Simple, Functional, Inexpensive Cell Extract forin vitroPrototyping of Proteins with Disulfide Bonds

Abstract

In vitro expression of proteins from E. coli extract is a useful method for prototyping and production of cytotoxic or unnatural products. However, proteins that have multiple disulfide bonds require custom extract that to date requires careful addition of exogenous, purified isomerase enzymes. Many important proteins such as enzymes, cytokines, and monoclonal antibodies require disulfide bonds for stabilization and/or proper activity. Currently the only solution to expressing these products is to purchase commercial kits (such as PUREfrex® (GeneFrontier) or PURExpress® (New England BioLabs)) or to grow up the KGK10 strain and supplement with purified enzymes (T7 RNA polymerase and disulfide bond isomerase C). This multistep process can limit the accessibility of such extract to some groups that wish to rapidly prototype proteins with disulfide bonds. In this work, we present a "one-pot" solution that does not require addition of supplemental enzymes. This is done using a commercially available SHuffle® T7 Express lysY strain of E. coli that can express both the needed T7 polymerase and DsbC isomerase enzymes. We find optimal growth conditions for our 1 L cultures in 2.5 L shake flasks (5.6 and 3.9 hr for harvest and induction respectively) using a luciferase (from Gaussia princeps) that contains 5 disulfide bonds as our reporter protein. The method presented here uses a continuous pass homogenizer and pilot scale lyophilizer to produce large volumes of extract; also, optimal reconstitution ratios of the dried extract are found. To show the broad applicability of the extract, three other enzymes containing ≥ 3 disulfide bonds (hevamine, endochitinase A, and periplasmic AppA) were also expressed from minimal genetic templates that had undergone rolling circle amplification.

Methodologies for preparation of prokaryotic extracts for cell-free expression systems

“…Commercially, Sutro Biopharma utilizes variants of the strain for producing cytokine rhGM-CSF at 200 L scale [ 61 ] and producing antibody fragment light and heavy chains [ 60 ]. A recent addition is the use of the commercial SHuffle T7 Express lysY strain of E. coli that expresses T7 RNAP and DsbC isomerase enzymes to rapidly prototype proteins with disulfide bonds [ 86 ].…”

Designing of an extract production protocol for industrial application of cell‐free protein synthesis technology: Building from a current best practice to a quality by design approach

“…The limited commercial use of CFPS means that there is no regulatory precedent to follow, and quality control approaches remain to be fully elucidated. Concerns around maintaining reagent [14][15][16] and reaction consistency in particular will need to be addressed. There is evidence to suggest that with automation, better process and environmental controls and process understanding derived from Multivariate Data Analysis (MVDA) [17,18] reaction-related variations can be minimised [19][20][21][22].…”

parSEQ:Probe and Rescue Sequencing for Advanced Variant Retrieval from DNA Pool

“…Unique sequences retrieved from this run were expressed in a PUREfrex 2.1 cell-free system 26 and screened for affinity against their antigen using bio-layer interferometry (BLI).…”