• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.183-187
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• 2008

Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. $VEGF_{165}$ is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human $VEGF_{165}$ $(rhVEGF_{165})$ protein. The production rate of the established CHO cells was over 80mg/l of $rhVEGF_{165}$ protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The $rhVEGF_{165}$ protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified $rhVEGF_{165}$ protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the $rhVEGF_{165}$ protein in CHO cells differed from that in insect cells. The purified $rhVEGF_{165}$ protein in this study was functionally active with a half-maximal effective concentration of 3.8ng/ml and specific activity of $2.5{\times}10^5U/mg$.

• Biomedical Science Letters
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• v.12 no.2
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• pp.81-89
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• 2006

The ischemia-responsive 94 gene (irp94) encoding a 94 kDa endoplasmic reticulum resident protein was investigated its molecular properties associated with unfoled protein responses. First, the expression of irp94 mRNA was tested after the reperfusion of the transient forebrain ischemia induction at the central nervous system in three Mongolian gerbils. Second, irp94 expression in PC12 cells, which are derived from transplantable rat pheochromocytoma cultured in the DMEM media, was tested at transcriptional and translational levels. The half life of irp94 mRNA was also determined In PC12 cells. Last, the changes of irp94 mRNA expression were investigated by the addition of various ER stress inducible chemicals (A23187, BFA, tunicamycin, DTT and $H_2O_2$) and proteasome inhibitors, and heat shock. High level expression of irp94 mRNA was detected after 3 hours reperfusion in the both sites of the cerebral cortex and hippocampus of the gerbil brain. The main regulation of irp94 mRNA expression in PC 12 cells was determined at the transcriptional level. The half life of irp94 mRNA in PC12 cells was approximately 5 hours after the initial translation. The remarkable expression of irp94 mRNA was detected by the treatment of tunicamycin, which blocks glycosylation of newly synthesized polypeptides, and $H_2O_2$, which induces apoptosis. When PC12 cells were treated with the cytosol proteasome inhibitors such as ALLN (N-acetyl-leucyl-norleucinal) and MG 132 (methylguanidine), irp94 mRNA expression was increased. These results indicate that expression of irp94 was induced by ER stress including oxidation condition and glycosylation blocking in proteins. Expression of irp94 was increased when the cells were chased after heat shock, suggesting that irp94 may be involved in recovery rather than protection against ER stresses. In addition, irp94 expression was remarkably increased when cytosol proteasomes were inhibited by ALLN and MG 132, suggesting that irp94 plays an important role for maintaining the ERAD (endoplasmic reticulum associated degradation) function.

• International Journal of Industrial Entomology and Biomaterials
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• v.30 no.2
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• pp.50-57
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• 2015

Porcine circovirus type 2 (PCV2) is an important infectious swine virus causing postweaning multisystemic wasting syndrome (PMWS). PCV2 capsid protein, encoded by ORF2 has type-specific epitopes, is very immunogenic, and is associated with the induction of neutralizing antibodies. For the efficient production of capsid protein, recombinant Autographa californica nucleopolyhedroviruses were generated to express ORF2 fused with two forms of a partial polyhedrin. Recombinant capsid protein was produced successfully with the partial polyhedrin fusion form and the yield was high, as was shown by SDS-PAGE. Production of recombinant capsid proteins in insect cells was confirmed by Western blot analysis using anti-His monoclonal antibody, anti-ORF2 monoclonal antibody, and anti-PCV2 porcine serum. Fusion expression with amino acids 19 to 110 of the polyhedrin increased the production of recombinant capsid protein, but fusion with amino acids 32 to 85 did not. Additionally, PCV2 capsid protein is a glycoprotein; however, the glycosylation of recombinant protein was not observed. The results of an Enzyme-linked immunosorbent assay (ELISA) showed that recombinant capsid proteins could be utilized as antigens for fast, large-scale diagnosis of PCV2-infected pigs. Our results suggest that the fusion expression of partial polyhedrin is able to increase the production of recombinant PCV2 capsid protein in insect cells.

