Optimum conditions for production and purification of human papillomavirus type 16 L1 protein from Saccharomyces cerevisiae.
Several prophylactic human papillomavirus (HPV) vaccines have been developed based on virus-like particles (VLPs) made from viral L1 proteins. A substantial number of VLPs is necessary for biochemical characterization and diagnostic test development. To establish the optimum conditions for production and purification of HPV L1 in the yeast expression system we varied the amount and nature of the carbon source and evaluated HPV 16 L1 recovery by three purification methods. Maximally threefold more HPV 16 L1 was produced with a 4% carbon source than with a 2% carbon source. In addition, the productivity of HPV 16 L1 varied by 25% depending on the combination of glucose and galactose in the 4% carbon source. We introduced an ammonium sulfate precipitation step in place of the ultracentrifugation using a sucrose cushion routinely used for HPV L1 purification, and optimized the purification by cation-exchange chromatography. Overall L1 protein recovery using the ammonium sulfate precipitation method was 30%, the highest recovery achieved so far. The purified HPV 16 L1 protein successfully self-assembled into VLPs. Purification by ammonium sulfate precipitation was maximally 15 times greater than ultracentrifugation on a sucrose cushion. We anticipate that our procedures for production and purification will reduce the cost, time and labor involved in obtaining sufficient yields of VLPs.
Park
Min-A
MA
College of Pharmacy, Chung-Ang University, 221 Huksuk-Dong, Dongjak-Ku, Seoul 156-756, South Korea.
Kim
Hyoung Jin
HJ
Kim
Hong-Jin
HJ
D000645:Ammonium Sulfate; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000648:ultrastructure; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods*; D003470:Culture Media; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D052162:Human papillomavirus 16 / Q000737:chemistry*; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014461:Ultracentrifugation
D036022:Capsid Proteins; D003470:Culture Media; D009856:Oncogene Proteins, Viral; C063317:L1 protein, Human papillomavirus type 16; D005947:Glucose; D000645:Ammonium Sulfate; D005690:Galactose