Expression and Characterization of P48E/R49I/G50L/M52V Mutant of Rhodostomin in Escherichia coli and Pichia pastoris

• Main Authors: Hsin-Wen Chi, 季欣文
• Other Authors: Hui-Liang Wang
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/47582332283754193347

碩士 === 國立高雄師範大學 === 生物科學研究所 === 90 === Abstract Integrins are a family of a、b heterodimeric cell surface receptors that involve in cellular adhesion, migration and signal transduction. Nearly half of integrins recognize the RGD（Arg-Gly-Asp）sequence in the extracellular matrix. Disintegrins are found in snake venoms, and they have high potency in inhibiting the function of integrins. Rhodostomin (Rho), a RGD sequence-containing disintegrin, is a snake venom protein isolated from Agkistodon rhodostoma, a Malayan pit viper. Rho consists of 68 amino acids including six disulfide bonds and a RGD sequence at positions of 49-51. In this study, the amino acids in the RGD and adjacent to RGD region of Rhodostomin were mutated to EILDV, and the EILDV mutant was constructed into E.coli and P. pastoris expression systems. The Rho EILDV mutant was also expressed in E.coli as well as P. pastoris, and the proteins were analyzed by SDS-PAGE. The protein was purified by Ni2+-chelating chromatography and Reverse Phase-High Performance Liquid Chromomatography (RP-HPLC), and the yield of the Rho EILDV mutant protein was 5 mg/L. The molecular weight of Rho EILDV mutant determined by Mass spectrometry was 8430.3 Da that was the same as the result of the prediction from the computer. According to the NOESY spectra of Rho EILDV mutant at pH 6.0 determined by NMR, there was no difference between wild type Rho and the Rho EILDV mutant and the folding of Rho EILDV mutant was correct.