Human Cysteine-Rich Intestinal Protein: cDNA Cloning and Expression of Recombinant Protein and Identification in Human Peripheral Blood Mononuclear Cells

Abstract

Cysteine-rich intestinal protein (CRIP) is a small, 8.5-kDa protein with one double zinc-finger motif called a LIM domain. It is very abundant in intestine and some immune cells in rodents, and expression is influenced by development and the immune response. We have cloned a human CRIP cDNA from human small intestine poly(A)+RNA by RT-PCR. Through sequencing, we found that the human intestinal CRIP protein (hCRIP) differed from the previously cloned rat CRIP by two amino acids (residues 8 and 58). hCRIP was expressed with the pET vector/bacterial system and isolated by gel filtration and ion-exchange chromatography. The protein was purified to homogeneity as confirmed by PAGE, Western blotting, and immunodetection. Recombinant hCRIP has a molecular mass of 8390 Da based on mass spectrum analysis. Southern analysis suggests that there are three copies of the CRIP gene in the human genome. hCRIP mRNA was detected by RT-PCR in human monocytes purified from peripheral blood and THP-1 cells, a human monocytic cell line. Incubation of THP-1 cells with65Zn and chromatography of the cytosol show that a significant amount of the radioactivity is associated with CRIP as was shown previously for rat intestine. The results are consistent with a functional role for CRIP in proliferation/differentiation of specific cell types, particularly those associated with host defense.