Objective   To construct the prokaryotic expression vector of M4-M5 between the fourth and fifth transmembrane domains of Na

+

K

+

-pumpα2 isoform

and express and purify it in E. coli.  Methods   PCR and enzyme digestion technology were used to construct a recombinant plasmid pET28b(+)-M4-5

which was transformed into E. coli Rosetta 2. The expression plasmid in soluble inclusion bodies was degenerated with 6 mol/L GuHCl

refolded through gradient dilution in a buffer system. The

target protein was purified by Ni

2+

-NTA affinity chromatography

and verified by Western blotting

and its ATPase activity was detected by inorganic phosphonium colourimetry.   Results   Restriction endonuclease digestion and recombinant plasmid sequencing demonstrated that the expression vector pET28b(+)-M4-5 was successfully constructed. After the target protein was induced into E.coli Rosetta 2

its relative molecular weight was about 52×10

3

as expected

accounting for about 30% of the total bacterial protein. SDS-PAGE analysis showed that the protein existed mostly in the form of inclusion bodies. After purification by affinity chromatography

the fusion protein had a purity of above 95%. Western blotting analysis showed that the expressed protein could specifically bind to anti-His antibody with an ATPase activity of 15.3±1.8）mmol ·g

-1

·h

-1

.   Conclusion  The prokaryotic expression vector pET28b(+)-M4-5 can be correctly constructed.The M4-M5 fusion protein can be expressed in E.coli Rosetta 2 and purified by affinity chromatography with a high purity.