Expression and self-assembly of Heterocapsa circularisquama RNA virus-like particles synthesized in Pichia pastoris

SPECIAL TOPIC Omics in Marine Biotechnology WU YuanZheng 2 3 KIM SHIN Hyun-Jae ) 2 Wonduck 1 KIM Si-Wouk 1 EOM Chi-Yong 0 YANG HeTong 3 0 Seoul Center, Korea Basic Science Institute , Seoul 136-713, Republic of Korea 1 Department of Environmental Engineering, Chosun University , Gwangju 501-759, Republic of Korea 2 Department of Chemical and Biochemical Engineering, Chosun University , Gwangju 501-759, Republic of Korea 3 Biotechnology Center of Shandong Academy of Sciences , Jinan 250014, China Heterocapsa circularisquama RNA virus (HcRNAV) is the first single-stranded RNA virus to be characterized that infects dinoflagellates. The ability of HcRNAV coat protein (HcRNAV CP) to self-assemble into virus-like particles (VLPs) in vitro suggested that heterologous expression was possible, and that the VLPs might be ideal nanocontainers for the targeted delivery of genes and chemicals. In this paper, we report the expression of a codon-optimized HcRNAV 109 CP gene in Pichia pastoris and the production of self-assembled HcRNAV VLPs using large-scale fermentation. The HcRNAV 109 CP gene was synthesized according to the codon preference of P. pastoris and cloned into a pPICZA vector. The recombinant plasmid pPICZA-CPsyns was transformed into P. pastoris by electroporation. The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS polyacrylamide gel electrophoresis. After large-scale fermentation, the yield of HcRNAV CPsyns reached approximately 2.5 g L1 within 4 d. The HcRNAV VLPs were purified using PEG precipitation followed by cesium chloride density gradient ultracentrifugation, and were subsequently analyzed using UV spectrophotometry and transmission electron microscopy. Fluorescence dye-labeled myoglobin was loaded into the cages of the HcRNAV VLPs and the encapsulation was confirmed by fluorescence spectroscopy. The results point to the possible utilization in pharmacology or nanotechnology of HcRNAV VLPs produced by P. pastoris fermentation. - Harmful algal blooms (HABs), also known as red tides, have had a negative impact on aquatic ecosystems and have increasingly become a threat to human and marine health [1,2]. Rapid increases in an algal population can lead to water discoloration, shading of submerged vegetation, disruption of food-web dynamics and oxygen depletion in the water. HABs are known to have damaged the fishing industry, and to have affected shoreline quality and local economies. The potent neurotoxins can concentrate in filterfeeding shellfish and poison human consumers [35]. Even non-toxic algae can be harmful when they amass in sufficient numbers. HABs occur in many regions around the world, including Scandinavia, the North Pacific, the Caribbean and the South Pacific [6,7]. The toxic or harmful phytoplanktons that cause HABs are commonly dinoflagellates, such as Alexandrium and Karenia [8]. Most dinoflagellates have a unique structure that includes a nucleus known as the dinokaryon within which the chromosomes are attached to the nuclear membrane. Many efforts have been made to control harmful algal blooms with little success [9]. Today, the biological control of HABs is considered to be feasible [10]. Viruses that are abundant in marine systems replicate rapidly and tend to be host-specific, suggesting that single algal species The Author(s) 2012. This article is published with open access at Springerlink.com could be targeted [11,12]. Parasites also have the potential to control algal bloom species, but their specific role in this regard is largely unknown [13]. Heterocapsa circularisquama RNA virus (HcRNAV) is the first single-stranded RNA virus to be characterized that infects dinoflagellates [14]. H. circularisquama Horiguchi, first observed in Uranouchi Bay, Japan, is a harmful bloomcausing dinoflagellate that specifically kills bivalves [15]. HcRNAV particles are polyhedral with a diameter of approximately 30 nm, and encapsulate a single positive-stranded 4.4 kb RNA genome. Two open reading frames (ORF-1 and ORF-2) were identified in the genome, ORF-2 coding for the viral coat protein [16]. HcRNAV targets and accumulates in the dinoflagellate nucleus. The virus clones have been divided into types CY and UA (HcRNAV109 and HcRNAV34, respectively), based on their host strain specificity [17]. The methylotropic yeast, Pichia pastoris, has been shown to be a suitable system for the heterologous expression of virus coat protein, which could then self assemble into viruslike particles (VLPs) in vitro. The successful examples include cowpea chlorotic mottle virus (CCMV), hepatitis B virus (HBV), and bacteriophage Qbeta [1820]. Besides its ability to express foreign proteins at a high level, P. pastoris has been shown to have several advantages over other expression systems. The fermentation period was usually 4 5 d, compared to the plant hosts in which production took weeks [21]. The synthesized VLPs were soluble and able to self-assemble, while production in prokaryote hosts, such as E. coli, often results in insoluble inclusions [22]. In this paper, we report on the heterologous synthesis of HcRNAV 109 coat protein by P. pastoris. The successful large-scale fermentation and purification of the self-assembled HcRNAV VLPs suggest their potential application as nanocontainers. To our knowledge, this is the first paper to report the heterologous expression of HcRNAV. Materials and methods 1.1 Synthesis of P. pastoris codon-optimized HcRNAV The sequence of the HcRNAV 109 CP gene, GenBank accession [AB218609] [23], was redesigned to substitute amino acid codons that are seldom used in P. pastoris with those more frequently used [24]. Chemical synthesis of the new CP gene (CPsyns) was accomplished with GenScript, USA. Restriction sites for EcoR I (GAATTC) and Not I (GCGGCCGC) were added upstream and downstream, respectively, of the CPsyns gene. The plasmid pUC57-CPsyns was transformed into E. coli Top10 for amplification and preservation. 1.2 Construction of the recombinant vector pPICZA The P. pastoris host strain GS115 (his4, histidine-requiring auxotroph) and the intracellular expression vector pPICZA were purchased from Invitrogen, USA. For the in vivo expression in P. pastoris, the 1.1-kb CPsyns gene was retrieved from the pUC57 vector as an EcoR I/Not I fragment and cloned into the corresponding EcoR I and Not I sites of the Pichia integrative vector, pPICZA. The resultant vector pPICZA-CPsyns was then transformed into E. coli Top10 for its amplification. 1.3 Transformation of P. pastoris and cultivation of HCRNAV 109 CPsyns The transformation and expression of the CPsyns gene in P. pastoris was performed using established procedures [25]. The recombinant plasmid pPICZA-CPsyns was linearized with Sac I and subsequently used to transform P. pastoris GS115 by electroporation (Multiporator, Eppendorf, Germany). The transformed yeast cells were incubated in YPD agar containing Zeocin at 30C for 23 d. Be (...truncated)