Person Calcium Cyclin-binding Protein (cacybp) Cloning And Functional Studies

Calcium is very important for the function of cells as the second signal molecule. Until now, many kinds of Ca²⁺-binding proteins has been found which function in regulation of Ca²⁺ concentration, regulation of cell cycle and signal transduction of apoptosis. S100 family is the largest subfamily of Ca²⁺-binding protein which consists of a group of 10-12 kDa acid protein. As a kind of S100 protein, calcyclin (also called S100A6) has been studied in the recent years. It was found that calcyclin is related to cell cycle. Tonini et al found that calcyclin is probably related to induced differentiation of nerve system tumors. In 1996 Filipek et al found a kind of new protein-p30 in mouse Ehrlich ascites cells could bind to calcyclin in vitro which he called calcyclin binding protein.We got a EST-T23 from human glioma cell line-BT325. The EST T23 was used as probe to screen human cDNA library and a coding full-length cDNA sequence was obtained. There is no same cDNA sequence in GenBank. The length of this cDNA sequence is 1 100 bp containing an open reading frame of 684 bp coding 228 amino acids. The predicted sequence of amino acid of this gene is 93% identical and 97% similar to mouse calcyclin binding protein which is shown by Blastp of nr database (http://www.ncbi.nlm.nih.gov). So this gene is named human calcyclin binding protein (CacyBP). The function of CacyBP was studied.Software analysis and protein database analysis of this gene suggests that it contains a nuclear locating site and multiple possible phosphorylation sites which are highly conserved in its mouse counterpart except the casein kinase II phosphorylation site in 183¹86 amino acids. Hydrophobicity analysis suggests that CacyBP is highly hydrophilic at the amino and carboxyl end and relatively hydrophobic in the middle. The secondary and tertiary structure of CacyBP was predicted using http://www.sbg.bio.ic.ac.uk/³dpssm. The secondary structure for CacyBP is 95% similar to co-chaperon p23 (Hsp20-like chaperon) and so the tertiary structure of this protein was predicted on this basis of similarity.In order to obtain anti-CacyBP polyclonal antibody so as to investigate this protein at the level of tissue and cell, the whole coding region was amplified by PCR and cloned into prokaryotic vector pET28c. pET28c-CacyBP was efficiently expressed in BL21 and the recombinant human CacyBP was purified by affinity chromatography using the N-terminal six-histidine epitope. Highly-purified CacyBP was got. The purified CacyBP...