600 of 0.8 (2 to 2.5 h), then cultures were cooled to 22 °C and gene expression was induced overnight (~16 h) with 0.25 mM IPTG. Cells were harvested in the next day by centrifugation at 4472× g, for 15 min at 4 °C. OD600 measurements were done in Ultrospec 10 Cell Density Meter. Cell pellets were re-suspended and homogenized in lysis buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 20 mM MgSO4, 1 mM TCEP), protease inhibitor EDTA-free cocktail (Thermo Scientific™, Waltham, USA; Catalog number: 88266) and 25 U/mL of Benzonase nuclease (Santa Cruz Biotechnology, Dallas, USA; Catalog number: sc-391121). Cell suspension was passed twice at 15,000 psi on a cell disruptor (Constant Systems Ltd., Daventry, UK). Membranes were collected by ultracentrifugation at 197,215× g, for 30 min at 4 °C. Membranes were manually homogenized using a Wheaton® glass homogenizer (DWK Life Sciences Limited, Stoke-on-Trent, UK) in 20 mM HEPES pH 7.5 and 200 mM NaCl, to which DDM was added to a final concentration of 1% (w/v). Membranes were solubilized for 2 h, with gentle agitation, at 4 °C. Soluble membrane fraction was collected by ultracentrifugation at 203,756× g for 30 min, at 4 °C. The supernatants were collected and incubated with 2 mL of equilibrated Ni-NTA agarose resin for 1.5 h at 4 °C, with gentle agitation. Imidazole was added to each sample to a final concentration of 10 mM, to prevent unspecific binding of contaminants. After incubation, the sample was loaded into a column for elution by gravity flow. The resin bed was washed with 10 column volumes (CV) with washing buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 0.1% DDM, 60 mM Imidazole), and the proteins were eluted with 4 CV of elution buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 0.05% DDM and 300 mM Imidazole). Eluted samples were concentrated and injected into a Superdex 200 column (Cytiva Europe GmbH, Freiburg, Germany) to assess protein dispersity. The collected fractions were run on SDS-PAGE: 10% polyacrylamide for 73–149 kDa targets (AftD, EmbA, EmbB and EmbC) and 12% polyacrylamide for those around 47–75 kDa (AftA, AftB and AftC).

3. Results

3.1. Genomic Expansion and High-Throughput Cloning of Arabinofuranosyltransferases

A set of 96 target genes was assembled from the genomic sequences coding for seven AraTs from Mtb (AftA, AftB, AftC, AftD, EmbA, EmbB and EmbC). To each “seed” sequence, a “cluster” of homolog sequences, from 14 different Mycobacterium genomes was expanded, coding for proteins likely to have similar structure as the seed protein [28] (Table A1). LIC was performed as described previously by Bruni and Kloss [27]. Briefly, all sequences were amplified by PCR, using genomic DNA available from ATCC® (Manassas, VA, USA) [https://www.lgcstandards-atcc.org (accessed on 26 March 2021)] and primer pairs compatible with LIC-adapted expression vectors (pNYCOMPS-N23 and pNYCOMPS-C23) that contained decahistidine affinity tag and Tobacco Etch Virus (TEV) protease cleavage site (ENLYFQS). 56 targets were successfully cloned into pNYCOMPS-N23 and 40 targets into pNYCOMPS-C23. Previous screening experiments had shown no expression for all constructs in pNYCOMPS-C23 vector (data not shown), therefore only the clones in pNYCOMPS-N23 were used for the HTP expression screening approach.

3.2. Small Scale High-Throughput Expression of Arabinofuranosyltransferases

All 56 positive clones in pNYCOMPS-N23 were transformed into C41, C43 and BL21 (DE3) pLysS E. coli strains. 24 deep-well plates were used to grow the positive clones simultaneously. Growth conditions, 2xYT rich medium, 0.25 mM IPTG and overnight post-induction at 22 °C, were established based on the results obtained in previous experiments. This allowed a fast and reliable comparison between different E. coli strains, also leaving room for optimization after target selection.
Cell harvesting by centrifugation and lysis were performed in 24 deep-well plates, maintaining a HTP downstream processing of the samples. Extraction of membrane proteins was achieved by adding detergent directly to each well, after cell lysis, incubating the plate at low temperature. The 24 deep-well plate is centrifuged again to clear the solubilized lysate from the cell debris. The solubilized lysate was transferred to a HisPurTM Ni-NTA Spin 96 well plate (Thermo Scientific™, Waltham, MA, USA) for affinity chromatography purification. In this step, the use of adjustable multichannel pipettes to transfer solutions from 24-well plate to 96-well plate was important for sake of speed and reproducibility/reliability, however, standard multichannel pipettes can also be used although not in an optimal manner. After a single Ni-NTA purification step, the amount of eluted target protein was too low to be detected by SDS-PAGE. Since the sample solution slowly flows from the filter plate by gravity during the incubation period, a second passage was deemed necessary to increase the contact time between the sample and Ni-NTA resin, after which the eluted AraTs could be visualized on the gel. The full pipeline is summarized in Figure 1.
In total, 17 out of 96 distinct proteins were produced and purified, resulting in 18% success rate of protein production (Table 1). All three different E. coli host strains were able to produce target proteins: 16 in C41, 6 in C43 and 8 in BL21 (DE3) pLysS (Table A2). AftB and EmbC proteins were not detected in any E. coli strain using this HTP method, suggesting that different, perhaps more tailored conditions may be needed to successfully produce these proteins. We found that a single His-tag purification step was not very efficient, considering that persistent contaminants from the host cell are present across all targets (Figure 2; see Figure A1 and Figure A2). Moreover, the production yields for the target proteins herein studied were low in all E. coli host strains. Nevertheless, we were still able to successfully identify bands in the SDS-PAGE that could correspond to our target proteins, based on their predicted molecular weight (MW) and considering the gel shifting for membrane proteins in denaturing protein gels [29]. Due to this anomalous migration pattern, bands related to membrane proteins in SDS-PAGE most often appear ~20–30% below their predicted MW.

