Expression and Purification of Human γD crystallin

• Main Authors: Ming-Chien Hsieh, 謝明諫
• Other Authors: 王勝仕
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/13862619233589325024

碩士 === 國立臺灣大學 === 化學工程學研究所 === 96 === Abstract In this thesis, we used E. coli M15(pREP4), which harbors the plasmid pQE1, to express human γD crystallin (HGDC). After expression, several purification methods were utilized to separate HGDC from the other unwanted proteins. In the first part, we investigated the parameters such as pH of medium, the concentration of IPTG, and the timing of IPTG addition, the dissolved oxygen concentration, the type of carbon source, induction with lactose, the composition of LB medium, and tried to obtain the optimum expression environment for HGDC in E. coli. In the second part, acetone precipitation, affinity chromatography, and ultra-filtration were employed to purify HGDC from the other proteins. In order to obtain the solution with high purity of HGDC, attempts were also made to examine the relationship between the protine yield and the purification methods. Our results can be summarized as follows: (1) The growth of E. coli was not significantly affedcted by the initial pH value of medium. However, in comparison with those obtained at other pH values, a slightly higher yield of HGDC was achieved as the initial pH was set at 7.0. (2) The optimum condition for IPTG addition was found to be OD600 = 0.6 and final concentration = 0.1 mM. (3) Better cell growth and yield of HGDC were observed when the amount of dissolved oxygen was higher. (4) No considerable enhancements in the cell growth and the HGDC production were perceived as glucose, galactose, or glycerol was used as the carbon source. (5) Comparable amouts of HGDC were abtained from the expressions of E. coli with IPTG induction and lactose induction. (6) Through increasing the concentrations of tryptone and yeast extract of LB medium, the productivity of HGDC and the cell density (or OD600) were able to be effectively enhanced. (7) About 90% of the yield and two-fold of the purity were obtained when the protein was purified via acetone precipitation. (8) Using affinity chromatography, the purity and the yield of HGDC were found to be ~92% and ~17-32%, respectively.(9) It was observed that almost 100% pure HGDC solution could be acquired if the product from affinity chromatography was further purified with the ultra-filtration method.