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

Abstract

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 L 1 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. Heterocapsa circularisquama

Construction of target‐specific virus‐like particles for the delivery of algicidal compounds to harmful algae

“…Although a native‐like recombinant HcRNAV34 capsid protein was obtained herein from an E. coli overexpression system by thiol‐induced cleavage of the intein fusion protein, the observed particle size of the in vitro ‐assembled VLP (∼ 23–25 nm in diameter) was substantially smaller than that of the capsid shell of the authentic HcRNAV34 virion (Miller et al ., ). The self‐assembled VLP was also smaller than the in vitro assembly products of the HcRNAV109 (Wu et al ., ) and HcRNAV34 (Wada et al ., ) capsid proteins, which are ∼ 34 nm in diameter. The difference in size may reflect various morphological forms that a virion may adopt during the maturation, in which conformational changes of the capsid protein and the rearrangement of the capsomers by altered intermolecular interaction may take place (Salunke et al ., ; Hyun et al ., ).…”

“…Previously, the capsid protein of HcRNAV was successfully expressed in yeast and self‐assembled into VLPs in vitro (Wu et al ., ). In addition, we recently developed the potent algicidal compound, thiazolidinedione 49 (TD49) (Kim et al ., ).…”

Highly secretory expression of recombinant cowpea chlorotic mottle virus capsid proteins in Pichia pastoris and in-vitro encapsulation of ruthenium nanoparticles for catalysis

“…Therefore, several heterologous expression systems have been designed to overcome those disadvantages. Of these, eukaryotic yeast-based systems (e.g., Pichia pastoris [P. pastoris]) for expressing CCMV CPs have attracted much attention in recent years [18][19][20][21]. Yeast cells have advantages similar to those of prokaryotic cells, such as convenient genetic manipulation, low cost, rapid growth rate, and high cell density for protein expression.…”

Omics in marine biotechnology

“…This topic contains 12 papers produced by research groups from China, Germany, and Korea. To help readers, we have arranged the papers according to their research topics: genomics [1-3]; transcriptomics [4]; proteomics [5][6][7][8][9]; metabolomics [10,11], and nutrigenomics [12].The objective of this topic is to offer a platform for marine biological scientists to present recent advances in the discovery and development of omics in marine biotechnology, providing a large-scale, holistic approach to understanding life in encapsulated "omes". In this special topic, several genomes, including major histocompatibility complex genes [2], phytochrome genes [3] and immune-and antiviral-related genes [4], have been systematically investigated based on functional genomic studies, providing targets for further structural and functional analyses.…”

“…This topic contains 12 papers produced by research groups from China, Germany, and Korea. To help readers, we have arranged the papers according to their research topics: genomics [1-3]; transcriptomics [4]; proteomics [5][6][7][8][9]; metabolomics [10,11], and nutrigenomics [12].…”