• Journal of Microbiology and Biotechnology
• /
• 제18권2호
• /
• pp.383-391
• /
• 2008

The glycans linked to the insect cell-derived glycoproteins are known to differ from those expressed in mammalian cells, partly because of the low level or lack of glycosyltransferase activities. GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennarytype complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector. The glycosyltransferases, expressed as a fusion form with the IgG-binding domain, were secreted into the culture media and purified using IgG sepharose resin. The enzyme assay, performed using a pyridylaminated-sugar chain as an acceptor, indicated that the purified glycosyltransferases retained their enzyme activities. Human erythropoietin expressed in T. ni cells (rhEPO) was subjected to in vitro glycosylation by using recombinant glycosyltransferases and was converted into complex-type glycan with terminal sialic acid. The presence of Nacetylglucosamine, galactose, and sialic acid on the rhEPO moiety was detected by a lectin blot analysis, and the addition of galactose and sialic acid to rhEPO was confirmed by autoradiography using $UDP-^{14}C-Gal\;and\;CMP-^{14}C-Sia$ as donors. The in vitro glycosylated rhEPO was injected into mice, and the number of reticulocytes among the ed blood cells was counted using FACS. A significant increase in the number of reticulocytes was not observed in the mice injected with in vitro glycosylated rhEPO as compared with those injected with rhEPO.

• 한국미생물·생명공학회지
• /
• 제36권1호
• /
• pp.72-81
• /
• 2008

제니스테인의 미백제로서 응용 가능성을 알아보기 위해 멜라닌 생성에 미치는 영향을 in vitro 및 in vivo 실험을 통해 알아보았다. Melan-a 세포에 제니스테인을 처리하여 멜라닌 양을 측정한 결과 멜라닌 생성이 농도 의존적으로 감소되었다. 멜라닌 생성 억제가 tyrosinase의 활성 저해와 관련되는지를 확인해보고자 하였다. 그 결과 제니스테인은 tyrosinase에 직접 작용하여 활성을 저해하지는 않았으나, 세포내의 tyrosinase의 활성은 농도 의존적으로 저해하였다. 제니스테인이 tyrosinase의 발현에는 영향을 미치지 않았는데 이는 세포내 tyrosinase의 활성 저해가 발현과는 다른 기전에 의해 일어난다는 것을 의미한다. 그리고 제니스테인은 ${\alpha}$-glucosidase를 저해하였으며, 이를 통해 N-linked glycoprotein인 tyrosinase의 glycosylation을 저해하여 tyrosinase의 세포내 이동이나 활성을 억제할 수 있음을 알 수 있었다. 또한 제니스테인은 brown guinea pig에서 자외선에 의해 유도되는 피부흑화를 농도 의존적으로 개선하는 미백 효과가 있었다. 인공색소반에 제니스테인을 1%, 2% 도포한 결과 5주차에 대조군과 비교하여 유의적인 미백 효과를 확인할 수 있었다. 멜라닌에 대한 F-M 염색 결과를 살펴보면, 제니스테인 2%를 도포한 부위의 멜라닌 함량이 대조군 도포 부위에 비해 상당히 감소하였음을 확인할 수 있었다. 이상의 결과에서 제니스테인은 미백제로서 유용하게 활용할 가치가 있는 것으로 사료된다.

• 한국자원식물학회지
• /
• 제26권5호
• /
• pp.526-532
• /
• 2013

본 연구에서는 동해안에서 채집된 88종의 해조류에 대하여 미백제로서 응용 가능성을 알아보기 위해 멜라닌 생성에 미치는 영향을 in vitro 실험을 통해 알아보았다. Melan-a 세포에 해조류 추출물을 처리하여 멜라닌 양을 측정한 결과, 고리매, 미역쇠, 왜모자반, 감태, 톳추출물에서 멜라닌 생성이 감소되었으며, 특히 미역쇠추출물을 처리하였을 때 가장 유효하게 농도의존적으로 멜라닌 생성이 감소되었다. 멜라닌 생성 억제 효과가 tyrosinase 활성 저해와 관련 있는지를 확인한 결과, 약한 활성을 가지지만 직접 작용하여 활성을 저해하지는 않으면서, 세포 내의 tyrosinase 활성을 농도의존적으로 저해하였다. 이는 세포 내 tyrosinase 활성저해가 직접적이 아닌 다른 기전에 의해 일어난다는 것을 의미하며, 그 기전 중의 하나인 ${\alpha}$-glucosidase활성을 측정한 결과 농도의존적으로 활성을 저해하였으며, 세포 내 tyrosinase 활성 저해와 높은 상관도를 보임을 확인하였다. 즉 미역쇠추출물은 N-linked glycoprotein인 tyrosinase의 glycosylation을 억제하여 tyrosinase의 세포 내 이동이나 활성을 억제할 수 있음을 알 수 있었다. 결론적으로, 미역쇠추출물은 피부 미백에 유효한 활성을 나타내는 천연 유용자원으로 활용할 가치가 있는 것으로 사료된다.

