Study of Gamma-Echistatin from Scratch: Gene Synthesis, Protein Expression, Purification, Structure, and Function

• Main Authors: Li, Der-Shiang, 李德祥
• Other Authors: Wu, Shih-Hsiung
• Format: Others
• Language: en_US
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/14398440168595758509

碩士 === 國立臺灣大學 === 生化科學研究所 === 86 === A gene encoding an RGD-containing platelet aggregation inhibitor, gam ma-echistatin, has been synthesized through PCR method using four overla pping oligonucleotides. The synthetic gene has Hind III sites at both ends for cloning into pQE-30 expression vector and an (Asp)4-Lys coding seque nce recognized by enterokinase to cleave the fusion protein. The recombinant expression vector was transferred into M15[pREP4] competent cells, t he positive clones were identified by PCR and verified by DNA sequence analy sis. After over-expressio n by inducing with IPTG, crude gamma-echistatin fusion protein was purified through Ni-NTA column. The crude fusion protein was first denatured an d reduced to prevent mis-linkage of disulfidebonds. Then gamma-echistatin fusi on protein was cleaved by enterokinase and refolded. The recombinant, matur e gamma-echistatin was purified to homogeneity by HPLC, and verified by CD spectrum and mass spectrometry. This recombinant gamma-echistatin was also assayed for inhibiting platelet aggregation and found to be identical to that of native gamma-echistatin. We also constructed the mutants of gamma-ech istatin, K45E and R24N, by site directed mutagenesis. As our previous resea rch pointed out, Lys45 might play an important role in platelet aggregation i nhibition because the inhibitory potency of des(46-49)-gamma-echistatin dec rease 1.7-fold whereas that of des(45-49)-gamma-echistatin is 15-fold less than native gamma-echistatin. Furthermore, on the basis of the structural m odel of gamma-echistatin, Lys45 is situated near the RGD loop and facing t he same side of the molecule. Nevertheless, the inhibitory potency of K45E mu tant is only 2-fold less than the wild type. It implies that Lys45 need to cooperate with other C-terminal residues for the effect of platelet aggregation inhibition instead of acting alone. The R24N mutant, as exp ected, had little activity in inhibiting platelet aggregation.