Optimizing functional versus total expression of the human mu-opioid receptor in Pichia pastoris.
The expression of the EGFP-human mu-opioid receptor fusion protein in the methylotrophic yeast Pichia pastoris was optimized and monitored using both fluorescence and ligand-binding experiments. A set of parameters, including gene copy number, strain type, temperature, pH, and methanol inducer levels, was studied for its effect on the production of the recombinant protein. We show here that the expression level is optimal after 10 h of promoter induction and that the maximum is reached at a lower temperature and a higher pH than normally used. The optimized conditions have allowed a fourfold increase of the ligand-binding active form of the receptor, whereas the total expression level determined by EGFP fluorescence measurements was not modified.
Sarramegna
Valérie
V
Institut de Pharmacologie et de Biologie Structurale, UMR 5089, Toulouse, France.
Demange
Pascal
P
Milon
Alain
A
Talmont
Franck
F
D003001:Cloning, Molecular / Q000379:methods; D066246:ErbB Receptors / Q000096:biosynthesis / Q000235:genetics*; D018628:Gene Dosage; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D010843:Pichia; D011401:Promoter Regions, Genetic; D017450:Receptors, Opioid, mu / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics*; D013696:Temperature
D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors; D000432:Methanol