A method for removing contaminating protein during purification of human papillomavirus type 18 L1 protein from Saccharomyces cerevisiae

Abstract

Human papillomavirus (HPV) types 16 and 18 are the main targets in the field of prophylactic vaccines for preventing cervical cancer. L1 protein, the major capsid protein of HPV, selfassembles into virus-like particles (VLP), which are the major component of prophylactic vaccines. To obtain highly purified L1 protein, contaminants must be removed by several chromatography steps. However, this requires a great deal of time and labor, and results in loss of large amounts of the target protein. Therefore, we have sought to develop an efficient method for removing contaminants prior to chromatography during the purification of HPV18 L1 protein from Saccharomyces cerevisiae. For this purpose the contaminating proteins were removed by an ammonium sulfate precipitation step and further removed by a removal of precipitated contaminants step. Purification of the L1 protein by chromatography was significantly improved by the removal of precipitated contaminants step. In the present work we developed two one-step chromatography methods (heparin and cation-exchange chromatography), and HPV18 L1 proteins purified by both methods self-assembled into VLP. The two chromatographic purification methods are simpler and more convenient than previous methods and are widely applicable to work with VLPs.

Interaction of human papillomavirus type 16 particles with heparan sulfate and syndecan-1 molecules in the keratinocyte extracellular matrix plays an active role in infection

“…ECM-bound HPV16 was washed intensively and incubated at 37 uC in CM. Media were collected after various time points, centrifuged to remove cell debris, and released PsVs were captured with heparin-agarose beads (Sigma-Aldrich) for 1 h, 4 uC (Joyce et al, 1999;Kim et al, 2009;Faust et al, 2010). Beads were washed three times with PBS, resuspended with SDS sample buffer, and analysed by SDS-PAGE and immunoblot for HPV16 L1.…”

Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using thePichia pastoris PGK1Promoter

“…It has been argued that creating optimum conditions for the production and purification of HPV VLPs is a strategy that can reduce the production costs of vaccines [ 31 , 40 ], since it could require less time and labor in industrial production. In this regard, different procedures have been explored related to VLP purification steps, such as ultracentrifugation, size-exclusion chromatography, and cation-exchange chromatography or even their combination [ 39 – 41 ], as well as the findings about how cell culture conditions can be optimized [ 31 , 38 ]. We believe that secretion of HPV L1 in the culture media could improve the downstream process.…”

Current status and future challenges in transitioning to continuous bioprocessing of virus‐like particles

“…It has been shown to generate higher VLP yield compared to ultracentrifugation. 49,50 However, this technique has not been applied for commercial application. Ammonium sulfate precipitation can easily be transitioned to continuous plug flow reactors and requires further consideration.…”

“…Ammonium sulfate precipitation has been used for purification of HPV VLPs and hepatitis B VLPs from yeast. It has been shown to generate higher VLP yield compared to ultracentrifugation 49,50 . However, this technique has not been applied for commercial application.…”