3.3. Validation of HTP Target Selection by Large-Scale Protein Production

Based on SDS-PAGE analysis, we selected one target from each cluster for large-scale production: AftA and AftC from M. neoaurum, AftD from M. abscessus 1948 F5/8, EmbA from M. marinum M. and EmbB from M. vanbaalenii PYR-1, all produced in E. coli C41. Growth conditions were similar to the ones used in the HTP screening, although cell lysis and membrane extraction steps were modified according to the cell mass. Most importantly, the incubation time of solubilized membranes with Ni-NTA resin was increased to improve protein binding and purification yield. All chosen targets were successfully produced in large scale, thus validating the selection made from the HTP screening. Size exclusion chromatography (SEC) was performed after affinity chromatography to further purify the protein and as tool for preliminary biophysical characterization of each protein (Figure 3). Although all targets show some aggregation in the presence of DDM, it was still possible to identify heterogeneous protein populations in most target samples. Upon SDS-PAGE analysis of the SEC elution fractions, we observed that the dominant protein bands correspond to the desired targets, however there were still contaminants present. AftA (Figure 3A), EmbA (Figure 3D) and EmbB (Figure 3E) showed the least amount of contaminant proteins.
For EmbB, a second SEC step was performed (Figure 4), running each population separately. We could observe that the high molecular weight EmbB population behaves as a stable monodisperse population (Figure 4A,B), while the low molecular weight EmbB population splits into the same two populations observed in the first SEC run (Figure 3E), suggesting that EmbB monomers are prone to form an equilibrium with stable EmbB dimers.

4. Discussion

The need to screen the expression of a large number of membrane protein targets, as well as the selection of optimal conditions for production and purification of desired targets, led to the development of several HTP strategies. The strategy used in this study is not novel and was intended to setup a protocol to search for the best candidates to pursue functional and structural studies on AraTs from Mycobacteria. Embs are targets of ethambutol, whereas Afts are potential targets to develop new drugs to treat tuberculosis. Nevertheless, the protocol herein described can also be applied to evaluate the expression and purification of other membrane proteins.
The methodology involved the selection of 13–14 orthologue genes of each AraT sub-family (EmbA-C, AftA-D) from a variety of host genomes, gene expression with vectors harboring a poly-histidine affinity tag at either N- or C-terminus, transformation into three different E. coli strains, membrane extraction and protein solubilization by DDM detergent, and purification by Ni-NTA chromatography. By using this simple combinatorial approach, we were able to clone 56 genes at pNYCOMPS-N23 and 40 at pNYCOMPS-C23, and produce 17 proteins out of 96 chosen targets, corresponding to a success rate of 18%. Such rate is not surprising, considering that membrane proteins are often difficult to express and purify [7,30]. Heterologous expression of mycobacterial proteins in E. coli has previously been reported not to exceed 40% [30,31,32].
No expression of AraTs cloned into pNYCOMPS-C23 was observed (data not shown). It is well known that type and location of the fused affinity tag has a significant effect at all stages of protein production [33], however it is not possible to know a priori the impact caused by tag addition. This unpredictability is somehow the foundation of HTP approaches—try as many conditions as is reasonably possible and assess what works to proceed with further studies.
Concerning the host organism, M. smegmatis could be a viable alternative for the heterologous expression of Mb proteins [31], yet we considered it not appropriate for a HTP approach due to its slower growth rate compared to E. coli, and mostly due to its waxy surface [34], which promotes clumping, film formation and cell adhesion to surfaces, especially plastic, preventing an optimal use of 96- or 24-well plates for cell growth. Instead, we used E. coli C41 and C43 suited for overexpression of toxic and membrane proteins [35], and BL21 (DE3) pLysS for controlled expression [36]. The different expression levels observed among E. coli hosts suggests that the type of strain plays a pivotal role in the number of well-expressed AraTs, as also reported for other target proteins [37]. Indeed, regulation of T7 RNA polymerase expression either by mutations in its promoter (C41 and C43 Walker strains) or by its natural inhibitor T7 lysozyme (T7Lys, pLysS strain) can significantly influence membrane protein overexpression yields [38].
Large scale production (from 2 L of culture) of 5 targets, chosen based on the results of small-scale experiments, yielded purified proteins in milligram amounts, although differences are observed on the intensity of their respective bands (SDS-PAGE) in small and large-scale experiments. The shorter incubation time with the Ni-NTA agarose in the small-scale screening (2 × 15 min vs. 1.5 h) could account for this discrepancy. Moreover, the aeration rate related with the size and shape of the growth vessel (24-well plate vs. 2.5 L flasks) may also affect the overexpression levels. In addition, switching to a cobalt spin plate, instead of nickel, may increase binding specificity of the target protein and thus further improve the results for the small-scale screening. We expect similar results will be obtained on the scale-up production of other targets that showed expression on the HTP screening. Fusions with GFP tag could be advantageous to monitor the various steps of protein production by measuring fluorescence, a very sensitive detection method [13]. However, this methodology is not suitable for membrane proteins with periplasmic C-terminus [13,20,39], which is the case for most of the AraTs herein studied, so it was not considered.
Detergents are required to extract and purify target proteins and their choice is a key parameter on the entire process. We chose to use DDM since it is a mild detergent and one of the most commonly used for this purpose [20,27,33,40]. The aggregation detected in the large-scale production experiments suggests further detergent screens may be needed to select the best detergent formula for each individual target. We were able to separate two different populations of EmbB from M. vanbaalenii PYR-1, likely constituted by monomer and dimer, respectively. Despite the aggregation, all targets showed soluble populations in the SEC elution profiles, which represents a good starting point for optimization towards structural studies. Interestingly, 3D structures of both oligomeric states have been already characterized by single particle cryo-electron microscopy (cryo-EM) for EmbB from M. smegmatis [41,42]. Noteworthy, different detergents or solubilizing agents may be needed for structural studies. The cryo-EM structures of several AraTs, namely EmbA-EmbB complex, M. tuberculosis EmbB [43], M. smegmatis EmbB [42] and AftD [44], have been recently characterized and different solubilization agents were used, namely glyco-diosgenin (GDN) detergent, amphipols or nanodiscs. Interestingly, the structure of EmbC solubilized in DDM has been determined by X-ray crystallography [43].
On one hand, the production of AftB and EmbC targets was not achieved using the HTP workflow with “standard” conditions. Therefore, other parameters must be explored, such as growth media, temperature, incubation time and type of detergent, host strains or expression vectors, which will likely lead to better success rates. On the other hand, AftA and AftC from M. neoaurum were expressed and are attractive targets for drug development [45,46,47] and structural elucidation, since their structures are not yet known.
The presented protocol stands as a simplified approach based in previous HTP strategies developed at NYCOMPS [19,20] and by others [15], to identify the best candidates for further biochemical and structural studies in a fast and affordable manner for most laboratories. Most importantly, this methodology delivers similar results to conventional medium throughput approaches and, by reducing the variables throughout, it allows target optimization for large-scale protein production.

