Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery

Abstract

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, β, β’ and ω are simultaneously coexpressed. Appending a 3′ UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

“…Likewise, starvation response regulator cyaR repression did not affect the T7 lysis. RNase I encoded by rna localizes in the periplasm and is thus unlikely to interfere with T7 mRNA stability . Phosphoglycerate kinase ( pgk ) did not affect lysis timing or EOP, which is unexpected given the lysis delay and EOP drop induced by the enolase ( eno ) knockdown as both enzymes are part of the canonical glycolysis pathway …”

“…RNase I encoded by rna localizes in the periplasm and is thus unlikely to interfere with T7 mRNA stability. 46 Phosphoglycerate kinase (pgk) did not affect lysis timing or EOP, which is unexpected given the lysis delay and EOP drop induced by the enolase (eno) knockdown as both enzymes are part of the canonical glycolysis pathway. 47 eno was chosen as a CRISPRi target because of its roles in glycolysis, as part of the mRNA degradosome complex, and interactions between enolase and other phage.…”

“…To determine if in vivo effectors of T7 fitness had the same qualitative effect in cell-free systems, CRISPRi and overexpression strains were selected to prepare transcription/translation machinery (TXTL) for cell-free protein express systems (CFES) and cell-free bacteriophage synthesis (CFBS) based on prior research, indicating their usefulness in cell-free protein synthesis or positive effects on T7 fitness during in vivo studies. Exogenous IF-3 ( infC ) supplementation, recC deletion, and rna deletion each have been shown to improve CFES yields of GFP. ,, oxyS - and cyaR -overexpression strains were chosen as positive in vivo effectors, dgt -overexpression, and trxA- knockdown strains as likely negative effectors and trxA -overexpression for its strong negative effects. All lysates were harvested 4 h after induction with an appropriate inducer and harvested in mid log-phase with OD 600 ∼ 2 to 3 yielding total protein yields ranging from 15 to 25 mg/mL.…”

“…Exogenous IF-3 ( infC ) supplementation, recC deletion, and rna deletion each have been shown to improve CFES yields of GFP. 14 , 46 , 65 oxyS - and cyaR -overexpression strains were chosen as positive in vivo effectors, dgt -overexpression, and trxA- knockdown strains as likely negative effectors and trxA -overexpression for its strong negative effects. All lysates were harvested 4 h after induction with an appropriate inducer and harvested in mid log-phase with OD 600 ∼ 2 to 3 yielding total protein yields ranging from 15 to 25 mg/mL.…”

Antimikrobiyal Peptitlere Yönelik Güncel Eğilimler; Nanotaşıyıcı Sistemeler ve Farmasötik Uygulamalar

“…E. coli BL21 (DE3) sisteminin bir avantajı olarak, eksojen protein bozulmasını önleyen, Lon proteazında bir eksikliğe sahiptir. Ek olarak, dış membran proteazı OmpT'yi kodlayan gen, E. coli BL21 (DE3) genomunda bulunmaz, bu da hücre dışı proteinlerin bozulmasını önler (Snapyan vd., 2021). BL21 suşunun bir başka avantajı, lac, tac, trc, ParaBAD, PrhaBAD, T5 ve T7 gibi RNA polimeraz promotörlerinin kontrolü altındaki genleri eksprese etmesidir (Du vd., 2021).…”