Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours

Abstract

In this study, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hr from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell‐based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost‐optimized, scalably‐manufactured Escherichia coli extract and minimal supplement reagents to perform cell‐free protein synthesis (CFPS) of the template protein. We demonstrate the utility of this template design and 24 hr process with seven fluorescent proteins (sfGFP, mVenus, mCherry, and four GFP variants), three enzymes (chloramphenicol acetyltransferase, a chitinase catalytic domain, and native subtilisin), a capture protein (anti‐GFP nanobody), and 2 antimicrobial peptides (BP100 and CA(1–7)M(2–9)). We detected each of these directly from the CFPS reaction using colorimetric, fluorogenic, and growth assays. Of especial note, the GFP variant sequences were found from genomic screening data and had not been expressed or characterized before, thus demonstrating the utility of this approach for phenotype characterization of sequenced libraries. We also demonstrate that the rolling circle amplified version of the linear template exhibits expression similar to that of a complete plasmid when expressing sfGFP in the CFPS reaction. We evaluate the cost of this approach to be $61/mg sfGFP for a 4 hr reaction. We also detail limitations of this approach and strategies to overcome these, namely proteins with posttranslational modifications.

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

“…To investigate the SHuffle strain's ability to quickly prototype proteins, we chose to express three enzymes (hevamine, endochitinase A (ChitA), and periplasmic AppA which have 3, 7, and 4 disulfide bonds) using our previously established minimal, rapid-order genetic template method. 12 Hevamine is a class IIIa chitinase from the rubber tree Hevea brasiliensis and exhibits exochitinase activity. 50 ChitA is a class IV chitinase found primarily in developing maize kernels of Zea mays that exhibits endochitinase activity.…”

“…The production of minimal genetic templates for prototyping new enzymes has been described in previous work. 12 In brief, the gene of interest was codon optimized using IDT's codon optimization tool and purchased in the form of a gene fragment. The gene fragment was then amplified using OneTaq (NEB), digested to form sticky ends with HindIII (NEB), ligated with T4 ligase (NEB), and isothermally amplified with TempliPhi (GE Healthcare).…”

“…[9][10][11] Cell extract derived from E. coli is the most widely used with efficient extract protocols, multiple supplement recipes, and clear experimental protocols available in literature. [12][13][14][15][16][17][18] One major drawback to using E. coli extract for protein expression is the inability to perform post-translational modifications. There has been much work to engineer glycosylation 19,20 and disulfide bond-formation 21,22 pathways.…”

“…33,34 The PUREfrex® commercial system, made of purified, recombinantly expressed proteins, is another platform capable of reliably producing proteins with disulfide bonds but has higher cost per reaction. 35 In line with our goal to make CFPS a robust method readily transferable to any research group, [12][13][14] we desired to identify and optimize a single strain that could be used to grow a single batch ('one-pot') of cells to produce extract without need for supplementation with exogenous enzymes. We identified a commercially available E. coli strain that has been optimized to support in vivo disulfide bonded protein expression in the cytoplasm, T7 SHuffle® by New England BioLabs (NEB).…”

Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins

“…CF systems de-couple the cultivation of bacteria from protein expression, which is beneficial for toxic proteins and can reduce the time of the actual protein synthesis. Templates can be provided in form of DNA or RNA, and the expression of different constructs (e.g., fusions with different protein tags) can be performed in a high throughput manner [ 31 , 32 ]. One key feature of CF reactions is that they are open systems and can be tailored to the protein of interest.…”