STUDIES ON THE EXPRESSION OF A LOW MOLECULAR-WEIGHT PROTEIN IN E. COLI SYSTEM

• Main Authors: Kuai-Chieh Huang, 黃冠傑
• Other Authors: I-Ching Kuan
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/88089728507958147760

碩士 === 大同大學 === 生物工程學系(所) === 105 === In this study, E. coli was used to heterologously express a human low-molecular-weight protein, YS. In order to enhance YS expression, the DNA sequence of the corresponding gene was subject to different degrees of codon optimization, and two artifical genes, ys1 and ys2 were synthesized. After construction onto pET22b, pET23a and pET24a and transformation into E. coli BL21 (DE3), the complex medium, terrific buffer (TB) and chemically defined medium, medium A, were used for cultivation. E. coli BL21 (DE3) containing pET23YS1 (recombinant ys1-carrying pET23a) when cultured with medium A exhibited the best YS expression. On the other hand, part of the 5’terminal sequences of ys1 and ys2 were modified to generate ays1, bys1, cys1, ays2, bys2 and cys2. After construction onto pET23a, the recombinant plasmids were also transformed into E. coli BL21 (DE3) for the YS expression. Because E. coli BL21 (DE3) containing pBYS1 (recombinant bys1-carrying pET23a) in medium A had the best YS expression, this recombinant was used to further examine the effects of different concentrations and kinds of inducers as well as different amounts of inocula on the YS expression. SDS-PAGE analysis showed that the best YS expression was achieved with 0.4 mM IPTG induction and 1:50 inoculation. To evaluate the possibility of scale-up of the YS production, a 5 L fermenter containing 2 L medium A was used for cultivation. The use of glucose feeding and an increase in IPTG addition to 1 mM resulted in a significant improvement in the YS production.