Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein.
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 DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp 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 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) 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.
Ochocka
Anna-Maria
AM
Department of Molecular Medicine, Medical University of Gdańsk, Gdańsk, Poland.
Czyzewska
Marzena
M
Pawełczyk
Tadeusz
T
D000918:Antibody Specificity; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D020690:GTPase-Activating Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D006639:Histidine; D006801:Humans; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
C109971:ARHGAP6 protein, human; D017931:DNA Primers; D020690:GTPase-Activating Proteins; D011994:Recombinant Proteins; D006639:Histidine; D020741:rho GTP-Binding Proteins