Functional Expression of Human Peptidylglycine alpha-Amidating Monooxygenase

• Main Authors: Hou Ren Wang, 王厚仁
• Other Authors: C. T. Wang
• Format: Others
• Language: en_US
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/75255606011138746582

碩士 === 國立清華大學 === 生物技術研究所 === 91 === Abstract Many peptide hormones must be amidated at their carboxyl termini for full biological activity. In higher organisms, the enzymatic formation of C-terminally amidated peptides is a two-step process catalyzed by a single bifunctional enzyme, peptidylglycine alpha amidating monooxygenase (PAM). PAM is important in production of recombinant pharmaceutical peptides that require amidation. Although there are some chemical methods for peptide alpha amidation, several existing drawbacks make the enzymatic way the favorite one. However, recombinant PAM enzyme was not available commercially. Thus, we have applied different expression systems in this study to produce economical PAM for industrial use. The commercial human PAM (hPAM) cDNA was initially expressed in Chinese hamster ovary cell line (CHO-K1) to confirm the correct expression of desired proteins. A 98 kD protein band of expected size was detected by Western blotting. To provide a rapid and cost-effective expression of hPAM, a secretory system by using the leader sequence of yeast alpha mating factor was primary selected. However, neither PAM protein nor its activity was detected through this approach. Further, by deleting the signal sequence, cytosolic expression of hPAM in yeast resulted in insoluble and function-less enzyme. Finally, secretory expression of the truncated version of the bi-functional PAM (31aa-817aa) by fusing with the Bip signal sequence was performed in the Drosophila Schneider 2 (S2) cell line. Under the regulation of metallothionein promoter, 5 g/ml of hPAM31-817 was obtained with continuous induction of 10 M CdCl2 for five days and a simple metal chelate resin purification procedure.