Author Contributions

Conceptualization, M.A., F.M., B.K. and J.R.; methodology, J.R., V.T.A., A.L.R. and Y.Z.T.; data analysis, all authors; original draft preparation, J.R. and V.T.A.; review and editing, all; supervision and funding acquisition, M.A. and F.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Fundação para a Ciência e Tecnologia (FCT), Lisbon, Portugal; grants (PTDC/BIA-BQM/30421/2017 and PTDC/BIA-BQM/4056/2020 to M.A. and PD/BD/128261/2016 to J.R.), European Union’s Horizon 2020 research and innovation programme under grant agreements: No. 857203 (Twinning), No. 823780 (MSCA-RISE) and No. 731005, Instruct-ULTRA, a project to further develop the services of Instruct-ERIC (M.A, J.R. and V.T.A.). This work was also supported by National Institutes of Health (NIH), Bethesda, MD, USA; grants GM132120 (to F.M.) and GM116799 (to Wayne A. Hendrickson).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank Alessio Bortoluzzi, Catarina Paiva and Sandra Santos for advice and help with equipment for high throughput protein expression.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Table
Table A1. Protein targets used in the high-throughput screening and expression results for each E. coli strain tested.
Table A1. Protein targets used in the high-throughput screening and expression results for each E. coli strain tested.
IDGenBank IDOrganismPredicted ProteinC41C43BL21 (DE3) pLysS
A1SIU02450.1Mycobacterium bovis AF2122/97AftA−−−
A8EUA63955.1Mycobacterium abscessus 1948AftA−−+
C2ABP43658.1Mycobacterium gilvum PYR-GCKAftA+−−
C9AFC41461.1Mycobacterium intracellulare ATCC 13950AftA−−−
F1ABM16394.1Mycobacterium vanbaalenii PYR-1AftA+−+
F3AGP61782.1Mycobacterium yongonense 05-1390AftA−−−
F7CDQ43571.1Mycobacterium neoaurumAftA+−+
F8AAS02550.1Mycobacterium avium subsp. paratuberculosis K-10AftA−−−
A2SIU02464.1Mycobacterium bovis AF2122/97AftB−−−
A9EUA63936.1Mycobacterium abscessus 1948AftB−−−
C7CCP46634.1Mycobacterium tuberculosis H37RvAftB−−−
D9AGP61763.1Mycobacterium yongonense 05-1390AftB−−−
D10AFC41442.1Mycobacterium intracellulare ATCC 13950AftB−−−
D11ABM16411.1Mycobacterium vanbaalenii PYR-1AftB−−−
E2AIR19061.1 *Mycobacterium kansasii 662AftB−−−
E5CDQ43590.1Mycobacterium neoaurumAftB−−−
A3AMC65006.1Mycobacterium bovis AF2122/97AftC+−+
A10EUA61591.1Mycobacterium abscessus 1948AftC+−−
C11AFC44620.1Mycobacterium intracellulare ATCC 13950AftC++−
C12AGZ53302.1Mycobacterium kansasii ATCC 12478AftC++−
D1ABP46386.1Mycobacterium gilvum PYR-GCKAftC+++
D2CDQ43952.1Mycobacterium neoaurumAftC+++
E6EHB50241.1Mycobacterium rhodesiae JS60AftC−−−
E7AFM16967.1Mycobacterium chubuense NBB4AftC−−−
E8AAS05110.1Mycobacterium avium subsp. paratuberculosis K-10AftC++−
E9AGP64972.1Mycobacterium yongonense 05-1390AftC++−
F9ABM13300.1Mycobacterium vanbaalenii PYR-1AftC−−−
A4CAB5247947.1Mycobacterium bovis AF2122/97AftD−−−
D4AGZ51741.1Mycobacterium kansasii ATCC 12478AftD−−−
H11EUA63217.1Mycobacterium abscessus 1948 F5/8AftD+−+
A5SIU02452.1Mycobacterium bovis AF2122/97EmbA−−−
A11EUA63951.1Mycobacterium abscessus 1948EmbA+−−
B4AAC45280.1Mycobacterium tuberculosis H37RvEmbA−−−
B8ACC43760.1Mycobacterium marinum MEmbA+−−
C5ABP43656.1Mycobacterium gilvum PYR-GCKEmbA−−−
G5AAS02546.1Mycobacterium avium subsp. paratuberculosis K-10EmbA−−−
G11CDQ43576.1Mycobacterium neoaurumEmbA−−−
H1ABM16396.1Mycobacterium vanbaalenii PYR-1EmbA−−−
H2AGZ51276.1Mycobacterium kansasii ATCC 12478EmbA−−−
A6SIU02453.1Mycobacterium bovis AF2122/97EmbB−−−
A12EUA63949.1Mycobacterium abscessus 1948EmbB+−−
B3AAC45281.1Mycobacterium tuberculosis H37RvEmbB−−−
B7ACC43761.1Mycobacterium marinum MEmbB−−−
C4ABP43655.1Mycobacterium gilvum PYR-GCKEmbB−−−
