Synonymous rare arginine codons and tRNA abundance affect protein production and quality of TEV protease variant.

Jie Fang,Jun Fan,Lingling Zou,Beijiu Cheng,Xuan Zhou,Grzegorz Kudla

doi:10.1371/journal.pone.0112254

Abstract

It has been identified that a TEV protease (TEVp) variant, TEVp5M, displays improved solubility. Here, we constructed fifteen TEVp5M 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 TEVp5M. After purification, two other codon variants from both E. coli strains exhibited less activity than TEVp5M 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 TEVp5M. Supply of rare tRNAs caused the decrease of activity of TEVp5M 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.

Highlights

As the host, Escherichia coli is fundamentally important in the manufacture of a wide range of biotechnological and biomedical products, owing to its easy culture, PLOS ONE | DOI:10.1371/journal.pone.0112254 November 26, 2014The Specific Codons and tRNA Abundance Affect Protein Quality fast growth, simple fermentation and easy genetic manipulation [1]
Since TEVp5M is more soluble than the TEVpS219V variant [11], we introduced a series of synonymous mutations into six rare arginine codons in TEVp5M coding sequence converting them to abundant arginine codons found in E. coli
The codon variants constructed include M1–M4 with one or two rare arginine codons being replaced with the optimized arginine codons, and M5–M15, which combined to the mutations in M1–M4 with additional codons (Table 1)

Summary

Introduction

Escherichia coli is fundamentally important in the manufacture of a wide range of biotechnological and biomedical products, owing to its easy culture, PLOS ONE | DOI:10.1371/journal.pone.0112254 November 26, 2014The Specific Codons and tRNA Abundance Affect Protein Quality fast growth, simple fermentation and easy genetic manipulation [1]. High production of recombinant protein in E. coli is a combination of several factors such as efficiency of transcription, mRNA stability, mRNA folding, codon usage, protein solubility and folding [2]. In E. coli, tRNA levels for rare codons AGG, AGA, AUA, CCC, CUA and GGA restrain high-level production of the recombinant proteins [3]. Codon optimization could enhance soluble protein production [4, 5], but sometimes it impaired protein folding and decreased activity [6, 7]. Supply of deficient tRNAs increased expression levels of recombinant proteins, but decreased soluble production of some recombinant proteins [8]. Obtaining well-folded recombinant proteins with high yield remains a major challenge, as yield, solubility and conformational quality of soluble proteins could not be simultaneously optimized in E. coli [9]

Methods

Results

Conclusion