• BMB Reports
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• v.35 no.6
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• pp.595-603
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• 2002

A gene that encodes a homologue to baculoviral ODVP-6E/ODV-E56, a baculoviral envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). The ChfuGV odvp-6e/odv-e56 gene was located on an 11-kb BamHI subgenomic fragment using different sets of degenerated primers, which were designed using the results of the protein sequencing of a major 39 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1062 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 353 amino acids with a predicated molecular mass of 38.5 kDa. The amino acid sequence data that was derived from the nucleotide sequence in ChfuGV was compared to those of other baculoviruses. ChfuGV ODVP-6E/ODV-E56, along with othe baculoviral ODVP-6E/ODV-E56 proteins, all contained two putative transmembrane domains at their C-terminus. Several putative N-and O-glycosylation, N-myristoylation, and phosphorylation sites were detected in the ChfuGV ODVP-6E/ODV-E56 protein. A similar pattern was detected when a hydrophobicity-plots comparison was performed on ChfuGV ODVP-6E/ODV-E56 with other baculoviral homologue proteins. At the nucleotide level, a late promoter motif (GTAAG) was located at -14 nt upstream to the start codon of the GhfuGV odvp-6e/odv-e56 gene. a slight variant of the polyadenylation signal, AATAAT, was detected at the position +10 nt that is downstream from the termination signal. A phylogenetic tree for baculoviral ODVP-6E/ODV-E56 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV ODVP-6E/ODV-E56 is most closely related to those of Cydia pomonella granulovirus (CpGV) and Plutella xylostella granulovirus (PxGV).

• BMB Reports
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• v.48 no.1
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• pp.1-5
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• 2015

Transient Receptor Potential, Melastatin-related, member 4 (TRPM4) channels are $Ca^{2+}$-activated $Ca^{2+}$-impermeable cation channels. These channels are expressed in various types of mammalian tissues including the brain and are implicated in many diverse physiological and pathophysiological conditions. In the past several years, the trafficking processes and regulatory mechanism of these channels and their interacting proteins have been uncovered. Here in this minireview, we summarize the current understanding of the trafficking mechanism of TRPM4 channels on the plasma membrane as well as heteromeric complex formation via protein interactions. We also describe physiological implications of protein-TRPM4 interactions and suggest TRPM4 channels as therapeutic targets in many related diseases.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1397-1401
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• 2005

Bone morphogenetic protein-4 (BMP-4) is a signaling homodimeric molecule that acts as a morphogen to influence cell fate in a concentration-dependent manner. The limited supply of a pure preparation of BMP-4, due to very low level of their expression in vivo, makes it difficult not only to study the biological activities of BMPs, but also to use them as a clinical tool. For a large-scale production of BMP-4, human BMP-4 cDNA was expressed in Chinese hamster ovary (CHO) cells by a recently development vector system, which confers position-independent stable expression of the foreign genes. The CHO cell line expressing recombinant human BMP-4 (rhBMP-4) at the level of $7\;{\mu}g/ml$ could be obtained after stepwise selection with methotrexate. This level of expression is about 70 times higher than those previously reported. The partially processed form of BMP-4 as well as mature form could be detected, when the aliquots of culture media were analyzed by Western blot. The glycosylation pattern and biological activity of the rhBMP-4 were determined by glycosidase treatment and the induction rate of alkaline phosphatase in mouse osteoblastic cells.

• Korean Journal of Veterinary Research
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• v.43 no.3
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• pp.439-448
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• 2003

The VP2 gene of infectious bursal disease virus (IBDV) Chinju which was previously detected in Chinju, Korea was cloned and sequenced to establish the information for the development of genetically engineered vaccines and diagnostic reagents against IBDV. The nucleotide sequence of the entire Chinju VP2 gene consisted of 1,356 bases long encoding 452 amino acids in a single open reading frame (ORF). It consisted of 368 adenine (27.1%), 363 cytosine (26.8%), 339 guanine (25.0%) and 286 thymine (21.1%) residues. The predicted $M_r$ of the Chinju VP2 protein was 48 kDa, and the protein contained 13 phosphorylation sites by protein kinase C, casein kinase II or tyrosine kinase, whereas 3 asparagine-linked glycosylation sites were recognized. The nucleotide sequence of Chinju VP2 ORF had a very close phylogenetic relationship with 98-99% homology to that of the very virulent IBDVs (vvIBDVs) HK46, OKYM, D6948, UK661, UPM97/61 and BD3/99. Also, the Chinju VP2 protein revealed a very close phylogenetic relationship with 99-100% homology to that of these vvIBDVs. The Chinju VP2 protein had 100% amino acid identity in the variable region of residues 206-360 with that of the D6948, HK46, OKYM and UK661, as well as 100% identity in two hypervariable regions of residues 212-224 and 314-324 with those of the D6948, HK46, OKYM, UK661, UPM97/61 and BD3/99. The amino acid sequence of the chinju VP2 protein contained a serine-rich heptapeptide of SWSASGS as in these vvIBDVs.

