Efficient Extracellular Expression of Phospholipase D in Escherichia Coli with an Optimized Signal Peptide

5th Annual International Conference on Material Science and Environmental Engineering

材料科学;生态环境科学

Yang, Leyun^1,2 ; Xu, Yu^1,2 ; Chen, Yong^1,2 ; Ying, Hanjie^1,2

State Key Laboratory of Materials-Oriented Chemical Engineering, College of Life Science and Pharmaceutical Engineering, Nanjing Tech University, P.R. China, Nanjing, 210009, China^1

National Engineering Technique Research Center for Biotechnology, Nanjing

211816, China^2

关键词: Extracellular enzyme activity;    Phospholipase D;    SDS-PAGE analysis;    Signal peptide;    Signal sequence;    Structural gene;    Subcellular fractions;    Synthetic signals;   

Others  :  https://iopscience.iop.org/article/10.1088/1757-899X/301/1/012105/pdf
DOI  :  10.1088/1757-899X/301/1/012105

来源: IOP

【 摘 要 】

New secretion vectors containing the synthetic signal sequence (OmpA') was constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli Phospholipase D structural gene (Accession number:NC-018658) fused to various signal sequence were expressed from the Lac promoter in E. coli Rosetta strains by induction with 0.4mM IPTG at 28°C for 48h. SDS-PaGe analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of Phospholipase D (PLD) not only to the medium but also remained in periplasm of E. coli with OmpA' signal sequence at the N-terminus of PLD. Thus the study on the effects of various surfactants on PLD extracellular production in Escherichia coli in shake flasks revealed that optimal PLD extracellular production could be achieved by adding 0.4% Triton X-100 into the medium. The maximal extracellular PLD production and extracellular enzyme activity were 0.23mg ml-1and 16U ml-1, respectively. These results demonstrate the possibility of efficient secretory production of recombinant PLD in E. coli should be a potential industrial applications.