title: Synonymous rare arginine codons and tRNA abundance affect protein production and quality of TEV protease variant.
abstract: It has been identified that a TEV protease (TEVp) variant, TEVp(5M), displays improved solubility. Here, we constructed fifteen TEVp(5M) variants with one or more of six rare arginine codons in the coding sequence replaced with abundant E. coli arginine codons. These codon variants expressed in either E. coli BL21 (DE3) or Rossetta (DE3) showed different solubility and activity. Supply of rare tRNAs increased the tendency of certain codon variants to form insoluble aggregates at early induction stage, as determined by the fused S-tag. About 32% increase in soluble protein production of M5 variant with four synonymously mutated arginine codons was identified in Rossetta (DE3) cells using GFP fusion reporter, comparable to that of TEVp(5M). After purification, two other codon variants from both E. coli strains exhibited less activity than TEVp(5M) on cleaving the native or modified recognition sequence incorporated between GST and E. coli diaminopropionate ammonialyase by enzyme-coupled assay, whereas purified M5 variant showed activity similar to the TEVp(5M). Supply of rare tRNAs caused the decrease of activity of TEVp(5M) and M5 by about 21%. Our results revealed that engineering of highly soluble TEVp variants can be achieved by the combined mutations of amino acid residues and optimization of specific rare codons, whereas simple augment of rare tRNAs abundance resulted in partial loss of activity.
authors.affiliation: Key Laboratory of Crop Biology of Anhui Province, School of Life Science, Anhui Agricultural University, Hefei, Anhui, PR China.
authors.forename: Jie
authors.initials: J
authors.lastname: Fang
authors.affiliation: Key Laboratory of Crop Biology of Anhui Province, School of Life Science, Anhui Agricultural University, Hefei, Anhui, PR China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Zou
authors.affiliation: Key Laboratory of Crop Biology of Anhui Province, School of Life Science, Anhui Agricultural University, Hefei, Anhui, PR China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Crop Biology of Anhui Province, School of Life Science, Anhui Agricultural University, Hefei, Anhui, PR China.
authors.forename: Beijiu
authors.initials: B
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Crop Biology of Anhui Province, School of Life Science, Anhui Agricultural University, Hefei, Anhui, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Fan
mesh_terms: D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D003062:Codon / Q000737:chemistry* / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: D003062:Codon; D007527:Isoenzymes; D011994:Recombinant Proteins; D012343:RNA, Transfer; D001120:Arginine; D010450:Endopeptidases; C099488:TEV protease