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Isozymes delta of phosphoinositide-specific phospholipase C and their role in signal transduction in the cell.

100%

Ochocka A.-M., Pawelczyk T.

2003

vol. 50

issue 4

1097-1110

Phospholipase C (PLC, EC 3.1.4.11) is an enzyme crucial for the phosphoinositol pathway and whose activity is involved in eukaryotic signal transduction as it generates two second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). There are four major types of phospholipase C named: β, γ, δ and the recently discovered ε, but this review will focus only on the recent advances for the γ isozymes of PLC. So far, four d isozymes (named γ1-4) have been discovered and examined. They differ with regard to cellular distribution, activities, biochemical features and involvement in human ailments.

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

81%

Ochocka A.-M., Czyżewska M., Pawełczyk T.

2003

vol. 50

issue 1

239-247

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 DH5α 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 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.

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2 Acta Biochimica Polonica

2 2003

2 Ochocka A.-M.

1 Czyżewska M.

1 Pawelczyk T.

1 Pawełczyk T.

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Isozymes delta of phosphoinositide-specific phospholipase C and their role in signal transduction in the cell.

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