• Journal of Microbiology and Biotechnology
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• v.26 no.7
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• pp.1163-1172
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• 2016

The Ω-loop is a nonregular and flexible structure that plays an important role in molecular recognition, protein folding, and thermostability. In the present study, molecular dynamics simulation was carried out to assess the molecular stability and flexibility profile of the porcine trypsin structures. Two Ω-Loops (fragment 57-67 and fragment 78-91) were confirmed to represent the flexible region. Subsequently, glycosylation site-directed mutations (A73S, N84S, and R104S) were introduced within the Ω-loop region and its wing chain based on its potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences) and structure information to improve the thermostability of trypsin. The result demonstrated that the half-life of the N84S mutant at 50℃ increased by 177.89 min when compared with that of the wild-type enzyme. Furthermore, the significant increase in the thermal stability of the N84S mutant has also been proven by an increase in the T_m values determined by circular dichroism. Additionally, the optimum temperatures of the wild-type enzyme and the N84S mutant were 75℃ and 80℃, respectively. In conclusion, we obtained the thermostability-improved enzyme N84S mutant, and the strategy used to design this mutant based on its structural information and N-linked glycosylation modification could be applied to engineer other enzymes to meet the needs of the biotechnological industry.

• International Journal of Industrial Entomology and Biomaterials
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• v.36 no.1
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• pp.15-24
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• 2018

N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (${\alpha}1AGP$) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the ${\alpha}1AGP$ to be a better model for studying glycosylation. The modified ${\alpha}1AGP$ (${\alpha}1AGP{\Delta}$) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the ${\alpha}1AGP$. Subsequently, we confirmed the detailed profile of N-glycan on the ${\alpha}1AGP{\Delta}$ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant ${\alpha}1AGP{\Delta}$ could be usable as a better model glycoprotein of N-glycosylation research in BES.

• Journal of Life Science
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• v.20 no.3
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• pp.365-373
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• 2010

To monitor newly emerged influenza virus variants and to investigate the prevalence pattern, our laboratory performed isolation of the viruses from surveillance sentinel hospitals. In the present study, we analysed influenza A/H1N1, A/H3N2, B viruses isolated in Busan during the 2006/07 and 2007/08 seasons by sequence analysis of the hemagglutinin (HA1 subunit) and neuraminidase (NA) genes. The isolates studied here were selected by the stratified random sample method from a total of 277 isolates, in which 15 were A/H1N1, 16 were A/H3N2 and 29 were B. Based on the phylogenetic tree, the HA1 gene showed that A/H1N1 isolates had a 96.7% to 97.7% homology with the A/Brisbane/59/2007, A/H3N2 isolates had a 98.4% to 99.7% homology with the A/Brisbane/10/2007, and B isolates had a 96.5% to 99.7% homology with the B/Florida/4/2006(Yamagata lineage), which are all the vaccine strains for the Northern Hemisphere in 2008~2009 season. In the case of the NA gene, A/H1N1 isolates had 97.8% to 98.5% homologies, A/H3N2 isolates had 98.9% to 99.4% homologies, and B isolates had 98.9% to 100% homologies with each vaccine strain in the 2008~2009 season, respectively. Characterization of the hemagglutinin gene revealed that amino acids at the receptor-binding site and N-linked glycosylation site were highly conserved. These results provide useful information for the control of influenza viruses in Busan and for a better understanding of vaccine strain selection.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.143-144
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.143-144
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• 2005

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.381-388
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• 2001

The present study was designed to obtain porcine growth hormone binding protein (pGHBP) improved biological activation as derived mutation in binding site with growth horlnone (GH). A 756 bp of fragment encoding the extracellular domain of pGHBP gene was cloned from the total RNA of porcine fat tissue by reverse transcriptase polymerase chain reaction (RT-PCR) and created mutation in positions 26 and 122 using site-directed mutagenesis method. Position 26 is one and it is near to get on five potential N-linked glycosylation sites located in the extracellular domain of porcine growth hormone receptor known to have a direct influence on combination with GH. Position 122 is known as one of conformational epitope in bovine. It was over-expressed in E. coli using pET-32(c) expression vector and precisely purified by S-protein agarose and enterokinase. In our results, we was obtained pmGHBP of 30 kDa. It suggests to study the effects of the pmGHBP on cell proliferation in vitro and growth rate in vivo after administration.

