Development of a Cold-Adapted Pseudoalteromonas Expression System for the Pseudoalteromonas Proteins Intractable for the Escherichia coli System.
Although the Escherichia coli expression system is the most commonly used expression system, some proteins are still difficult to be expressed by this system, such as proteins with high thermolability and enzymes that cannot mature by autoprocessing. Therefore, it is necessary to develop alternative expression systems. In this study, a cold-adapted Pseudoalteromonas expression system was developed. A shuttle vector was constructed, and a conjugational transfer system between E. coli and psychrophilic strain Pseudoalteromonas sp. SM20429 was established. Based on the shuttle vector, three reporter vectors were constructed to compare the strength of the cloned promoters at low temperature. The promoter of xylanase gene from Pseudoalteromonas sp. BSi20429 was chosen due to its high activity at 10-15°C. An expression vector pEV containing the chosen promoter, multiple cloning sites and a His tag was constructed for protein expression and purification. With pEV as expression vector and SM20429 as the host, a cold-adapted protease, pseudoalterin, which cannot be maturely expressed in E. coli, was successfully expressed as an active extracellular enzyme when induced by 2% oat spelt xylan at 15°C for 48 h. Recombinant pseudoalterin purified from the culture by Ni affinity chromatography had identical N-terminal sequence, similar molecular mass and substrate specificity as the native pseudoalterin. In addition, another two cold-adapted enzymes were also successfully expressed by this system. Our results indicate that this cold-adapted Pseudoalteromonas expression system will provide an alternative choice for protein expression, especially for the Pseudoalteromonas proteins intractable for the E. coli system.
State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; Marine Biotechnology Research Center, Shandong University, Jinan, China.
Zi-Chao
ZC
Yu
State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; Marine Biotechnology Research Center, Shandong University, Jinan, China.
Bai-Lu
BL
Tang
State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; Marine Biotechnology Research Center, Shandong University, Jinan, China.
Dian-Li
DL
Zhao
State Key Laboratory of Microbial Technology, Shandong University, Jinan, China.
Xiuhua
X
Pang
State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; Marine Biotechnology Research Center, Shandong University, Jinan, China.
Qi-Long
QL
Qin
Marine Biotechnology Research Center, Shandong University, Jinan, China.
Bai-Cheng
BC
Zhou
State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; Marine Biotechnology Research Center, Shandong University, Jinan, China.
Xi-Ying
XY
Zhang
State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; Marine Biotechnology Research Center, Shandong University, Jinan, China.
Xiu-Lan
XL
Chen
State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; Marine Biotechnology Research Center, Shandong University, Jinan, China.
Yu-Zhong
YZ
Zhang
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D031161:Pseudoalteromonas / Q000378:metabolism*
D001426:Bacterial Proteins