Cloning and Expression of the cDNA Coding for Rat Peptidylglycine alpha-Amidating Monooxygenase.
The dDNA of genes encoding rat peptidylglycine alpha-aminating monooxygenase(rPAM) were cloned and their expression in E. coli studied. Three DNA fragments were isolated from the rat brain cDNA library using the methods of plaque hybridization and PCR. DNA sequencing showed that they contained the total coding sequence for rPAM-2. By using the sited-directed mutation and PCR recombination methods, we obtained intact genes coding for the rPHM domain, the rPAL domain and rPAM, respectively. Different plasmids of these genes controlled under T(7) or P(L) promoter were constructed and transformed into E. coli. The high-level expression of rPAM-N260 in E. coli was first observed and its antiserum was prepared for the immunoassay of natural or recombinant PAMs. By the analysis with SDS-PAGE and Western blot, the products of rPHM and rPAM in E. coli were detected, and the amount of rPHM reached over 10% of the total bacterial proteins. It was found that low temperature and copper ion obviously increased the stability and solubility of the rPHM expressed in E. coli.
Jiang
Zhi-Hong
ZH
Shanghai Institute of Biochemistry, Academia Sinica, Shanghai 200031, China.
Yang
Yi
Y
Tu
Hong-Ming
HM
Yang
Xin-Ying
XY
Xia
Qi-Chang
QC
Li
Bo-Liang
BL
Wang
De-Bao
DB