Effect of the DnaK chaperone on the conformational quality of JCV VP1 virus‐like particles produced in Escherichia coli

Protein nanoparticles such as virus‐like particles (VLPs) can be obtained by recombinant protein production of viral capsid proteins and spontaneous self‐assembling in cell factories. Contrarily to infective viral particles, VLPs lack infective viral genome while retaining important viral properties like cellular tropism and intracellular delivery of internalized molecules. These properties make VLPs promising and fully biocompatible nanovehicles for drug delivery. VLPs of human JC virus (hJCV) VP1 capsid protein produced in Escherichia coli elicit variable hemagglutination properties when incubated at different NaCl concentrations and pH conditions, being optimal at 200 mM NaCl and at pH range between 5.8 and 7.5. In addition, the presence or absence of chaperone DnaK in E. coli cells influence the solubility of recombinant VP1 and the conformational quality of this protein in the VLPs. The hemagglutination ability of hJCV VP1 VLPs contained in E. coli cell extracts can be modulated by buffer composition in the hemagglutination assay. It has been also determined that the production of recombinant hJCV VP1 in E. coli is favored by the absence of chaperone DnaK as observed by Western Blot analysis in different E. coli genetic backgrounds, indicating a proteolysis targeting role for DnaK. However, solubility is highly compromised in a DnaK^? E. coli strain suggesting an important role of this chaperone in reduction of protein aggregates. Finally, hemagglutination efficiency of recombinant VP1 is directly related to the presence of DnaK in the producing cells. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:744–748, 2014     

Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells.

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.     

Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.     

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy’s disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy’s disease and vehicles for the delivery of drugs.     

Expression of human papillomavirus type 11 L1 protein in insect cells: in vivo and in vitro assembly of viruslike particles.

The L1 coat protein of human papillomavirus type 11 (HPV-11) was expressed in Sf-9 insect cells with the recombinant baculovirus vector Ac11L1. Viruslike particles (VLPs) were identified by electron microscopy in the nucleus and cytoplasm of Sf-9 cells infected with Ac11L1. The L1 protein was purified from Ac11L1-infected insect cells. The purified protein spontaneously assembled in vitro into various aggregates, including particles appearing similar to empty virions. Reaction of VLP-containing insect cell extracts with antisera directed against either denatured or nondenatured capsid epitopes in Western blot (immunoblot) and immuno-dot blot assays suggested that conformational epitopes present in native HPV-11 infectious virions were also present on the baculovirus-produced HPV-11 VLPs. Immuno-dot blot assays using human sera obtained from individuals with biopsy-proven condyloma acuminatum correlated closely with results previously obtained in HPV-11 whole virus particle-based enzyme-linked immunosorbent assays. These morphologic and immunologic similarities to native HPV-11 virions suggest that recombinant VLPs produced in the baculovirus system may be useful in seroepidemiology and pathogenesis studies of genital HPV infection and that they may also be potential candidates for vaccine development.     

Induction of Humoral and Cell-Mediated Immune Responses by Hepatitis B Virus Epitope Displayed on the Virus-Like Particles of Prawn Nodavirus

Hepatitis B virus (HBV) is a deadly pathogen that has killed countless people worldwide. Saccharomyces cerevisiae-derived HBV vaccines based upon hepatitis B surface antigen (HBsAg) is highly effective. However, the emergence of vaccine escape mutants due to mutations on the HBsAg and polymerase genes has produced a continuous need for the development of new HBV vaccines. In this study, the “a” determinant within HBsAg was displayed on the recombinant capsid protein of Macrobrachium rosenbergii nodavirus (MrNV), which can be purified easily in a single step through immobilized metal affinity chromatography (IMAC). The purified protein self-assembled into virus-like particles (VLPs) when observed under a transmission electron microscope (TEM). Immunization of BALB/c mice with this chimeric protein induced specific antibodies against the “a” determinant. In addition, it induced significantly more natural killer and cytotoxic T cells, as well as an increase in interferon gamma (IFN-γ) secretion, which are vital for virus clearance. Collectively, these findings demonstrated that the MrNV capsid protein is a potential carrier for the HBV “a” determinant, which can be further extended to display other foreign epitopes. This paper is the first to report the application of MrNV VLPs as a novel platform to display foreign epitopes.     

