Efficient expression of membrane-bound water channel protein (Aquaporin Z) in Escherichia coli.
In order to explore the possibility of preparing a high-efficiency aquaporin-based biofilter, an efficient approach for expression of membrane-bound Aquaporin Z (AqpZ) in E. coli was proposed. The AqpZ gene was amplified by means of PCR, and two expression vectors (pET28-AqpZ and pET32-AqpZ) were constructed. The channel protein of interest was synthesized in E. coli BL21(DE3)/pET32-AqpZ as an insoluble fusion protein linked with trxA. However, with BL21(DE3)/pET28-AqpZ, significant amount of AqpZ fused only with 6-His (6-His-AqpZ) could be expressed, correctly folded and targeted into the membrane. Under the optimized culture conditions, the highest expression level (9.05 mg/l) of membrane-bound 6-His-AqpZ was achieved with BL21(DE3)/pET28-AqpZ, and an additional amount (2.35 mg/l) was expressed concomitantly as the inclusion body form. This expression result was 3.5 times higher than that in the previous studies.
Lian
Jiazhang
J
Institute of Bioengineering, Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China.
Fang
Xiangming
X
Cai
Jin
J
Chen
Qixing
Q
Zheng
Qiang
Q
Kai
Lei
L
Xu
Zhinan
Z
D020346:Aquaporins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D020346:Aquaporins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C106425:aqpZ protein, E coli