High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli

Abstract

BackgroundDisulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited.ResultsHere we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization.ConclusionsOur results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB-/gor- strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.

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

“…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 'generic' 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 proteins 11 . Using DsbC fusions and high throughput methods, within a week the soluble expression of multiple targets can be observed 11 and then additional variables, such as those discussed in the introduction, can be screened in subsequent rounds on those targets that require further optimization.…”

“…We have identified DsbC as being a generally applicable fusion partner for the soluble expression of disulfide-rich peptides and proteins 11 . Using DsbC fusions and high throughput methods, within a week the soluble expression of multiple targets can be observed 11 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.…”

“…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 tested 11 . 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.…”

“…Potential variables to be optimized include, but are not limited to, varying expression strains 1,2 , temperature 3,4 , media 2,3 , target variants 5 , fusion partners [6][7][8][9][10][11][12][13] , co-expression with chaperones 14,15 , cytoplasmic or periplasmic expression [16][17][18] , and purification buffer components 3 . 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.…”

“…We recently used our high throughput screening approach for the expression of soluble disulfide-rich proteins 11 . 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.…”