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

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.