• Journal of Microbiology and Biotechnology
• /
• 제13권5호
• /
• pp.710-716
• /
• 2003

Gonadotropin (GTH) is a pituitary glycoprotein hormone that regulates gonadal development in vertebrates. In teleosts, two types of gonadotropins, GTH-I and GTH-II, are produced in the pituitary, and they comprised of common ${\alpha}$ and distinct ${\beta}$ subunits. In the present study, the cDNAs encoding GTH ${\alpha}\;and\;GTH-II{\beta}$ subunits were cloned and sequenced from flounder (Paralichthys olivaceus) pituitary cDNA library. The nucleotide sequence of the a subunit was 619 bp long, encoding 124 amino acids, and that of the $GTH-II{\beta}$ subunit was 538 bp long, encoding 145 amino acids. GTH subunits had well conserved cysteines, when aligned with other members of the glycoprotein family. The ${\beta}$ subunit of gonadotropin II ($GTH-II{\beta}$) had a different N-linked glycosylation site. RT-PCR analysis showed an increase of GTH II mRNA levels in association with gonadal development, and also showed that the mRNA expression of the ${\alpha}$ subunit was detected only in tissues from pituitary glands.

• 한국식품영양과학회지
• /
• 제44권7호
• /
• pp.993-999
• /
• 2015

본 연구에서는 선정한 생약재 및 복합처방단의 ${\alpha}$-glucosidase 저해 활성을 알아보았으며, 이 방법이 미백 소재 스크리닝을 위한 유용한 평가법인지를 알아보았다. 한의학과 민간에서 당뇨의 개선 및 치료 효과가 우수하다고 알려진 생약재 및 처방 중 죽력, 귀전우, 적양, 연자육, 마인, 청심연자음의 ${\alpha}$-glucosidase 활성 저해 효과는 식후 혈당조절제인 acarbose와 비교하여 볼 때 우수한 효과를 나타내었다. 미백 효과가 알려진 연자육을 함유한 청심연자음 hydrolyzed EtOAc layer는 $100{\mu}g/mL$ 농도에서 약 50% 멜라닌 생성 저해 효과를 보였다. 또한 청심연자음 hydrolyzed EtOAc layer는 ${\alpha}$-glucosidase 활성 저해 효과가 우수하였으나 mushroom tyrosinase 활성 저해 효과는 나타나지 않았다. 이로써 청심연자음 hydrolyzed EtOAc layer는 ${\alpha}$-glucosidase 활성을 저해시켜 tyrosinase의 glycosylation을 저해함으로써 멜라닌 생성 억제 효과가 나타나는 것으로 생각된다. 이상의 결과로 볼 때 ${\alpha}$-glucosidase 활성 억제 효과가 있으면서 당뇨병에 효과가 있는 생약재들은 N-linked glycoprotein인 tyrosinase의 glycosylation을 저해하여 tyrosinase의 세포 내 이동이나 활성을 억제함으로써 멜라닌 생성을 억제할 것으로 사료되며, 본 연구에서 선정된 생약재들은 당뇨병 치료를 위한 목적뿐만 아니라 화장품에서 새로운 미백 소재로서의 활용가치가 있을 것으로 판단된다. 또한 미백에 효과가 있는 소재 스크리닝을 위해 현재 널리 사용되고 있는 mushroom tyrosinase 활성 저해 효과와 다른 접근 방법으로써 ${\alpha}$-glucosidase 활성 측정 방법도 하나의 평가법으로 유용할 것으로 생각된다.

• Journal of Microbiology and Biotechnology
• /
• 제19권10호
• /
• pp.1098-1102
• /
• 2009

Gamma-linolenic acid (GLA, C18:3 ${\Delta}^{6,9,12}$) is synthesized by a delta-6 fatty acid desaturase using linoleic acid (LA, C18:2 ${\Delta}^{9,12}$) as a substrate. To enable the production of GLA in the conventional yeast Pichia pastoris, we have isolated a cDNA encoding the delta-6 fatty acid desaturase from Cunninghamella echinulata MIAN6 and confirmed its function by heterogeneous expression in P. pastoris. Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,404 bp, which encodes a 52 kDa peptide of 468 amino acids. This sequence has 64% identity to the previously reported delta-6 fatty acid desaturase from Rhizopus oryzae. The polypeptide has a cytochrome b5 domain at the N-terminus including the HPGG motif in the heme-binding region, as reported for other delta-6 fatty acid desaturases. In addition, this enzyme differs from other desaturases by the presence of three possible N-linked glycosylation sites. Analysis of the fatty acid composition demonstrated the accumulation of GLA to the level of 3.1% of the total fatty acids. Notably, the amounts of ginkgolic acid (C17:1) and palmitic acid (C16:0) were increased from 1.3% to 29.6% and from 15% to 33%, respectively. These results reveal that the modification of the fatty acid biosynthetic pathway by genetic manipulation in order to produce specific polyunsaturated fatty acids in P. pastoris is a promising technique.