• Journal of Ginseng Research
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• v.44 no.5
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• pp.680-689
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• 2020

Background: Peptides have diverse and important physiological roles in plants and are ideal markers for species identification. It is unclear whether there are specific peptides in Panax quinquefolius L. (PQ). The aims of this study were to identify Quinetides, a series of diverse posttranslational modified native peptides of the ribonuclease-like storage protein (ginseng major protein), from PQ to explore novel peptide markers and develop a new method to distinguish PQ from Panax ginseng. Methods: We used different fragmentation modes in the LTQ Orbitrap analysis to identify the enriched Quinetide targets of PQ, and we discovered Quinetide markers of PQ and P. ginseng using ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. These "peptide markers" were validated by simultaneously monitoring Rf and F11 as standard ginsenosides. Results: We discovered 100 Quinetides of PQ with various post-translational modifications (PTMs), including a series of glycopeptides, all of which originated from the protein ginseng major protein. We effectively distinguished PQ from P. ginseng using new "peptide markers." Four unique peptides (Quinetides TP6 and TP7 as markers of PQ and Quinetides TP8 and TP9 as markers of P. ginseng) and their associated glycosylation products were discovered in PQ and P. ginseng. Conclusion: We provide specific information on PQ peptides and propose the clinical application of peptide markers to distinguish PQ from P. ginseng.

• Journal of Life Science
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• v.13 no.6
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• pp.805-813
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• 2003

cDNA encoding somatolactin (SL) was obtained by RT-PCR from pituitary glands of rock bream (Oplegnathus fasciatus). The full length cDNA of rock bream somatolactin (rbSL) is 1636 bp long. It contains a 696 bp open reading frame encoding a signal peptide of 24 amino acids (an) and a mature protein of 207 aa. rbSL has seven cysteine residues$(Cys^{5},\; Cys^{15},\; Cys^{42},\; Cys^{65},\; Cys^{181},\; Cys^{198}\; $and $Cys^{206})$ and two potential N-glycosylation sites at positions $Asn^{121}$and $Asn^{153}$. The rbSL shares 61.1∼92.6% amino acid sequence similarities and 63∼92.6% nucleotide sequence identities with other teleost SLs, except for goldfish and channel catfish SL. Amino acid sequence alignment revealed that rbSL has four conserved domains $(A_{SL},\; B_{SL},\; C_{SL}and\; D_{SL})$ common to all SLs. Out of these domains, $(A_{SL},\; B_{SL},\; C_{SL}and\; D_{SL})$, are also conserved in all teleost growth hormones and prolactins. The cDNA of rbSL has been cloned into pET expression vector in order to produce recombinant rbSL in E. coli BL2l(DE3) cells. The recombinant protein showed a molecular weight of 27 kDa in SDS-PAGE.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.96-103
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• 2010

Oligosaccharyltransferase (OTase) catalyzes the transfer of a lipid-linked oligosaccharide (LLO) to the nascent polypeptide. Most eukaryotes have an OTase composed of a multisubunit protein complex. However, the kinetoplastid Leishmania major and the bacterium Campylobacter jejuni have only a single subunit for OTase activity, Stt3p and PglB, respectively. In this study, a new in vitro assay for OTase was developed by using a fluorescent peptide containing N-glycosylation sequon, Asn-Xaa-Thr/Ser, where Xaa can be any amino acid residue except Pro. L. major Stt3p and C. jejuni PglB as a model OTase enzyme demonstrated the formation of glycopeptides from a fluorescent peptide through OTase activities. For separation and measurement of the glycopeptides produced by the OTases, Tricine-SDS-PAGE, a lectin column and fluorospectrophotometer, and HPLC were applied. Comparison of these assay methods for analyzing a fluorescent glycopeptide showed HPLC analysis is the best method for separation of glycopeptides and nonglycosylated peptides as well as for quantify the peptides than other methods.