Application of High-Throughput Methodologies to the Expression of Recombinant Proteins in E. coli

Background: The key issue in the development of novel antimicrobials is a rapid expansion of new bacterial strains resistant to current antibiotics. Indeed, World Health Organization has reported that bacteria commonly causing infections in hospitals and in the community, e.g. E. Coli, K. pneumoniae and S. aureus, have high resistance vs the last generations of cephalosporins, carbapenems and fluoroquinolones. During the past decades, only few successful efforts to develop and launch new antibacterial medications have been performed. This study aims to identify new class of antibacterial agents using novel high-throughput screening technique. Methods: We have designed library containing 125K compounds not similar in structure (Tanimoto coeff.< 0.7) to that published previously as antibiotics. The HTS platform based on double reporter system pDualrep2 was used to distinguish between molecules able to block translational machinery or induce SOS-response in a model E. coli system. MICs for most active chemicals in LB and M9 medium were determined using broth microdilution assay. Results: In an attempt to discover novel classes of antibacterials, we performed HTS of a large-scale small molecule library using our unique screening platform. This approach permitted us to quickly and robustly evaluate a lot of compounds as well as to determine the mechanism of action in the case of compounds being either translational machinery inhibitors or DNA-damaging agents/replication blockers. HTS has resulted in several new structural classes of molecules exhibiting an attractive antibacterial activity. Herein, we report as promising antibacterials. Two most active compounds from this series showed MIC value of 1.2 (5) and 1.8 μg/mL (6) and good selectivity index. Compound 6 caused RFP induction and low SOS response. In vitro luciferase assay has revealed that it is able to slightly inhibit protein biosynthesis. Compound 5 was tested on several archival strains and exhibited slight activity against gram-negative bacteria and outstanding activity against S. aureus. The key structural requirements for antibacterial potency were also explored. We found, that the unsubstituted carboxylic group is crucial for antibacterial activity as well as the presence of bulky hydrophobic substituents at phenyl fragment. Conclusion: The obtained results provide a solid background for further characterization of the 5'- (carbonylamino)-2,3'-bithiophene-4'-carboxylate derivatives discussed herein as new class of antibacterials and their optimization campaign.

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High-throughput microbioreactor provides a capable tool for early stage bioprocess development

Scientific Reports ◽

10.1038/s41598-021-81633-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mathias Fink ◽

Monika Cserjan-Puschmann ◽

Daniela Reinisch ◽

Gerald Striedner

Keyword(s):

High Throughput ◽

Process Development ◽

Early Stage ◽

Explanatory Power ◽

Batch Mode ◽

E Coli ◽

Speed Up ◽

Bioreactor System ◽

Cell Densities ◽

Microbial Systems

AbstractTremendous advancements in cell and protein engineering methodologies and bioinformatics have led to a vast increase in bacterial production clones and recombinant protein variants to be screened and evaluated. Consequently, an urgent need exists for efficient high-throughput (HTP) screening approaches to improve the efficiency in early process development as a basis to speed-up all subsequent steps in the course of process design and engineering. In this study, we selected the BioLector micro-bioreactor (µ-bioreactor) system as an HTP cultivation platform to screen E. coli expression clones producing representative protein candidates for biopharmaceutical applications. We evaluated the extent to which generated clones and condition screening results were transferable and comparable to results from fully controlled bioreactor systems operated in fed-batch mode at moderate or high cell densities. Direct comparison of 22 different production clones showed great transferability. We observed the same growth and expression characteristics, and identical clone rankings except one host-Fab-leader combination. This outcome demonstrates the explanatory power of HTP µ-bioreactor data and the suitability of this platform as a screening tool in upstream development of microbial systems. Fast, reliable, and transferable screening data significantly reduce experiments in fully controlled bioreactor systems and accelerate process development at lower cost.

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A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00537-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5995-6002 ◽

Cited By ~ 7

Author(s):

Kristin R. Baker ◽

Bimal Jana ◽

Henrik Franzyk ◽

Luca Guardabassi

Keyword(s):

Escherichia Coli ◽

High Throughput ◽

High Throughput Screening ◽

Gram Negative Bacteria ◽

Chromogenic Substrate ◽

Intrinsic Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

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CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics

The Analyst ◽

10.1039/c4an00228h ◽

2014 ◽

Vol 139 (13) ◽

pp. 3314-3323 ◽

Cited By ~ 39

Author(s):

Thomas Beneyton ◽

Faith Coldren ◽

Jean-Christophe Baret ◽

Andrew D. Griffiths ◽

Valérie Taly

Keyword(s):

Directed Evolution ◽

High Throughput ◽

Recombinant Enzyme ◽

Cell Analysis ◽

E Coli ◽

Cota Laccase

A high-throughput cell analysis and sorting platform using droplet-based microfluidics is introduced for directed evolution of recombinant CotA laccase expressed in E. coli.

