Paper Citing NAMD - Abstract
Zhang, Yunfeng; Ling, Zhenmin; Du, Guocheng; Chen, Jian; Kang, Zhen
Improved Production of Active Streptomyces griseus Trypsin with a Novel Auto-Catalyzed Strategy
SCIENTIFIC REPORTS, 6 Art. No. 23158, MAR 17 2016
N-terminal sequences play crucial roles in regulating expression, translation, activation and enzymatic properties of proteins. To reduce cell toxicity of intracellular trypsin and increase secretory expression, we developed a novel auto-catalyzed strategy to produce recombinant trypsin by engineering the N-terminus of mature Streptomyces griseus trypsin (SGT). The engineered N-terminal peptide of SGT was composed of the thioredoxin, glycine-serine linker, His(6)-tag and the partial bovine trypsinogen propeptide (DDDDK). Furthermore, we constructed a variant TLEI with insertion of the artificial peptide at N-terminus and site-directed mutagenesis of the autolysis residue R145. In fed-batch fermentation, the production of extracellular trypsin activity was significantly improved to 47.4 +/- 1.2 U.ml(-1) (amidase activity, 8532 +/- 142.2 U.ml(-1) BAEE activity) with a productivity of 0.49 U.ml(-1).h(-1), which was 329% greater than that of parent strain Pichia pastoris GS115-SGT. This work has significant potential to be scaled-up for microbial production of SGT. In addition, the N-terminal peptide engineering strategy can be extended to improve heterologous expression of other toxic enzymes.