G3AFC41455.1Mycobacterium intracellulare ATCC 13950EmbB−−−
G6AAS02545.1Mycobacterium avium subsp. paratuberculosis K-10EmbB−−−
G9AGP61776.1Mycobacterium yongonense 05-1390EmbB−−−
H7KEP38884.1Mycobacterium kansasiiEmbB−−−
H9ABM16397.1Mycobacterium vanbaalenii PYR-1EmbB+−+
B1EUA63954.1Mycobacterium abscessus 1948EmbC−−−
C3ABP43657.1Mycobacterium gilvum PYR-GCKEmbC−−−
G4AFC41460.1Mycobacterium intracellulare ATCC 13950EmbC−−−
G7AAS02549.1Mycobacterium avium subsp. paratuberculosis K-10EmbC−−−
G10AGP61781.1Mycobacterium yongonense 05-1390EmbC−−−
H4CDQ43572.1Mycobacterium neoaurumEmbC−−−
H5AGZ51274.1Mycobacterium kansasii ATCC 12478EmbC−−−
* Record removed.
Table
Table A2. List of genes coding for AraTs from several Mycobacterium species selected for cloning and expression screening.
Table A2. List of genes coding for AraTs from several Mycobacterium species selected for cloning and expression screening.
IDGenBank IDOrganismPredicted Protein% Identity
(M. tuberculosis)		Predicted Molecular Weight (kDa)Predicted
Transmembrane Helixes
A1SIU02450.1Mycobacterium bovis AF2122/97AftA1007013
A2SIU02464.1Mycobacterium bovis AF2122/97AftB99699
A3AMC65006.1Mycobacterium bovis AF2122/97AftC99498
A4CAB5247947.1Mycobacterium bovis AF2122/97AftD991469
A5SIU02452.1Mycobacterium bovis AF2122/97EmbA9911613
A6SIU02453.1Mycobacterium bovis AF2122/97EmbB9911813
A7SIU02451.1Mycobacterium bovis AF2122/97EmbC9911813
A8EUA63955.1Mycobacterium abscessus 1948AftA656813
A9EUA63936.1Mycobacterium abscessus 1948AftB677110
A10EUA61591.1Mycobacterium abscessus 1948AftC64478
A11EUA63951.1Mycobacterium abscessus 1948EmbA6511412
A12EUA63949.1Mycobacterium abscessus 1948EmbB68738
B1EUA63954.1Mycobacterium abscessus 1948EmbC6811711
B2AAC45279.1Mycobacterium tuberculosis H37RvEmbC10011713
B3AAC45281.1Mycobacterium tuberculosis H37RvEmbB10011812
B4AAC45280.1Mycobacterium tuberculosis H37RvEmbA10011613
B5CCP42964.1Mycobacterium tuberculosis H37RvAftD1001469
B6ACC43759.1Mycobacterium marinum MEmbC8611714
B7ACC43761.1Mycobacterium marinum MEmbB8911612
B8ACC43760.1Mycobacterium marinum MEmbA8711813
B9AFM19671.1Mycobacterium chubuense NBB4EmbB7211513
B10AFM19669.1Mycobacterium chubuense NBB4EmbC7511612
B11AFM19670.1Mycobacterium chubuense NBB4EmbA6911513
B12AFM19668.1Mycobacterium chubuense NBB4AftA677110
C1ACC43758.1Mycobacterium marinum MAftA837013
C2ABP43658.1Mycobacterium gilvum PYR-GCKAftA686713
C3ABP43657.1Mycobacterium gilvum PYR-GCKEmbC7411514
C4ABP43655.1Mycobacterium gilvum PYR-GCKEmbB7011513
C5ABP43656.1Mycobacterium gilvum PYR-GCKEmbA6911412
C6AIU11367.1Mycobacterium smegmatis str. MC2 155AftA686712
C7CCP46634.1Mycobacterium tuberculosis H37RvAftB100699
C8AAS02532.1Mycobacterium avium subsp. ParatuberculosisAftB82709
C9AFC41461.1Mycobacterium intracellulare ATCC 13950AftA776813
C10ABK72123.1Mycobacterium smegmatis str. MC2 155AftC70498
C11AFC44620.1Mycobacterium intracellulare ATCC 13950AftC84508
C12AGZ53302.1Mycobacterium kansasii ATCC 12478AftC89498
D1ABP46386.1Mycobacterium gilvum PYR-GCKAftC70489
D2CDQ43952.1Mycobacterium neoaurumAftC70488
D3ABK71542.1Mycobacterium smegmatis str. MC2 155AftD7114812
D4AGZ51741.1Mycobacterium kansasii ATCC 12478AftD8214813
D5ACC38960.1Mycobacterium marinumAftD801467
D6AFM15049.1Mycobacterium chubuense NBB4AftD7014512
D7ADT97050.1Mycobacterium gilvum Spyr1AftD7014813
D8ABP43645.1Mycobacterium gilvum PYR-GCKAftB71709
D9AGP61763.1Mycobacterium yongonense 05-1390AftB827210
D10AFC41442.1Mycobacterium intracellulare ATCC 13950AftB827210
D11ABM16411.1Mycobacterium vanbaalenii PYR-1AftB70729
D12EHB54870.1Mycobacterium rhodesiae JS60AftB687310