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Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols

Applied and Environmental Microbiology ◽

10.1128/aem.00239-06 ◽

2006 ◽

Vol 72 (8) ◽

pp. 5225-5231 ◽

Cited By ~ 21

Author(s):

Emmanuel Frachon ◽

Vincent Bondet ◽

Hélène Munier-Lehmann ◽

Jacques Bellalou

Keyword(s):

Recombinant Protein ◽

Recombinant Proteins ◽

Protein Production ◽

Batch Cultures ◽

E Coli ◽

Working Volume ◽

Versatile Tool ◽

Labview Program ◽

Medium Formulation ◽

Conventional Medium

ABSTRACT A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

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Rapid E. coli-based cloning and expression system for production of recombinant proteins

Journal of Biotechnology ◽

10.1016/j.jbiotec.2014.07.237 ◽

2014 ◽

Vol 185 ◽

pp. S70

Author(s):

Boguslaw Lupa ◽

Krzysztof Stawujak ◽

Igor Rozanski ◽

Justyna Stec-Niemczyk

Keyword(s):

Recombinant Proteins ◽

Expression System ◽

Cloning And Expression ◽

E Coli

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Modular automated bottom-up proteomic sample preparation for high-throughput applications v1

10.17504/protocols.io.bwxypfpw ◽

2021 ◽

Author(s):

Yan Chen ◽

Nurgul Kaplan Lease ◽

Jennifer Gin ◽

Tad Ogorzalek ◽

Paul D. Adams ◽

...

Keyword(s):

Sample Preparation ◽

High Throughput ◽

Tryptic Digestion ◽

Gram Negative Bacteria ◽

Bottom Up ◽

Preparation Methods ◽

Gram Negative ◽

E Coli ◽

Protocol Development ◽

Set Up

Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimised step-by-step protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of peptides for 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 E. coli and R. toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with peptide variance (CVs) below 15%.

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Expression and bioactivity of recombinant segments of human perforinThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

Biochemistry and Cell Biology ◽

10.1139/o07-017 ◽

2007 ◽

Vol 85 (2) ◽

pp. 203-208 ◽

Cited By ~ 2

Author(s):

Hongmei Dong ◽

Xiaohu Xu ◽

Mohong Deng ◽

Xiaojun Yu ◽

Hu Zhao ◽

...

Keyword(s):

Recombinant Proteins ◽

Fusion Proteins ◽

Target Genes ◽

Review Process ◽

Peer Review Process ◽

Nitrilotriacetic Acid ◽

E Coli ◽

Recombinant Plasmids ◽

Expression Plasmids ◽

Selection Of

The aim of the study was to prepare an active recombinant human perforin by comparing 5 candidate segments of human perforin. Full-length perforin, MAC1 (28–349 aa), MAC2 (166–369 aa), C-100, and N-60 of human perforin were selected as candidate active segments and designated, respectively, HP1, HP2, HP3, HP4, and HP5. The target genes were amplified by PCR and the products were individually subcloned into pGEM-T. The genes for HP1, HP2, HP3, and HP5 were subcloned into pET-DsbA, whereas pET-41a (+) was used as the expression vector of HP4. The fusion proteins were expressed in Escherichia coli BL21pLysS(DE3) and purified using nickel nitrilotriacetic acid (NTA) agarose affinity chromatography. The hemolysis microassay was used as an activity assay of fusion protein. From this study, we obtained the recombinant plasmids pGEM-T-HP1, -HP2, -HP3, -HP4 and -HP5, consisting of 1600, 960, 600, 300bp, and 180, respectively. From these recombinant plasmids, expression plasmids were successfully constructed and expressed in E. coli BL21pLysS(DE3). The resultant fusion proteins, affinity purified using Ni–NTA, were ~80, 58, 45, 44, and 30 kDa, respectively. The recombinant proteins were assayed for activity on hemolysis. HP2 and HP5 were the only recombinant proteins that were active in hemolysis, and the hemolytic function was concentration dependent. These results demonstrate that active recombinant forms of perforin can be synthesized in a prokaryote model. The recombinant N-60 and MAC1 (28–349 aa) of human perforin have the function of forming pores. Our study provides the experimental basis for further investigation on the application of perforin.

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