牛病毒性腹泻病毒1型病毒样颗粒的制备及其对豚鼠的免疫原性分析

为了获得预防牛病毒性腹泻病毒1型(bovine viral diarrhea virus 1,BVDV-1)感染的病毒样颗粒,扩增C-Ems-E1-E2编码区段并克隆至pFastBacDaul载体,转化大肠杆菌DH10Bac感受态细胞与Bacmid重组获得Bacmid-BVDV-1,转染至Sf9细胞,获得重组杆状病毒B...     

Processing of infectious bursal disease virus (IBDV) polyprotein and self-assembly of IBDV-like particles in Hi-5 cells

The capsid of infectious bursal disease virus (IBDV), with a size of 60-65 nm, is formed by an initial processing of polyprotein (pVP2-VP4-VP3) by VP4, subsequent assemblage of pVP2 and VP3, and the maturation of VP2. In Sf9 cells, the processing of polyprotein expressed was restrained in the stage of VP2 maturation, leading to a limited production of capsid, i.e., IBDV-like particles (VLPs). In the present study, another insect cell line, High-Five (Hi-5) cells, was demonstrated to efficiently produce VLPs. Meanwhile, in this system, polyprotein was processed to pVP2 and VP3 protein and pVP2 was further processed to the matured form of VP2. Consequently, Hi-5 cells are better in terms of polyprotein processing and formation of VLPs than Sf9. In addition to the processing of pVP2, VP3 was also degraded. With insufficient intact VP3 protein present for the formation of VLPs, the excessive VP2 form subviral particles (SVPs) with a size of about 25 nm. The ratio of VLPs to SVPs is dependent on the multiplicity of infections (MOIs) used, and an optimal MOI is found for the production of both particles. VLPs were separated from SVPs with a combination of ultracentrifugation and gel-filtration chromatography, and a large number of purified particles of both were obtained. In conclusion, the insect cell lines and MOIs were optimized for the production of VLPs, and pure VLPs with morphology similar to that of the wild-type viruses can be effectively prepared. The efficient production and purification of VLPs benefits not only the development of an antiviral vaccine against IBDV but also the understanding of the structure of this avian virus that is economically important.     

N端融合外源蛋白的兔出血症病毒衣壳蛋白自聚能力的研究

将兔出血症病毒衣壳蛋白VP6 0基因插入杆状病毒转移载体pBLUEBACHIS2_B的 6 HIS表达标签下游 ,与线性化野生型杆状病毒基因组DNA共转染Sf9昆虫细胞 ,经蚀斑纯化后获克隆化重组杆状病毒pBLUEBACHIS2B_VP6 0。以重组杆状病毒感染Sf9细胞 ,经SDS_PAGE和Westernblot检测显示高效表达一分子量为 6 9kD的重组蛋白 ,并且该蛋白可被兔抗RHDV高免血清识别。血凝试验表明 ,该重组蛋白可以凝集人“O”型红细胞 ,血凝价达 2 1 6 ,同时 ,该血凝性可被抗RHDV的高免血清所抑制。经电镜观察 ,重组病毒表达的融合有 6 HIS表达标签的衣壳蛋白仍可在昆虫细胞内自聚成不包裹核酸的、与天然RHDV病毒粒子在物理形态上相似的病毒样颗粒 (VLPs) ,并且该VLPs与兔抗RHDV高免血清作用后于电镜下可见凝集成团的现象 ,表明其与天然RHDV病毒粒子在抗原性上也极为相似     

Immunogenicity of plant‐produced porcine circovirus‐like particles in mice

Porcine circovirus type 2 (PCV‐2) is the main causative agent associated with a group of diseases collectively known as porcine circovirus‐associated disease (PCAD). There is a significant economic strain on the global swine industry due to PCAD and the production of commercial PCV‐2 vaccines is expensive. Plant expression systems are increasingly regarded as a viable technology to produce recombinant proteins for use as pharmaceutical agents and vaccines. However, successful production and purification of PCV‐2 capsid protein (CP) from plants is an essential first step towards the goal of a plant‐produced PCV‐2 vaccine candidate. In this study, the PCV‐2 CP was transiently expressed in Nicotiana benthamiana plants via agroinfiltration and PCV‐2 CP was successfully purified using sucrose gradient ultracentrifugation. The CP self‐assembled into virus‐like particles (VLPs) resembling native virions and up to 6.5 mg of VLPs could be purified from 1 kg of leaf wet weight. Mice immunized with the plant‐produced PCV‐2 VLPs elicited specific antibody responses to PCV‐2 CP. This is the first report describing the expression of PCV‐2 CP in plants, the confirmation of its assembly into VLPs and the demonstration of their use to elicit a strong immune response in a mammalian model.     

