상세 정보

underline
Metadata Downloads : dc(xml) or Excel
Cited 0 time in scopus ci

Title 

Enhancing functional expression of heterologous proteins through random substitution of genetic codes in the 5' coding region

Authors 

Dae Eun CheongKyong-Cheol KoYunjon HanHo Geun JeonBong Hyun SungG J KimJong Hyun ChoiJae Jun Song

Publisher 

Wiley-Blackwell

Issue Date 

2015

Citation 

Biotechnology and Bioengineering, vol. 112, no. 4, pp. 822-826

Keywords 

Aggregation-prone proteinEscherichia coliFunctional expressionSynonymous codon

Abstract 

Recent studies using heterologous protein expression systems suggest that synonymous codons affect not only the expression but also the properties of the expressed protein. However, practical application of this information is challenging, and to date, efforts to employ bioinformatics tools to design synonymous codon mixes have been only marginally successful. Here, we sought to enhance the functional expression of heterologous protein in Escherichia coli through completely random substitution of the first ten codons with synonymous codons, using a previously isolated exocellulase CelEdx-SF301 as the model protein. Synonymous codon variants were generated by PCR using forward primers with mixed nucleotides at the third position in each codon and a conventional reverse primer. The resulting PCR products were inserted upstream of the fluorescent protein mCherry without linkers. After transformation and cultivation, colonies exhibiting red fluorescence were selected, and the activity of SF301-mCherry fusion proteins was tested. Synonymous codon variant fusion proteins exhibited 35- to 530-fold increases in functional expression compared with wild-type controls. Unlike results from other reports, we found that the stability of mRNA secondary structure in the 5' untranslated region and codon rarity were not correlated with functional expression level. Our work demonstrates that a completely random mixed of synonymous codons effectively enhances functional expression levels without the need for amino acid substitutions.

ISSN 

0006-3592

Link 

http://dx.doi.org/10.1002/bit.25478

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


There are no files associated with this item.
qrcode

FusionCharts.
DSpace Software Coptright(c) 2010 MIT and Hewleft-Packard  /  KRIBB-REPOSITORY ( Email:jakim@kribb.re.kr)