mRNA secondary structure at start AUG codon is a key limiting factor for human protein expression in Escherichia coli

Journal Article · Fri Oct 13 04:00:00 EDT 2006 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/j.bbrc.2006.07.209· OSTI ID:20854511

Weici, Zhang ^[1]; Weihua, Xiao ^[2]; Haiming, Wei ^[1]; Jian, Zhang ^[3]; Zhigang, Tian ^[2]

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1. Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China)
2. Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China) and School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012 (China)
3. School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012 (China)

Codon usage and thermodynamic optimization of the 5'-end of mRNA have been applied to improve the efficiency of human protein production in Escherichia coli. However, high level expression of human protein in E. coli is still a challenge that virtually depends upon each individual target genes. Using human interleukin 10 (huIL-10) and interferon {alpha} (huIFN-{alpha}) coding sequences, we systematically analyzed the influence of several major factors on expression of human protein in E. coli. The results from huIL-10 and reinforced by huIFN-{alpha} showed that exposing AUG initiator codon from base-paired structure within mRNA itself significantly improved the translation of target protein, which resulted in a 10-fold higher protein expression than the wild-type genes. It was also noted that translation process was not affected by the retained short-range stem-loop structure at Shine-Dalgarno (SD) sequences. On the other hand, codon-optimized constructs of huIL-10 showed unimproved levels of protein expression, on the contrary, led to a remarkable RNA degradation. Our study demonstrates that exposure of AUG initiator codon from long-range intra-strand secondary structure at 5'-end of mRNA may be used as a general strategy for human protein production in E. coli.

• OSTI ID: 20854511
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 1 Vol. 349; ISSN BBRCA9; ISSN 0006-291X
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
ESCHERICHIA COLI
GENES
INTERFERON
OPTIMIZATION
RNA
STABILITY