Production of the virus‐like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus‐like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti‐myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high‐performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038–1045, 2016     

Production of porcine parvovirus virus-like particles using silkworm larvae

Porcine parvovirus (PPV) is a significant causative agent of porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a nonenveloped virus, and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2, is the main target for PPV neutralizing antibodies and vaccine development. In this study, PPV-VP2 protein was expressed in silkworm larvae, and its antigenicity and production were compared with those in B. mori cells (Bm5). The recombinant VP2 protein was expressed successfully in silkworm larvae and Bm5 cells with a size of approximately 64 kDa. The formation of virus-like particles (VLPs) by recombinant PPV-VP2 was confirmed through transmission electron microscopy. The recombinant PPV-VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm. The antigenicity of PPV-VLPs was comparatively analyzed between Bm5 cells and silkworm larvae by ELISA, hemagglutination and hemagglutination inhibition assays. Consequently, it was confirmed that the PPV-VLPs produced in the silkworm larvae were more antigenic than VLPs produced in Bm5 cells. Therefore, it is expected that economical and effective vaccine development will be possible by mass production of PPV-VLPs in silkworm larvae.     

Suitability and perspectives on using recombinant insect cells for the production of virus-like particles

Virus-like particles (VLPs) can be produced in recombinant protein production systems by expressing viral surface proteins that spontaneously assemble into particulate structures similar to authentic viral or subviral particles. VLPs serve as excellent platforms for the development of safe and effective vaccines and diagnostic antigens. Among various recombinant protein production systems, the baculovirus–insect cell system has been used extensively for the production of a wide variety of VLPs. This system is already employed for the manufacture of a licensed human papillomavirus-like particle vaccine. However, the baculovirus–insect cell system has several inherent limitations including contamination of VLPs with progeny baculovirus particles. Stably transformed insect cells have emerged as attractive alternatives to the baculovirus–insect cell system. Different types of VLPs, with or without an envelope and composed of either single or multiple structural proteins, have been produced in stably transformed insect cells. VLPs produced by stably transformed insect cells have successfully elicited immune responses in vivo. In some cases, the yield of VLPs attained with recombinant insect cells was comparable to, or higher than, that obtained by baculovirus-infected insect cells. Recombinant insect cells offer a promising approach to the development and production of VLPs.     

Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production.     

杆状病毒表达EV71病毒样颗粒的装配、制备纯化与鉴定

将EV71P1和3CD基因片段克隆入同一杆状病毒穿梭质粒Bacmid中,构建出重组杆状病毒表达质粒Bac-mid-P1-3CD;脂质体介导其转染Sf9昆虫细胞获得共表达P1和3CD的重组杆状病毒(AcMNPV-P1-3CD)。用IFA和Western-blot法对表达产物进行鉴定和分析。电镜结果显示P1经3CD切割装配成了大小约为27nm的类球形颗粒(即EV71VLPs)。进一步分析影响杆状病毒表达系统的因素以对表达条件进行优化,结果显示MOI值和时间均可影响目的蛋白的表达,其中时间是主要因素。选择优化后条件利用无血清培养基对贴壁Sf9细胞在多层细胞培养器中进行VLPs的大量表达,密度梯度离心法纯化,SDS-PAGE结果可见三条大小约为39kD、34kD和26kD的VP1、VP0和VP3特异性条带。纯化后EV71VLPs颗粒结构完好,为下一步EV71蛋白结构的基础研究和基因工程疫苗的研究奠定了基础。     

Expression and Assembly Mechanism of the Capsid Proteins of a Satellite Virus (XSV) Associated with Macrobrachium rosenbergii Nodavirus

The extra small virus (XSV) is a satellite virus associated with Macrobrachium rosenbergii nodavirus (MrNV) and its genome consists of two overlapping ORFs, CP17 and CP16. Here we demonstrate that CP16 is expressed from the second AUG of the CP17 gene and is not a proteinase cleavage result of CP17. We further expressed CP17 and several truncated CP17s (in which the N- or C-terminus or both was deleted), respectively, in Escherichia coli. Except for the recombinant plasmid CP17ΔC10, all recombinant plasmids expressed soluble protein which assembled into virus-like particles (VLPs), suggesting that the C-terminus is important for VLP formation.     

