Characterizing and Improving pET Vectors for Cell-free Expression

Abstract

Cell-free protein synthesis (CFPS) is an in vitro process that enables diverse applications in research, biomanufacturing, point-of-care diagnostics, therapeutics, and education using minimal laboratory equipment and reagents. One of the major limitations of CFPS implementation is its sensitivity to plasmid type. Specifically, plasmid templates based on commonly used vector backbones such as the pET series of bacterial expression vectors result in the inferior production of proteins. To overcome this limitation, we have evaluated the effect of expression cassette elements present in the pET30 vector on protein production across three different CFPS systems: NEBExpress, PURExpress, and CFAI-based E. coli extracts. Through the systematic elimination of genetic elements within the pET30 vector, we have identified elements that are responsible for the poor performance of pET30 vectors in the various CFPS systems. As a result, we demonstrate that through the removal of the lac operator (lacO) and N-terminal tags included in the vector backbone sequence, a pET vector can support high titers of protein expression when using extract-based CFPS systems. This work provides two key advances for the research community: 1) identification of vector sequence elements that affect robust production of proteins; 2) evaluation of expression across three unique CFPS systems including CFAI extracts, NEBexpress, and PURExpress. We anticipate that this work will improve access to CFPS by enabling researchers to choose the correct expression backbone within the context of their preferred expression system.

Amino acid balancing for the prediction and evaluation of protein concentrations in cell‐free protein synthesis systems

“…For the systems of the other two organisms considered, comparison of protein concentrations and fractional yields demonstrates that consideration of achieved concentrations alone does not necessarily reveal an accurate picture of production performance. For example, although the E. coli system of Jew et al 27 results in higher protein concentrations than in this study, the yields in terms of absolute achievable concentrations are almost identical. The case is different for the two best V. natriegens systems.…”

“…For this optimization, not only the synthesis system under consideration can play a role, but also the design of the DNA template and the target protein itself. For example, it has been shown that the choice of the expression vector plays a critical role in CFPS, as vectors may contain elements that negatively affect the protein expression 27 . To date, the highest protein concentration achieved in batch‐mode and described in literature is about 4 mg mL −1 of deGFP and was synthesized by an E. coli ‐based system 45 .…”

“…For example, it has been shown that the choice of the expression vector plays a critical role in CFPS, as vectors may contain elements that negatively affect the protein expression. 27 To date, the highest protein concentration achieved in batch-mode and described in literature is about 4 mg mL À1 of deGFP and was synthesized by an E. coli-based system. 45 The applied amino acid concentrations differed in this synthesis depending on the amino acid in a range of 1.5 to 3 mM.…”

Development of Solid-State Storage for Cell-Free Expression Systems

“…DNA constructs purified for this research include pJL1-sfGFP, pY71-mRFP1, pJL1-SpCas9, and pJL1-mRFP1 gRNA. ,, DNA constructs pJL1-sfGFP, pJL1-SpCas9, and pJL1-mRFP1 gRNA were purified from DH5α cells using an Invitrogen PureLink HiPure Plasmid Maxiprep Kit and stored at −20 °C. DNA construct pY71-mRFP1 was purified from BL21*DE3 cells using an Invitrogen PureLink HiPure Plasmid Miniprep Kit and transformed into chemically competent NEB5alpha cells, a derivative of DH5α cells.…”

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

“…The modular nature of cell-free systems allows for the optimization of each reaction component. The DNA template purification method (e.g., miniprep vs ethanol precipitation), structure (circular vs linear), and design (orientation of gene regulatory elements) all contribute to CFES yields and reproducibility. − Energy buffers can be to enhance protein synthesis, primarily by adjusting magnesium (Mg 2+ ) and potassium (K + ) salt concentrations as well as by supplementation with stabilizers or enzymatic cofactors. , The TXTL can be modified to improve yields by altering growth conditions (e.g., media composition, temperature, flask vs bioreactor) of donor strains, lysate preparation method (e.g., French-press vs sonication, runoff reactions, lysate dialysis), or genetic engineering of donor stains to improve protein/mRNA stability or transcription/translation rates. , CFBS builds upon CFES to produce phage using purified phage genomes as templates and an energy buffer supplemented with polyethylene glycol (PEG) as a molecular crowder and dNTPs to enable phage genome replication (Figure S1b). …”