Production and purification of human papillomavirus type 33 L1 virus-like particles from Spodoptera frugiperda 9 cells using two-step column chromatography.
The major capsid protein L1 of human papillomavirus (HPV) is essential in construction of recombinant antigen vaccines against cervical cancer. HPV type 33 accounts for about 10% of all HPV infections in Asia. The gene encoding the major capsid protein L1 of the high-risk HPV type 33 was isolated from a Korean patient and expressed in Sf-9 insect cells using a baculovirus expression system. HPV33 L1 protein was isolated by two-step chromatographic purification using strong-cation exchange and ceramic hydroxyapatite chromatography. Strong-cation-exchange chromatography was performed to achieve initial purification of HPV33 L1 and to remove most contaminating proteins, and secondary ceramic hydroxyapatite chromatography yielded pure HPV33 L1 virus-like particles (VLPs). Ceramic hydroxyapatite columns are particularly useful in the purification of antibodies, antigens, human viruses, and VLPs, and we thus used this system. The expression of HPV L1 protein in Sf-9 cells was examined by SDS-PAGE, Western-blotting, and ELISA analyses, and the data showed that HPV33 L1 VLPs were determined to > 98% purity and 58.7% recovery by a quantitative immuno-ELISA assay. Transmission electron microscopy analysis revealed that the HPV VLPs were approximately 50-60 nm in diameter and created by self-assembly of HPV L1 protein. The efficient and simple purification process described here should be useful in production of a cervical cancer vaccine.
Baek
Jin-Oh
JO
Microbe-based Fusion Technology Research Center, Jeonbuk Baranch Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup, Jeonbuk 580-185, South Korea.
Seo
Jeong-Woo
JW
Kim
Ik-Hwan
IH
Kim
Chul Ho
CH
D000818:Animals; D036022:Capsid Proteins / Q000737:chemistry / Q000302:isolation & purification; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007723:Korea; D008854:Microscopy, Electron; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000235:genetics / Q000276:immunology / Q000821:virology; D053918:Papillomavirus Vaccines / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D018411:Spodoptera; D014462:Ultrafiltration; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000276:immunology / Q000821:virology; D058425:Vaccines, Virus-Like Particle / Q000737:chemistry / Q000302:isolation & purification; D019065:Virus Assembly
D036022:Capsid Proteins; D009856:Oncogene Proteins, Viral; D053918:Papillomavirus Vaccines; D011994:Recombinant Proteins; D058425:Vaccines, Virus-Like Particle