HUMAN CYSTEINE-RICH INTESTINAL PROTEIN - CDNA CLONING AND EXPRESSION OF RECOMBINANT PROTEIN AND IDENTIFICATION IN HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS

Cysteine-rich intestinal protein (GRIP) is a small, 8.5-kDa protein wi th one double zinc-finger motif called a LIM domain. It is very abunda nt in intestine and some immune cells in rodents, and expression is in fluenced by development and the immune response. We have cloned a huma n GRIP cDNA from human small intestine poly(A)(+) RNA by RT-PCR. Throu gh sequencing, we found that the human intestinal GRIP protein (hCRIP) differed from the previously cloned rat GRIP by two amino acids (resi dues 8 and 58). hCRIP was expressed with the pET vector/bacterial syst em and isolated by gel filtration and ion-exchange chromatography. The protein was purified to homogeneity as confirmed by PAGE, Western blo tting, and immunodetection. Recombinant hCRIP has a molecular mass of 8390 Da based on mass spectrum analysis. Southern analysis suggests th at there are three copies of the GRIP gene in the human genome. hCRIP mRNA was detected by RT-PCR in human monocytes purified from periphera l blood and THP-1 cells, a human monocytic cell line, Incubation of TH P-1 cells with Zn-65 and chromatography of the cytosol show that a sig nificant amount of the radioactivity is associated with GRIP as was sh own previously for rat intestine. The results are consistent with a fu nctional role for GRIP in proliferation/differentiation of specific ce ll types, particularly those associated with host defense. (C) 1997 Ac ademic Press.