Cloning, expression, mutagenesis and characterization of crocalbin, a binding protein for crotoxin

• Main Authors: Li-Fen Huang, 黃麗芬
• Other Authors: Mu-Chin Tzeng
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/30891704860319011540

碩士 === 國立臺灣大學 === 生化科學研究所 === 89 === Crocalbin, a crotoxin binding protein, was previously purified from porcine brain by crotoxin affinity column. The mature crocalbin consists of 296 amino acid residues and is most closely related to the "CREC family" of calcium binding proteins. It contains six EF-hand domains capable of interacting with calcium. In the present study, E. coli expression systems using GST-fusion protein and 6x His-tag were constructed to overexpress crocalbin and its truncated forms. These proteins were purified and used to search for the crotoxin binding site of crocalbin and the inhibition the PLA2 activity of crotoxin by crocalbin. In the GST system, the overexpressed GST-crocalbin was purified by affinity chromatography with glutathione resin. GST was then removed by cleavage with thrombin to obtain crocalbin. The final yield was about 3 mg per liter culture. In the 6x His-tagged system, however, the yield after purification with Ni-NTA column was only about 1 mg per liter culture. The 45Ca overlay assay was used to check the calcium binding ability of crocalbin and its truncated forms. Calcium was observed to bind to crocalbin and its truncated forms containing all six EF-hand motifs. In a further assay, crotoxin affinity resin was used to check the crotoxin binding ability of crocalbin and to obtain some information about the crotoxin binding site of crocalbin. The expressed crocalbin was able to bind crotoxin and the domain in crocalbin for binding to crotoxin appears to be in the middle part which also contains the six EF-hand motifs. Moreover, the PLA2 activity of crotoxin was found to be inhibited by crocalbin and its truncated forms containing the six EF-hand motifs. These results indicated that the binding of crocalbin or some of its fragments to crotoxin blocked the active site of crotoxin or induce changes in the conformation of crotoxin to inhibit PLA2 activity of crotoxin. The relationship between the crocalbin binding and toxicity of crotoxin, and the detail about the crotoxin binding site requires further studies. In the future, the overexpressed crocalbin may be immobilized for used in affinity chromatography to find out proteins that interact with crocalbin, thereby gaining understanding of possible functions of crocalbin.