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Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein

100%

Ochocka A. M., Czyzewska M., Pawelczyk T.

Acta Biochimica Polonica

2003

tom 50

nr 1

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5a cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Δibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22°C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A600 about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.

Ograniczanie wyników

1 Acta Biochimica Polonica

1 Czyzewska M.

1 Ochocka A. M.

1 Pawelczyk T.

1 2003

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Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein