American Journal of Medical Sciences and Medicine
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American Journal of Medical Sciences and Medicine. 2014, 2(1), 1-6
DOI: 10.12691/ajmsm-2-1-1
Open AccessArticle

Orthogonal Test Design for Optimization of the Expression of Thymosin α1 and Thymosin α1- iRGD Gene in Engineered E.coli BL21 Strain

Xingzhen Lao1, Meng Liu1, Fang Zhang2 and Heng Zheng1,

1School of Life Sciences and Biotechnology, China Pharmaceutical University, Nanjing, Jiangsu, PR China

2School of Pharmacy, Nanjing University of Traditional Chinese Medicine, Nanjing, Jiangsu, PR China

Pub. Date: December 30, 2013

Cite this paper:
Xingzhen Lao, Meng Liu, Fang Zhang and Heng Zheng. Orthogonal Test Design for Optimization of the Expression of Thymosin α1 and Thymosin α1- iRGD Gene in Engineered E.coli BL21 Strain. American Journal of Medical Sciences and Medicine. 2014; 2(1):1-6. doi: 10.12691/ajmsm-2-1-1

Abstract

In order to increase the expression of fusion protein of thymosin α1(Tα1) and thymosin α1-iRGD (Tα1-iRGD) by the engineered E. coli BL21 strain, containing pET32a-Trx-Tα1 and pET32a-Trx-Tα1-iRGD plasmid, respectively. The key parameters that influenced the expression of Tα1 and Tα1-iRGD were optimized by employing an orthogonal experiment [L25(5)3], including OD600nm before induction, lactose concentration and induction time, each with five levels. The intensity of target protein band was scanned as a quantitative measure method of the protein expression, after electrophoresis separation of total soluble protein on SDS-PAGE. For Tα1 fusion protein, the optimal conditions were OD600nm=0.6 before induction, 2.5 mmol/L lactose concentration, 4 hours induction time. Target protein expression levels could be achieved 32.8% of the total soluble proteins. For Tα1-iRGD fusion protein, the optimal conditions were OD600nm=0.8 before induction, 7.5mmol/L lactose concentration, 4 hours for induction. Under the condition, the amount of expressed protein could reach 33.8% of the total soluble proteins. Whereas before optimization, the expression level of Tα1 and Tα1-iRGD fusion proteins were 20.6% and 9.0% of the total soluble proteins, respectively. The orthogonal test was proved to be an effective method to optimize the expression of target proteins.

Keywords:
thymosin α1 thymosin-iRGD gene expression orthogonal experiment anticancer

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