Importance of expression system in the production of unnatural recombinant proteins in Escherichia coli

Abstract

^Äëíê~Åí= In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in bëÅÜÉêáÅÜá~=Åçäá. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while Lhomopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LC-MS/MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system. © KSBB hÉóïçêÇëW=ìåå~íìê~ä= êÉÅçãÄáå~åí= éêçíÉáåI= ÉñéêÉëëáçå= ëóëíÉãI= êÉëáÇìÉ= ëéÉÅáÑáÅ= ìåå~íìê~ä=~ãáåç=~ÅáÇ= áåÅçêéçê~íáçåI= iJ Üçãçéêçé~êÖóäÖäóÅáåÉ=

Deletional Protein Engineering Based on Stable Fold

“…The genes of n-GFP variants and s-GFP variants were respectively PCR amplified from pQE80-GFP [25] and pQE-80L-GFPhs1 [22] using the designed primers and cloned into Nde I and Xho I restriction sites of pET30b(+). The internal deletions were made using overlap extension PCR [26].…”

Engineering Escherichia coli to sense acidic amino acids by introduction of a chimeric two-component system

“…used to amplify the gfp gene from GFP-mut3.1, which was previously cloned in the pET expression system [12]. The ompC promoter was then ligated with the gfp gene, after which the product was cloned into pUC19 to construct pOGFP1.…”

Engineered fumarate sensing Escherichia coli based on novel chimeric two-component system

“…Primers for the amplification of the dcuS, envZ, dcuB promoter, and ompC promoter genes were designed based on the reported E. coli genome sequence (Blattner et al, 1997) and primers for the amplification of the gfp gene (Miller et al, 2000) and the gfp mut3.1b gene (Ayyadurai et al, 2009) were designed based on the reported sequences. The genomic region containing 570 bp of the dcuB promoter gene was amplified from the chromosomal DNA of E. coli XL1-Blue with oligonucleotides DcuB F SalI and DcuB R BamHI ( Table 2).…”

“…This part of the promoter contains all three known binding sites for OmpR-P (Nara et al, 1986) and four of the nine LRP-binding sites present in the full-length chromosomal promoter (Ferrario et al, 1995). The gfp gene encoding GFP-mut3.1b was simultaneously amplified from the pET21a plasmid (Ayyadurai et al, 2009) with oligonucleotides GFPm F BamHI and GFPm R SalI (Table 2). Then the ompC promoter was amplified by PCR using OmpC F EcoRI and OmpC R SalI ( Table 2).…”