E1CDM79377.1Mycobacterium marinum E11AftB857310
E2AIR19061.1 *Mycobacterium kansasii 662AftB867310
E3ABK75671.1Mycobacterium smegmatis str. MC2 155AftB73709
E4AFM19681.1Mycobacterium chubuense NBB4AftB687211
E5CDQ43590.1Mycobacterium neoaurumAftB676711
E6EHB50241.1Mycobacterium rhodesiae JS60AftC71497
E7AFM16967.1Mycobacterium chubuense NBB4AftC71499
E8AAS05110.1Mycobacterium avium subsp. paratuberculosis K-10AftC83498
E9AGP64972.1Mycobacterium yongonense 05-1390AftC83508
E10ACC40492.1Mycobacterium marinumAftC87508
E11AAS06236.1Mycobacterium avium subsp. paratuberculosis K-10AftD791459
E12EHB55421.1Mycobacterium rhodesiae JS60AftD7114913
F1ABM16394.1Mycobacterium vanbaalenii PYR-1AftA676813
F2CDQ42439.1Mycobacterium neoaurum F5/8AftD6714813
F3AGP61782.1Mycobacterium yongonense 05-1390AftA776813
F4EHB54850.1Mycobacterium rhodesiae JS60AftA716613
F5AGZ51273.1Mycobacterium kansasii ATCC 12478AftA847013
F6AGP66385.1Mycobacterium yongonense 05-1390AftD8014613
F7CDQ43571.1Mycobacterium neoaurumAftA676711
F8AAS02550.1Mycobacterium avium subsp. paratuberculosis K-10AftA797511
F9ABM13300.1Mycobacterium vanbaalenii PYR-1AftC69489
F10ABM11102.1Mycobacterium vanbaalenii PYR-1AftD7014710
F11AFP42646.1Mycobacterium smegmatis str. MC2 155EmbA6911713
F12ABK72840.1Mycobacterium smegmatis str. MC2 155EmbB6911713
G1ABK72375.1Mycobacterium smegmatis str. MC2 155EmbC7511510
G2AFC41456.1Mycobacterium intracellulare ATCC 13950EmbA8311514
G3AFC41455.1Mycobacterium intracellulare ATCC 13950EmbB8511512
G4AFC41460.1Mycobacterium intracellulare ATCC 13950EmbC8511413
G5AAS02546.1Mycobacterium avium subsp. paratuberculosis K-10EmbA8311714
G6AAS02545.1Mycobacterium avium subsp. paratuberculosis K-10EmbB8411512
G7AAS02549.1Mycobacterium avium subsp. paratuberculosis K-10EmbC8511713
G8AGP61777.1Mycobacterium yongonense 05-1390EmbA8311614
G9AGP61776.1Mycobacterium yongonense 05-1390EmbB8511512
G10AGP61781.1Mycobacterium yongonense 05-1390EmbC8511413
G11CDQ43576.1Mycobacterium neoaurumEmbA6911613
G12EHB54852.1Mycobacterium rhodesiae JS60EmbA7211613
H1ABM16396.1Mycobacterium vanbaalenii PYR-1EmbA7011613
H2AGZ51276.1Mycobacterium kansasii ATCC 12478EmbA8811513
H3AEV72559.1Mycobacterium rhodesiae NBB3EmbC7611414
H4CDQ43572.1Mycobacterium neoaurumEmbC7211513
H5AGZ51274.1Mycobacterium kansasii ATCC 12478EmbC8811714
H6ABM16395.1Mycobacterium vanbaalenii PYR-1EmbC7311610
H7KEP38884.1Mycobacterium kansasiiEmbB9011711
H8AEV72557.1Mycobacterium rhodesiae NBB3EmbB7611413
H9ABM16397.1Mycobacterium vanbaalenii PYR-1EmbB7011513
H10AHC23140.2Mycobacterium neoaurum VKM Ac-1815DEmbB6911513
H11EUA63217.1Mycobacterium abscessus 1948 F5/8AftD6214912
H12AFC46048.1Mycobacterium intracellulare ATCC 13950AftD7914613
* Record removed.
Table
Table A3. Optical Density at 600 nm (OD600) of small-scale cultures. In-plate target coordinates.
Table A3. Optical Density at 600 nm (OD600) of small-scale cultures. In-plate target coordinates.
AftAA1A8C2C9F1F3F7F8
AftBA2A9C7D9D10D11E2E5
AftCA3A10C11C12D1D2E6E7E8E9F9
AftDA4D4H11
EmbAA5A11B4B8C5G5G11H1H2
EmbBA6A12B3B7C4G3G6G9H7H9
EmbCB1C3G4G7G10H4H5
Table
Table A4. OD600 upon induction of expression. Color scheme: green color—OD600 interval (0.5 −1.0), red color—OD600 below 0.5 or above 1.0. The black contoured squares show the values obtained for the targets selected for large scale.
Table A4. OD600 upon induction of expression. Color scheme: green color—OD600 interval (0.5 −1.0), red color—OD600 below 0.5 or above 1.0. The black contoured squares show the values obtained for the targets selected for large scale.
OD of Induction
BL21 (DE3) pLysSAftA0.920.991.040.851.031.050.801.10
AftB0.820.871.000.750.780.820.580.65
