[Gene optimization is necessary to express HPV type 6 L1 protein in the methylotrophic yeast Pichia pastoris].
OBJECTIVE
Human papillomavirus 6 (HPV 6) causes genital warts, a common sexually transmitted disease. L1-capsids protein is a highly promising vaccine candidate that has entered phase II clinical trial. But the existing methodologies for producing L1-capsids in insect cells is expensive for use in developing countries. As methylotrophic yeast,the Pichia pastoris expression system offers economy,and high expression levels. Over-expression of HPV6-L1 protein in P. pastoris was the purpose of this study.


METHODS
The whole L1 gene with preferred codons for P. pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5K,electroporation of KM71, in vivo screen of multiple inserts by G418 resistance, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins.


RESULTS
Three clones were found to produce L1 protein which can be identified with Western blot. Compared with L1 protein from E.coli, pichia-produced L1 has some glycosylation. Reacting strongly with MabH6B10.5 in indirect immunofluorescence assay indicated that L1 protein expressed in pichia cell holds its native conformational epitopes which is important for vaccine use. A total 125 microg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography.


CONCLUSIONS
HPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.
Li
Ping-chuan
PC
Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China.
Zhang
Xiao-guang
XG
Zhou
Ling
L
Zeng
Yi
Y
D036022:Capsid Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D005796:Genes; D027383:Papillomaviridae / Q000235:genetics*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins
D036022:Capsid Proteins; D014764:Viral Proteins; C091560:L1 protein, Human papillomavirus type 6