Papers by Janis Freivalds
LU Biomedicīnas pētījumu un studiju centrs Rātsupītes iela 1, Rīga, LV-1067 E-pasts: andris@biome... more LU Biomedicīnas pētījumu un studiju centrs Rātsupītes iela 1, Rīga, LV-1067 E-pasts: [email protected] Ar gēnu inženierijas metodēm tika konstruēta virkne himēru B hepatīta vīrusa nukleokapsīda proteīna (HBc) gēnu, kuri ietvēra tumorspecifiskā MAGE-3 antigēna epitopu. Ekspresējot šos gēnus Escherichia coli šūnās, tikai viens no septiņiem himērajiem HBc-MAGE proteīniem saglabāja spēju veidot vīrusveidīgas daļiņas (virus-like particles, VLP). Himērās VLP tika attīrītas no E. coli proteīniem, lai inkorporētu to sastāvā imūnstimulējošus oligonukleotīdus. Šādas struktūras varētu tikt izmantotas kā antitumoru vakcīnas. Atslēgvārdi: vīrusveidīgas daļiņas, B hepatīta vīrusa nukleokapsīds, CTL epitopi, vakcīnas.
Protein Expression and Purification, 2011
Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are rou... more Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163 g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0 mg per 1 g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast.
Molecular Biotechnology, 2014
Virus-like particles (VLPs) generated by heterologous expression of viral structural genes have b... more Virus-like particles (VLPs) generated by heterologous expression of viral structural genes have become powerful tools in vaccine development. Recently, we and others have reported on the assembly of VLPs of the RNA bacteriophages MS2, Qβ, and GA in yeast. Here, we investigate the formation of VLPs of five additional phages in the yeasts Saccharomyces cerevisiae and Pichia pastoris, namely, the coliphages SP and fr, Acinetobacter phage AP205, Pseudomonas phage PP7, and Caulobacter phage φCb5. In all cases except SP, particle formation was detected, although VLP outcome varied from 0.2 to 8 mg from 1 g of wet cells. We have found that phage φCb5 VLPs easily dissociate into coat protein dimers when applied to strong anion exchangers. Upon salt removal and the addition of nucleic acid or its mimics and calcium ions, the dimers re-assemble into VLPs with high efficiency. A variety of compounds, including RNA, DNA, and gold nanoparticles can be packaged inside φCb5 VLPs. The ease with which phage φCb5 coat protein dimers can be purified in high quantities and re-assembled into VLPs makes them attractive for downstream applications including the internal packaging of nanomaterials and the chemical coupling of peptides of interest on the surface.
Journal of Biotechnology, 2006
Recombinant bacteriophage Q coat protein (CP), which has been proposed as a promising carrier of... more Recombinant bacteriophage Q coat protein (CP), which has been proposed as a promising carrier of foreign epitopes via their incorporation either by gene engineering techniques or by chemical coupling, efficiently self-assembles into virus-like particles (VLPs) when expressed in Escherichia coli. Here, we demonstrate expression and self-assembly of Q CP in yeast Saccharomyces cerevisiae and Pichia pastoris. Production reached 3-4 mg/1 g of wet cells for S. cerevisiae and 4-6 mg for P. pastoris, which was about 15-20% and 20-30% of the E. coli expression level, respectively. Q VLPs were easily purified by size-exclusion chromatography in both cases and contained nucleic acid, shown by native agarose gel electrophoresis. The obtained particles were highly immunogenic in mice and the resulting sera recognized both E. coli-and yeast-derived Q VLPs equally well.
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Papers by Janis Freivalds