AftC1.020.640.720.801.050.840.881.070.860.751.23
AftD0.931.000.61
EmbA0.870.720.790.740.930.931.161.110.89
EmbB1.060.910.890.880.970.820.560.900.300.70
EmbC1.030.471.120.961.060.460.40
C41AftA1.191.000.770.570.680.630.760.60
AftB0.430.460.780.750.810.790.720.60
AftC1.010.550.870.771.140.840.851.060.660.590.86
AftD0.550.750.62
EmbA0.510.720.640.740.510.520.620.530.58
EmbB0.580.560.370.670.400.430.500.530.740.68
EmbC0.760.970.700.630.810.630.40
C43AftA0.730.800.880.710.840.810.920.81
AftB0.810.750.520.830.780.850.790.76
AftC0.790.760.750.720.821.040.830.870.800.780.83
AftD0.660.520.74
EmbA0.710.650.620.700.670.610.570.600.56
EmbB0.740.660.680.700.480.670.680.710.600.82
EmbC0.880.780.720.610.480.630.65
Table
Table A5. Variation of OD600 between the induction time and cell harvesting. A graduated scale of three colors was applied to these values, indicating average values in white, the lower values in red and higher values in green. The black contoured squares show the values obtained for the targets selected for large scale.
Table A5. Variation of OD600 between the induction time and cell harvesting. A graduated scale of three colors was applied to these values, indicating average values in white, the lower values in red and higher values in green. The black contoured squares show the values obtained for the targets selected for large scale.
OD Variation
BL21(DE3) pLysSAftA9.366.438.447.873.824.131.478.71
AftB7.948.338.266.765.517.117.805.70
AftC8.946.737.391.557.176.976.966.827.207.448.25
AftD5.156.755.44
EmbA8.546.517.858.087.788.307.717.607.58
EmbB6.928.335.195.358.207.707.188.375.231.58
EmbC6.767.656.417.387.138.147.94
C41AftA9.148.196.409.168.979.077.708.41
AftB7.439.6510.6714.877.489.076.126.45
AftC9.158.8910.069.608.4510.177.937.389.279.067.52
AftD5.627.525.03
EmbA7.096.396.575.758.329.606.293.406.45
EmbB6.677.674.155.015.285.414.005.227.186.98
EmbC6.568.907.968.699.389.457.72
C43AftA6.977.066.966.826.666.767.707.25
AftB7.316.8512.098.647.667.277.227.09
AftC7.249.827.9511.347.6212.737.838.028.807.647.38
AftD6.6110.167.21
EmbA6.7010.837.387.067.567.826.827.306.60
EmbB7.068.088.867.997.437.327.437.626.908.50
EmbC7.217.126.876.896.687.467.01
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Figure A1. Protein expression results from the HTP screening of 56 AraTs from Mycobacteria, overexpressed in E. coli C43 cells. A—AftA proteins: 1—A1; 2—A8; 3—C2; 4—C9; 5—F1; 6—F3; 7—F7; 8—F8. B—AftB proteins 1—A2; 2—A9; 3—C7; 4—D9; 5—D10; 6—D11; 7—E2; 8—E5. C—AftC proteins: 1—A3; 2—A10; 3—C11; 4—C12; 5—D1; 6—D2; 7—E6; 8—E7; 9—E8; 10—E9; 11—F9. D—AftD and EmbA proteins: 1—A4; 2—D4; 3—H11; 4—A5; 5—A11; 6—B4; 7—B8; 8—C5; 9—G5; 10—G11; 11—H1; 12—H2. E—EmbB proteins: 1—A6; 2—A12; 3—B3; 4—B7; 5—C4; 6—G3; 7—G6; 8—G9; 9—H7; 10—H9. F—EmbC proteins: 1—B1; 2—C3; 3—G4; 4—G7; 5—G10; 6—H4; 7—H5. Asterisks (⁎) indicate protein bands corresponding to the predicted MW of the corresponding target.
Figure A1. Protein expression results from the HTP screening of 56 AraTs from Mycobacteria, overexpressed in E. coli C43 cells. A—AftA proteins: 1—A1; 2—A8; 3—C2; 4—C9; 5—F1; 6—F3; 7—F7; 8—F8. B—AftB proteins 1—A2; 2—A9; 3—C7; 4—D9; 5—D10; 6—D11; 7—E2; 8—E5. C—AftC proteins: 1—A3; 2—A10; 3—C11; 4—C12; 5—D1; 6—D2; 7—E6; 8—E7; 9—E8; 10—E9; 11—F9. D—AftD and EmbA proteins: 1—A4; 2—D4; 3—H11; 4—A5; 5—A11; 6—B4; 7—B8; 8—C5; 9—G5; 10—G11; 11—H1; 12—H2. E—EmbB proteins: 1—A6; 2—A12; 3—B3; 4—B7; 5—C4; 6—G3; 7—G6; 8—G9; 9—H7; 10—H9. F—EmbC proteins: 1—B1; 2—C3; 3—G4; 4—G7; 5—G10; 6—H4; 7—H5. Asterisks (⁎) indicate protein bands corresponding to the predicted MW of the corresponding target.