Comparison of biological activity of recombinant woodchuck interferon gamma and tumor necrosis factor alpha produced in baculovirus and Escherichia coli expression systems

The full-length cDNAs of recombinant woodchuck interferon gamma (rwIFN gamma) and woodchuck tumor necrosis factor alpha (rwTNF alpha) were cloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The recombinant proteins secreted by the insect cells, bac-rwIFN gamma and bac-rwTNF alpha, were found to be functionally competent. Their biological activities were compared to those of rwIFN gamma and rwTNF alpha produced in the Escherichia coli (E. coli) expression system. The bac-rwIFN gamma demonstrated a 4.5-fold greater protective activity against encephalomyocarditis virus-induced cytolysis of woodchuck hepatocytes and that of class I MHC antigen presentation on the hepatocytes than rwIFN gamma derived from E. coli. The bac-rwTNF alpha was cytotoxic towards murine fibroblasts and able to upregulate class I MHC antigen display and these effects were about 18-fold greater than those triggered by rwTNF alpha from E. coli at a comparable protein level. In addition, the antiviral activity of bac-rwIFN gamma was inhibited by anti-wIFN gamma antibodies and the cytotoxicity of bac-rwTNF alpha neutralized by cross-reactive antibodies to murine TNF alpha. The study showed that the expression of rwIFN gamma and rwTNF alpha in the baculovirus system generated biologically active cytokines whose potency was considerably greater than those produced in E. coli.     

利用昆虫-杆状病毒表达系统表达人乳头瘤病毒16/18/33/58亚型L1蛋白

目的:通过昆虫-杆状病毒表达系统获得人乳头瘤病毒(HPV)16/18/33/58亚型的主要衣壳蛋白L1。方法:克隆了HPV16/18/33/58亚型的L1蛋白基因,并采用密码子优化策略进行改造(记为HPV16/18/33/58亚型mL1),将优化基因片段插入pFastBac Dual载体获得重组载体,转化大肠杆菌DH10Bac感受态细胞后得到重组Bacmid,转染昆虫Sf9细胞,Western印迹和SDS-PAGE检测重组蛋白的表达。结果:获得了表达HPV16/18/33/58亚型mL1蛋白的重组杆状病毒;Western印迹和SDS-PAGE分析表明该重组杆状病毒感染昆虫Sf9细胞后表达mL1蛋白,且mL1蛋白主要分布在细胞中;优化了蛋白表达时间和感染复数,获得目的蛋白mL1的高效表达。结论:多个亚型HPV L1蛋白的克隆表达,为中国优势血清型疫苗的研制奠定了基础。     

人诺如病毒衣壳蛋白的密码子优化及在昆虫细胞中的表达

诺如病毒是当前在中国引起腹泻的主要人类杯状病毒,其中GGII4、GGII1和GGII3等为主要的流行遗传型。为了提高诺如病毒衣壳蛋白的表达量,我们将其编码基因按杆状病毒喜用密码子进行了优化设计和人工合成。以杆状病毒为载体,在昆虫细胞Sf9中对密码子优化后的诺如病毒GGII1、GGII3、GGII4和GGII7型衣壳蛋白基因进行了表达。结果显示,与野生型基因相比,经过密码子优化的基因在昆虫细胞中的表达水平得到明显提高,并可装配成病毒样颗粒。重组杆状病毒感染Sf9细胞72h表达量达到高峰。这些结果的取得,为我国人杯状病毒免疫学检测试剂和疫苗的开发打下了基础。     

Assembly of the capsid protein of red-spotted grouper nervous necrosis virus during purification,and role of calcium ions in chromatography

Currently virus-like particles (VLPs) are receiving much attention as platforms for next generation vaccines. However, chromatography-based methods for purifying VLPs remain challenging. Unlike traditional methods using density gradient for purifying VLPs, there have been few advances in explaining how assembled particles can be obtained by chromatography. Nervous necrosis virus (NNV) infects over 30 species of fish and leads to large economic losses in the farmed fish industry. Previously we developed a heparin chromatography-based method for purifying red-spotted grouper NNV (RGNNV) VLPs. However it is unclear how the assembled RGNNV VLPs are obtained by this method. It is known that assembly of NNV capsid proteins depends on calcium ions. In the present study, we found that the yield of purified RGNNV capsid protein in heparin chromatography was enhanced when calcium ions were present during binding. Also, it appears that the capsid protein of RGNNV undergoes partial disassembly and reassembly during sample preparation prior to heparin chromatography and the protein finally undergoes assembly during the chromatography. Therefore, our results indicated that heparin-binding affinity of RGNNV capsid protein is linked to its ability for VLP formation. The assembly of RGNNV capsid proteins recombinantly produced is a good model for explaining VLP formation during chromatography-based purification processes.