[Expression of xenogeneic homologous epidermal growth factor receptor ectodomain in Pichia pastoris].
OBJECTIVE
To develop a secreted expression system of xenogeneic homologous epidermal growth factor receptor (EGFR) ectodomain using Pichia pastoris (P. pastoris), thus providing a basis for subsequent antitumor immunological researches on EGFR subunit protein vaccine.


METHODS
In order to construct the secreted expression vector pPICZaA-mer and pPICZaA-her, we recombined the cDNA of xenogeneic (human) EGFR ectodomain (her) and the corresponding control mouse EGFR ectodomain (mer) with the empty plasmid pPICZaA, respectively. Then the recombinant plasmids were linearized by Sac I and transformed into P. pastoris strain GS115 by electroporation. The positive transformants were selected on YPDS plate including Zeocin and were induced to express by methanol. Consequently, the proteins in the culture supernatant were assayed with SDS-PAGE and Western-blot. So we could choose the high-level secreted expression recombinant strain to produce protein vaccine vastly.


RESULTS
We got the secreted expression of mouse and human EGFR ectodomain in P. pastoris. The relative molecular mass of the two aimed proteins is about 95 x 10(3). The Western-blot analysis demonstrated that the expression proteins have much good antigenicity and specificity.


CONCLUSIONS
The recombinant plasmids pPICZaA-mer and pPICZaA-her could be induced to express the EGFR aimed proteins by methanol in methanol-trophic yeast expression system.
Fang
Fang
F
Key Laboratory of Biotherapy of Human Diseases of Ministry of Education, Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China.
Li
Jiong
J
Wen
Yan-jun
YJ
Tian
Ling
L
Wei
Yu-quan
YQ
D000818:Animals; D003001:Cloning, Molecular; D018274:Electroporation; D066246:ErbB Receptors / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D011994:Recombinant Proteins; D066246:ErbB Receptors