Processes 09 00629 g0a1
Processes 09 00629 g0a2 550
Figure A2. Protein expression results from the HTP screening of 56 AraTs from Mycobacteria, overexpressed in E. coli BL21 (DE3) pLysS cells. A—AftA proteins: 1—A1; 2—A8; 3—C2; 4—C9; 5—F1; 6—F3; 7—F7; 8—F8. B—AftB proteins 1—A2; 2—A9; 3—C7; 4—D9; 5—D10; 6—D11; 7—E2; 8—E5. C—AftC proteins: 1—A3; 2—A10; 3—C11; 4—C12; 5—D1; 6—D2; 7—E6; 8—E7; 9—E8; 10—E9; 11—F9. D—AftD and EmbA proteins: 1—A4; 2—D4; 3—H11; 4—A5; 5—A11; 6—B4; 7—B8; 8—C5; 9—G5; 10—G11; 11—H1; 12—H2. E—EmbB proteins: 1—A6; 2—A12; 3—B3; 4—B7; 5—C4; 6—G3; 7—G6; 8—G9; 9—H7; 10—H9. F—EmbC proteins: 1—B1; 2—C3; 3—G4; 4—G7; 5—G10; 6—H4; 7—H5. Asterisks (⁎) indicate protein bands corresponding to the predicted MW of the corresponding target.
Figure A2. Protein expression results from the HTP screening of 56 AraTs from Mycobacteria, overexpressed in E. coli BL21 (DE3) pLysS cells. A—AftA proteins: 1—A1; 2—A8; 3—C2; 4—C9; 5—F1; 6—F3; 7—F7; 8—F8. B—AftB proteins 1—A2; 2—A9; 3—C7; 4—D9; 5—D10; 6—D11; 7—E2; 8—E5. C—AftC proteins: 1—A3; 2—A10; 3—C11; 4—C12; 5—D1; 6—D2; 7—E6; 8—E7; 9—E8; 10—E9; 11—F9. D—AftD and EmbA proteins: 1—A4; 2—D4; 3—H11; 4—A5; 5—A11; 6—B4; 7—B8; 8—C5; 9—G5; 10—G11; 11—H1; 12—H2. E—EmbB proteins: 1—A6; 2—A12; 3—B3; 4—B7; 5—C4; 6—G3; 7—G6; 8—G9; 9—H7; 10—H9. F—EmbC proteins: 1—B1; 2—C3; 3—G4; 4—G7; 5—G10; 6—H4; 7—H5. Asterisks (⁎) indicate protein bands corresponding to the predicted MW of the corresponding target.
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Figure 1. Schematic representation of the high-throughput screening for membrane protein production and purification.
Figure 1. Schematic representation of the high-throughput screening for membrane protein production and purification.
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Figure 2. Protein expression results from the HTP screening of 56 AraTs from Mycobacteria overexpressed in E. coli C41 cells. A—AftA proteins: 1—A1; 2—A8; 3—C2; 4—C9; 5—F1; 6—F3; 7—F7; 8—F8. B—AftB proteins 1—A2; 2—A9; 3—C7; 4—D9; 5—D10; 6—D11; 7—E2; 8—E5. C—AftC proteins: 1—A3; 2—A10; 3—C11; 4—C12; 5—D1; 6—D2; 7—E6; 8—E7; 9—E8; 10—E9; 11—F9. D—AftD and EmbA proteins: 1—A4; 2—D4; 3—H11; 4—A5; 5—A11; 6—B4; 7—B8; 8—C5; 9—G5; 10—G11; 11—H1; 12—H2. E—EmbB proteins: 1—A6; 2—A12; 3—B3; 4—B7; 5—C4; 6—G3; 7—G6; 8—G9; 9—H7; 10—H9. F—EmbC proteins: 1—B1; 2—C3; 3—G4; 4—G7; 5—G10; 6—H4; 7—H5. Asterisks (⁎) indicate protein bands corresponding to the predicted MW of the corresponding target.
Figure 2. Protein expression results from the HTP screening of 56 AraTs from Mycobacteria overexpressed in E. coli C41 cells. A—AftA proteins: 1—A1; 2—A8; 3—C2; 4—C9; 5—F1; 6—F3; 7—F7; 8—F8. B—AftB proteins 1—A2; 2—A9; 3—C7; 4—D9; 5—D10; 6—D11; 7—E2; 8—E5. C—AftC proteins: 1—A3; 2—A10; 3—C11; 4—C12; 5—D1; 6—D2; 7—E6; 8—E7; 9—E8; 10—E9; 11—F9. D—AftD and EmbA proteins: 1—A4; 2—D4; 3—H11; 4—A5; 5—A11; 6—B4; 7—B8; 8—C5; 9—G5; 10—G11; 11—H1; 12—H2. E—EmbB proteins: 1—A6; 2—A12; 3—B3; 4—B7; 5—C4; 6—G3; 7—G6; 8—G9; 9—H7; 10—H9. F—EmbC proteins: 1—B1; 2—C3; 3—G4; 4—G7; 5—G10; 6—H4; 7—H5. Asterisks (⁎) indicate protein bands corresponding to the predicted MW of the corresponding target.
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Figure 3. Size-exclusion chromatography (SEC) elution profiles of large-scale expression experiments of AraTs and respective SDS-PAGE. Vertical arrows indicate void volume; asterisks (*) and (⁑) indicate different populations observed in the SEC elution profiles. Ni: Ni-NTA elution samples; HTP: Small-scale HTP results; horizontal arrows indicate the bands corresponding to the target protein of interest. (A) AftA from M. neoaurum, (B) AftC from M. neoaurum, (C) AftD from M. abscessus 1948 F5/8, (D) EmbA from M. marinum M. and (E) EmbB from M. vanbaalenii PYR-1.
