| Aquaporin Z (AqpZ) is a hydrophobic transmembrane protein located on the cell membrane of Escherichia coli. Aquaporin Z (AqpZ) is a water specific channel protein and belongs to the major intrinsic protein (MIP) family. Due to its extremely high water permeability, extremely high stability and low activation energy, AqpZ has attracted many attentions to develop the biomimetic water filtration technology. However, the requirement of sufficient functional AqpZ was the key bottleneck to limit such applications.In the present study, membrane-associated AqpZ was expressed in E. coli by using the maltose binding protein/polyhistidine superhydrophilicity dual affinity tags fusion system. First, The 8×HIS tag encoding oligonucleotides was successfully introduced into downstream of AqpZ gene by PCR method. Then this gene cassette was ligated into pMAL-p2 and pMAL-c5e, and the dual affinity tagged AqpZ fusion protein expression plasmids pMAL-p2-AqpZ-HIS and pMAL-c5e-AqpZ-HIS were successfully constructed. The effects of different host strains and the expression conditions on the production of such fusion protein were systematically investigated. The results showed that the recombinant expression of MBP-AqpZ-HIS was nontoxic in E.coli, and the highest productivity of 262 mg/L was achieved under optimized conditions. MBP-AqpZ-HIS was almost expressed as membrane-associated form. According to the characteristics of the fusion protein, two strategies to purify the AqpZ-HIS through affinity chromatography was developed. The high purity AqpZ-HIS was obtained after the final step affinity chromatography. The related SDS-PAGE and Trp fluorescence emission analyses illustrated that the purified AqpZ-HIS assembled as a homotetramer with high thermostability, suggesting the correct folding of AqpZ.Our results indicated that maltose binding protein/polyhistidine dual affinity tags system was very efficient for overexpression, membrane targeting and purification of MIP channel protein in E.coli. |
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