Abstract: Objective To develop a simple chromatography purification process of human papillomavirus (HPV) type 58 L1 virus-like particle (VLP) produced by insect cells baculovirus expression vector system (IBEVS). Methods A previously constructed mutant of HPV-58 L1 with a high expression level in insect baculovirus system were used to prepare the cell lysates.The L1 containing sample was used for the establishment of optimal protein purification process which consisting of an initial process of ammonium sulfate precipitation (ASP) and two polishing chromatography purifications. Purified L1 protein was dialyzed for VLP reassembly and the resultant VLPs were characterized physicochemically and their immunogenicity was then analyzed in mice. Results L1 protein was purified by 30% ASP with good purity (9.34%) and recovery (72%). When bound to anion exchange resin and eluted by 300 mmol/L NaCl-contained buffer, the L1 protein was further purified with ＞90% of recovery and 13.6% of purity. The resultant L1 protein mainly flowed through the ceramic hydroxyapatite column with a recovery of ＞80% and purity was over 99.1% by HCP analysis. With the three-step purification, the total recovery of L1 was about 55.7% and the yield was 57-65 mg/L. Purified L1 proteins self-assembled into homogenous VLPs with an average diameter of 55 nm as proved by TEM and DLS, and these VLPs induced high titer of neutralizing antibodies, comparable to that of VLPs purified by ultracentrifugation. Conclusions The chromatography process for producing HPV-58 L1 VLPs showed good purity, yield, and immunogenicity and is believed to be a time-saving, easy-performing, fully scalable, and low-cost method. It may be used to produce low-cost and multivalent HPV VLP vaccine by IBEVS.

Key words: human papillomavirus type 58, major capsid protein L1, virus-like particle(VLP), insect cell, purification