Figure 3. Size-exclusion chromatography (SEC) elution profiles of large-scale expression experiments of AraTs and respective SDS-PAGE. Vertical arrows indicate void volume; asterisks (*) and (⁑) indicate different populations observed in the SEC elution profiles. Ni: Ni-NTA elution samples; HTP: Small-scale HTP results; horizontal arrows indicate the bands corresponding to the target protein of interest. (A) AftA from M. neoaurum, (B) AftC from M. neoaurum, (C) AftD from M. abscessus 1948 F5/8, (D) EmbA from M. marinum M. and (E) EmbB from M. vanbaalenii PYR-1.
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Figure 4. Size-exclusion chromatography elution profiles of EmbB from M. vanbaalenii PYR-1. (A) SEC elution profiles of EmbB: after Ni-NTA (full black line), peak (⁎) after 1st SEC (red dashed lines), peak (⁑) after 1st SEC (spotted blue line). (B) Normalized SEC elution profiles of EmbB: after Ni-NTA (full black line), peak (⁎) after 1st SEC (red dashed lines), peak (⁑) after 1st SEC (spotted blue line). (C) SDS-PAGE analysis of peak (⁎) and (⁑) SEC. Vertical arrows indicate void volume. Colored asterisks correspond to the peaks observed in each SEC run, respective to different EmbB populations. Arrow indicates the corresponding band of EmbB (115 kDa).
Figure 4. Size-exclusion chromatography elution profiles of EmbB from M. vanbaalenii PYR-1. (A) SEC elution profiles of EmbB: after Ni-NTA (full black line), peak (⁎) after 1st SEC (red dashed lines), peak (⁑) after 1st SEC (spotted blue line). (B) Normalized SEC elution profiles of EmbB: after Ni-NTA (full black line), peak (⁎) after 1st SEC (red dashed lines), peak (⁑) after 1st SEC (spotted blue line). (C) SDS-PAGE analysis of peak (⁎) and (⁑) SEC. Vertical arrows indicate void volume. Colored asterisks correspond to the peaks observed in each SEC run, respective to different EmbB populations. Arrow indicates the corresponding band of EmbB (115 kDa).
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Table
Table 1. Summary of high-throughput screening of membrane protein production.
Table 1. Summary of high-throughput screening of membrane protein production.
NumberSuccess Rate (%)
Targets96-
Positive Clones (pNYCOMPS-N23)5658
1 Positive Clones (pNYCOMPS-C23)4042
Proteins purified (total)1718
E. coli C41::pNYCOMPS-N231617
E. coli C43::pNYCOMPS-N2366
E. coli BL21 (DE3) pLysS::pNYCOMPS-N2388
1 Expression of these constructs was not determined in this study.
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Rodrigues, J.; Almeida, V.T.; Rosário, A.L.; Tan, Y.Z.; Kloss, B.; Mancia, F.; Archer, M. High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria. Processes 2021, 9, 629. https://doi.org/10.3390/pr9040629

AMA Style

Rodrigues J, Almeida VT, Rosário AL, Tan YZ, Kloss B, Mancia F, Archer M. High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria. Processes. 2021; 9(4):629. https://doi.org/10.3390/pr9040629

Chicago/Turabian Style

Rodrigues, José, Vanessa T. Almeida, Ana L. Rosário, Yong Zi Tan, Brian Kloss, Filippo Mancia, and Margarida Archer. 2021. "High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria" Processes 9, no. 4: 629. https://doi.org/10.3390/pr9040629

APA Style

Rodrigues, J., Almeida, V. T., Rosário, A. L., Tan, Y. Z., Kloss, B., Mancia, F., & Archer, M. (2021). High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria. Processes, 9(4), 629. https://doi.org/10.3390/pr9040629

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