Efficient extracellular expression of transpeptidase sortase A in Pichia pastoris.
In order to achieve efficient extracellular expression of Sortase A (SrtA), various strategies in Pichia pastoris system were applied in this study. Among different constructed recombinant strains, the SMD1168 strain integrated 5.7 copies of srtA gene under control of AOX1 promoter was proved to be the best strain for the extracellular SrtA expression. After the optimization of fermentation conditions (induction 72 h at 28 °C, initial pH 6.0, supplemented with 1.5% methanol), the highest yield and activity of extracellular SrtA reached 97.8 mg/L and 131.9 U/mL at the shake-flask level, respectively. This is the first report on the efficient secretory expression of SrtA in P. pastoris and the yield of SrtA is the maximum compared with previous reports. In addition, the transpeptidation activity of extracellular SrtA was confirmed by the successful immobilization of enhanced green fluorescent protein (EGFP) onto Gly3-polystyrene beads.
The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road, Wuxi 1800, China.
Xinrui
X
Zhao
The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road, Wuxi 1800, China.
Haofei
H
Hong
The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road, Wuxi 1800, China; State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China. Electronic address: zwu@jiangnan.edu.cn.
Zhimeng
Z
Wu
D019881:Aminoacyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000097:blood / Q000737:chemistry / Q000235:genetics; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
Extracellular expression; Immobilization; Optimization; Pichia pastoris; Sortase A