Production of Recombinant Human Papillomavirus Type 52 L1 Protein in Hansenula polymorpha Formed Virus-Like Particles

Abstract

Human papillomavirus (HPV) type 52 is a high-risk HPV responsible for cervical cancer. HPV type 52 is common around the world and is the most common in some Asian regions. The available prophylactic HPV vaccines protect only from HPV types 16 and 18. Supplementing economical vaccines that target HPV type 52 may satisfactorily complement available prophylactic vaccines. A codon-adapted HPV 52 L1 gene was expressed in the methylotrophic yeast Hansenula polymorpha, which is used as an industrial platform for economical hepatitis B surface antigen particle production in China. We found that the recombinant proteins produced in this expression system could form virus-like particles (VLPs) with diameters of approximately 50 nm. This study suggests that the HPV 52 VLPs produced in this platform may satisfactorily complement available prophylactic vaccines in fighting against HPVs prevalent in Asia.

Introduction

Human papillomavirus (HPV) type 16 is the most common high-risk HPV responsible for cervical cancer, and HPV type 18 is the second most common [19]. Other types may be more prevalent in some regions [17]. HPV type 52 is a common type of HPV around the world [2,18], being the second most common in South Taiwan, Korea, and New Zealand [13,15,20] and the most common in Japan, northern Vietnam, and some regions in China [3,4,10,14,21,22].

Recombinant prophylactic cervical cancer vaccines that target HPV were developed because HPV does not grow in cell culture systems, whereas recombinant major structural protein L1 forms virus-like particles (VLPs), which mimic the ability of virions to elicit protective immune responses [8,12]. Cervarix (GlaxoSmithKline, Rixensart, Belgium) protects from HPV types 16 and 18, which are L1 proteins expressed in insect cells, and Gardasil (Merk and Co., Bluebell, PA, USA) protects from HPV types 16, 18, 6, and 11, which are L1 proteins expressed in Saccharomyces cerevisiae. HPV types 6 and 11 are low-risk HPVs that do not cause cervical cancer, and the above cervical cancer prophylactic vaccines mainly target HPV types 16 and 18. HPV type 52 is particularly common in Asian regions, which contain nearly half of the population of the planet. Supplementing economical vaccines that target HPV type 52 may be a good complement to available prophylactic vaccines. The methylotrophic yeast Hansenula polymorpha, similar to S. cerevisiae, is used as an industrial platform for producing hepatitis B surface antigen (HBsAg) particles, which are currently available as an inexpensive vaccine [1,6]. The recombinant HPV 52 L1 protein expression in H. polymorpha is described in this study.

 

Materials and Methods

Codon-Adapted Gene Synthesis and Expression Plasmid Construction

The gene that is translated to HPV type 52 late protein L1 was codon-adapted using JCat (http://www.jcat.de/) and was synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). The recombinant plasmid was cloned between the SnaB and EcoRI sites of the vector pMOX-HARS [16], and the recombinant plasmid was copied by Escherichia coli that survived 100 µg/ml kanamycin sulfate (Sigma) on LB agar plates. Plasmids in the E. coli were extracted and linearized with NdeI for transformation in H. polymorpha.

Yeast Transformation and Recombinant Protein Expression

The GenePulser Xcell system (BIO-RAD) was used to transform 1 µg of NdeI-linearized plasmids into the H. polymorpha host strain (ATCC26012) by using a 2 mm cuvette after a 1.5 kV/cm, 50 µF, and 129 Ω electric field pulse (5 ms resulting pulse length) [7]. The clones were subjected to static culture for 36 h at 37℃ on YPD agar plates containing 100 µg/ml zeocin, and the clones that survived were grown in test tubes (15 ml) with 2 ml of YPG at 37℃, 280 rpm for 36 to 48 h. Cells were harvested (1,500 ×g, 5 min), washed once with 2 ml of YPM/MM/BMMY, subsequently resuspended with 2 ml of YPM/MM/BMMY, and incubated at 28℃ or 37℃, 280 rpm for 24, 48, and 72 h to induce recombinant protein expression. The culture was added with 100% methanol every 24 h to a 0.5% final concentration to maintain recombinant protein induction. The media used are listed in Table 1.

Table 1.Medium used for recombinant protein expression in Hansenula polymorpha.

The pH of the medium was tested every 24 h during induction (pH/mv meter; Denver Instrument). The OD600 of the yeasts was tested at the end of induction (Biophotometer, Eppendorf).

SDS–PAGE and Western Blot Analysis

The samples (approximately 2.5 × 105 cells) were boiled in a water bath for 10 min with 2× SDS–PAGE sample loading buffer and were subjected to 12% SDS–PAGE and stained with Coomassie blue. Proteins were also transferred to a PVDF membrane (BioTrace PVDF, Life Science) by using the Trans-Blot SD Semi-Dry Transfer Cell (Bio-rad) at 150 mA for 1 h. The membrane was probed with anti-HPV 16 L1 monoclonal antibody (1:1,000, sc-47699; Santa Cruz) and secondary antibody (1: 10,000, goat anti-mouse IgG-HRP sc-2005; Santa Cruz) afterward.

