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

Abstract

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 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 ( www.venomics.eu ), 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.

Epitope Spreading of Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy

“…DsbC-His-HA-PLA2R1 fusion proteins were produced in E. coli essentially as described previously. 36 The detailed procedure will be published elsewhere. PLA2R1 domains with optimized codons for E. coli expression were added in frame with the leaderless DsbC open reading frame followed by His and HA tags for purification and detection, respectively.…”

Sequence homolog-based molecular engineering for shifting the enzymatic pH optimum

“…However, it should be noticed that this biostatistics method rely on well-characterized data for a large number of enzymes, which is still lacking for most of the enzyme families. Therefore, besides further enriching sequence information of enzyme families, developing high throughput techniques for expression and characterization of new enzymes would also make this method feasible and consummate [65] , [66] , [67] , [68] , [69] , [70] . The data showed in this study will help elucidate the mechanism that confer pH adaptation and thus may pave the way for redesigning biocatalysts with desired pH optimum for cell-free synthetic biology systems.…”

Recombinant antibody production evolves into multiple options aimed at yielding reagents suitable for application-specific needs

“…The consequence is that one has access to success stories but knows very little about conditions that resulted in failures and that would be useful to know in order to avoid similar mistakes. Systematic comparisons based on combinatorial approaches [ 114 ] and benchmarking initiatives [ 115 , 116 ] are extremely useful to evaluate methods, experimental sets or reagents but are still rare in the field of recombinant antibody production [ 27 , 60 , 78 , 86 , 117 ]. Another observed limit is represented by the publication of papers in which controls and characterization data necessary to assess the reliability of the claimed conclusions are missing.…”