• Journal of the Korean Wood Science and Technology
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• v.33 no.4 s.132
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• pp.45-52
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• 2005

The manganese peroxidase (mnp5) from white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. The majority of the rMnP5 (recombinant MnP5) produced by P. pastoris exhibited an approximate molecular mass 45 kDa considerably larger than that of the predicting mnp5 due to two glycosylation sites of mnp5. After site direct mutation treatment, the effect of N-linked hyperglycosylation was examined by enzyme activity. Analysis by sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with a molecular mass of 37 kDa. Enzyme activity of M-rMnP5 (mutant recombinant MnP5) was similar to that of rMnP5, indicating that hyperglycosylation did not affect the active site. In this work, active mnp5 was successfully expressed in P. pastoris, suggesting that P. pastoris has potential capability of producing active heme-containing proteins.

• Journal of Life Science
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• v.17 no.11
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• pp.1497-1504
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• 2007

To investigate the function and secretion of human thrombopoietin (TPO) in mammalian cells, hTPO cDNA was cloned using human liver cDNA, and recombinant hTPO (rec-hTPO) was produced in CHO cell lines. In addition, six N-linked glycosylation sites were substituted for Ala to elucidate the role of each carbohydrate chain. To analyze the biological activity, rec-hTPO protein was injected subcutaneously. Blood was withdrawn for platelet determination. The metabolic clearance rate (MCR) was also analyzed at the 1, 4, 10 and 24 hr after tail vein injection. Wild-type TPO (WT) was efficiently secreted into the medium. However, a hTPO mutant with 116 deleted nucleotides detected by PCR cloning was not secreted. The N-linked glycosylation sites had nearly the same expression quantity as rec-hTPO WT apart from mutants 3 and 4. The glycosylation site of mutant 4 appeared to be an indispensable site for hTPO secretion. Also characterized was the biological activity through an injection with rec-hTPO (10 ng) to ICR mice (7 weeks). The result of the blood analysis showed a considerable increase in the platelet number six days after He injection. To analyze the pharmacokinetics, rec-hTPO was injected into the tail vein (5 ng). The result was 200 pg/ml 1hr after this injection. Following this, it dramatically decreased and virtually disappeared 10 hours after the injection. Thus, rec-hTPO may be a treatment for thrombopenia by the production of the high active rec-hTPO. In addition, hTPO can permit the development of potent new analogues that stimulate the platelet value.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.150-155
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• 1995

Carboxypeptidase Z(CPZ) cDNA of Absidia zychae was experssed in Saccharomyces cerevisiae. The expressed CPZ(YCPZ) was secreted about 30 mg/l into the medium and has a little higher molecular weight than the wild type CPZ in SDS-PAGE. By the result of N-terminal amino acid sequencing, YCPZ has additional 15 amino acids residues in N-terminus of CPZ. But YCPZ shows no difference with CPZ in enzyme activity and substrate specificity. For the identification of processing mechanism of YCPZ, 36-Arg was changed to 36-Thr by site specific mutagenesis. Mutant YCPZ does not processed at 36-Thr. It was, therefore, concluded that the YCPZ was processed by KEX2. According to endo F treatment, high amount of carbohydrate was N-glycosylated in YCPZ.

• Molecules and Cells
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• v.38 no.6
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• pp.496-505
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• 2015

A variant peak was detected in the analysis of RP-HPLC of rHu-EPO, which has about 7% relative content. Fractions of the main and the variant peaks were pooled separately and further analyzed to identify the molecular structure of the variant peak. Total mass analysis for each peak fraction using ESI-TOF MS shows differences in molecular mass. The fraction of the main peak tends to result in higher molecular masses than the fraction of the variant. The detected masses for the variant are about 600-1000 Da smaller than those for the main peak. Peptide mapping analysis for each peak fraction using Asp-N and Glu-C shows differences in O-glycopeptide profiles at Ser126. The O-glycopeptides were not detected in the fraction of the variant. It is concluded that the variant peak is non-O-glycosylated rHu-EPO and the main peak is fully O-glycosylated rHu-EPO at Ser126.