Cell Lysate Preparation and Transmission Electron Microscopy

Cell lysates were prepared as described previously [11], with slight modifications. Yeast cultures (30 ml) were harvested by centrifugation at 1,500 ×g for 5 min, resuspended in 15 ml of disruption buffer (10 mM NaH2PO4, 150 mM NaCl, 1.7 mM ethylenediaminetetraacetic acid, and 0.01% Tween 80, pH 7.2), mixed with 0.5 mm diameter glass beads (15 g), and disrupted using a vortex mixer (Labnet International, Inc., USA) for 30 min. Cell debris were removed by centrifugation at 1,500 ×g for 5 min. The supernatants were re-centrifuged at 12,000 ×g for 10 min, collected, and precipitated using 40% saturated (NH4)2SO4 for 30 min at room temperature. The precipitated samples were collected by centrifugation at 12,000 ×g for 10 min, washed twice in 1 ml of 20% saturated (NH4)2SO4 using a pipette, collected by centrifugation at 12,000 ×g for 10 min, and dissolved in 100 µl of disruption buffer.

The samples were absorbed on carbon-coated copper grids and negatively stained with 2% phosphotungstic acid. The grids were air dried prior to examination under a transmission electron microscope (Hitachi, H-7650) with 80 kV acceleration voltage [9].

The GenBank accession number of the sequence that is translated to HPV type 52 late protein L1 reported in this paper is KJ778071.1.

 

Results and Discussion

Effects of Media Nutrition on Biomass and HPV 52 L1 Production

HPV 52 L1 was expressed well in both YPM and BMMY media, whereas its expression in MM was quite weak (Fig. 1). Yeasts grown in BMMY, the medium with the most nutrition, generated the most biomass after induction. MM is a minimal medium that may not have enough nutrition to support H. polymorpha growth during induction, unlike complex broth media (YPM and BMMY). Biomass may be a factor that affects HPV 52 L1 production, but it is not the determinant factor because the yeasts grown in MM also generated considerable amounts of biomass (Fig. 2A).

Fig. 1.Western blot analysis of HPV 52 L1 expression in Hansenula polymorpha in different induction media (YPM, MM, or BMMY) under 28℃ and 37℃. Approximately 2.5 × 105 cells were subjected to 12% SDS–PAGE for western blot analysis at 24, 48, and 72 h.

Fig. 2.Analysis of H. polymorpha biomass after induction. Seven different yeast strains from the same starting inocula (OD600 = 3) were fermented in different induction media (YPM, MM, or BMMY) under 28℃ and 37℃ for protein expression. The OD600 of the yeasts was measured after induction.

Effects of Temperature on Biomass and HPV 52 L1 Production

Yeasts grown under 28℃ generated more biomass than those grown at 37℃ in all media, even though their 52 L1 expression levels were similar at 24 h. This finding may be attributed to the fact that the optimal growth temperature of H. polymorpha is about 28℃. Nearly all of the OD600 located in the lines of yeasts grown in MM under 37℃ and in BMMY under 28℃ (Fig. 2B), which indicates that both medium nutrition content and temperature are key factors for generating biomass during induction.

Temperature had slight effects on 52 L1 expression. The yeasts grown in YPM expressed similar recombinant protein levels at 28℃ and at 37℃ after 24 h of induction in YPM and BMMY, respectively. HPV 52 L1 levels decreased in BMMY and YPM under both 28℃ and 37℃ after 24 h, but this decrease seems to be slower in the YPM under 28℃ (Fig. 1). This result may be caused by the fact that protease inhibitors were not added during recombinant protein induction, and low temperatures may be good for the stability of the expressed HPV 52 L1.

Effects of pH on Biomass and HPV 52 L1 Production

The pH may be a key factor of the low HPV 52 L1 level in the yeasts cultured in MM. Minimal medium is used for the intracellular expression of the desired proteins in Pichia pastoris systems. The pH of this medium was sharply decreased in the recombinant protein induction (Fig. 3) because it does not contain buffers. The pH of the buffered medium BMMY remained at 6 during recombinant protein induction, which may be important for the stability of the expressed recombinant proteins. The pH of the unbuffered YPM medium increased from 6 to 7.5 after 24 h of recombinant protein induction and stayed at 7.5 afterward. How the pH outside the yeast cells affects recombinant protein stability or expression levels inside the cells is yet to be determined. The acidic medium may be not good for the intracellular HPV 52 L1 expression in H. polymorpha.

Fig. 3.Analysis of the pH of different induction media (YPM, MM, or BMMY) under 28℃ or 37℃ along recombinant protein expression. The pH values of the media were tested every 24 h during induction.

HPV Type 52 L1 Proteins Produced in H. polymorpha Formed Virus-Like Particles

The HPV 52 L1 proteins expressed in H. polymorpha that did not undergo further processing, except for ammonium sulfate precipitation, formed VLPs of around 50 nm diameter, which are similar to the natural virions (Fig. 4). Whether these VLPs are formed inside the H. polymorpha cells or follow precipitation is still uncertain. Nevertheless, this study proved that the HPV 52 L1 proteins expressed in H. polymorpha formed VLPs, which may be used as HPV prophylactic vaccines in the same way the VLPs expressed in either a baculovirus expression vector system or S. cerevisiae were used [5,23].

Fig. 4.TEM analysis of the HPV 52 L1 proteins. Virus-like particles are indicated by arrows. Bars for reference are located in the lower corner of each picture.

In conclusion, this is the first report of recombinant HPV 52 L1 production in H. polymorpha, and the first report that the HPV 52 L1 expressed in H. polymorpha forms VLPs, which may be used as prophylactic vaccines. H. polymorpha is used as an industrial platform for producing HBsAg particles, which are currently available as an inexpensive vaccine. This study established the implications that the HPV 52 VLPs produced in this platform complement available prophylactic vaccines against the HPVs prevalent in Asia.