• BMB Reports
• /
• 제36권6호
• /
• pp.572-579
• /
• 2003

An expressed sequence tag (EST) library was established from the hypopharyngeal glands of Apis cerana. Sixty-six recombinant clones, possessing inserts >500 bp, were randomly selected and unidirectional sequenced. Forty-two of these (63.6%) were identified as homologues of Major Royal Jelly Proteins families 1, 2, 3, and 4 of A. mellifera (AmMRJP) for which MRJP1 was the most abundant family. The open-reading frame of the MRJP1 homologue (AcMRJP1) was 1299 nucleotides that encoded 433 deduced amino acids with three predicted N-linked glycosylation sites. The AcMRJP1 sequence showed 93% and 90% homologies with nucleotide and deduced amino acid sequences of AmMRJP1, respectively. Two complete transcripts of apisimin, and one and two partial transcripts of $\alpha$-glucosidase and glucose oxidase, were also isolated. In addition, the royal jelly proteins of A. cerana were purified and characterized using Q-Sepharose and Sephadex G-200 column chromatography. The native forms of protein peaks A1, A2, B1, and C1 were 115, 55, 50, and 300 kDa, respectively. SDS-PAGE analysis indicated that A1 and C1 were dimeric and oligomeric forms of the 80 kDa and 50 kDa subunits, respectively. The ratio of the total protein quantities of A1 : A2 : B1 : C1 were 2.52 : 4.72 : 1 : 12.21. Further characterization of each protein, using N-terminal and internal peptide sequencing, revealed that the respective proteins were homologues of MRJP3, MRJP2, MRJP1, and MRJP1 of A. mellifera.

• Journal of Microbiology and Biotechnology
• /
• 제28권9호
• /
• pp.1547-1553
• /
• 2018

Hyaluronidases are a family of enzymes that catalyse the breakdown of hyaluronic acid, which is abundant in the extracellular matrix and cumulus oocyte complex. To investigate the activity of recombinant bovine sperm hyaluronidase 1 (SPAM1) and determine the effect of the Asn-X-Ser/Thr motif on its activity, the bovine SPAM1 open reading frame was cloned into the mammalian expression vector pCXN2 and then transfected to the HEK293 cell line. Expression of recombinant bovine hyaluronidase was estimated using a hyaluronidase activity assay with gel electrophoresis. Recombinant hyaluronidase could resolve highly polymeric hyaluronic acid and also caused dispersal of the cumulus cell layer. Comparative analysis with respect to enzyme activity was carried out for the glycosylated and deglycosylated bovine sperm hyaluronidase by N-glycosidase F treatment. Finally, mutagenesis analysis revealed that among the five potential N-linked glycosylation sites, only three contributed to significant inhibition of hyaluronic activity. Recombinant bovine SPAM1 has hyaluronan degradation and cumulus oocyte complex dispersion ability, and the N-linked oligosaccharides are important for enzyme activity, providing a foundation for the commercialization of hyaluronidase.

• 생명과학회지
• /
• 제17권9호통권89호
• /
• pp.1248-1254
• /
• 2007

Kluyveromyces marxianus 유래의 exoinulinase (INU1) 분비 서열과 GAL10 promoter를 이용하여 Clostridium thermocellum endoglucanase A gene (celA)의 과발현과 분비 성능 검증연구를 수행하였다. 자체 분비 서열을 가지는 pYEG- CT1 과 INU1 분비 서열을 가지는 pYInu-CT1, 두개의 plasmid는 S. cer-evisiae SEY2102와 S. cerevisiae 2805에 각각 형질전환시켜 ur-acil이 결핍된 배지에서 선별하였다. celA gene 자체 분비 서열보다 INU1 분비 서열을 사용했을 때 발현량과 분비효율은 각각 약 $18{\sim}22%$ 와 11% 향상되었다. 이 중 361 unit/l의 발현율과 89%의 plasmid 안정성, 그리고 70%의 분비효율을 보인 S. cerevisiae 2805/pYinu-CT1 효모 형질전환체를 cellolose를 효과적으로 분해하는 재조합 효모 생균체로 선별하였다. Galactose 배지내에서 S. cerevisiae 2805/ pYInu-CT1의 발효조 회분배양 결과, 총발현량과 분비효율은 각각 418 unit/l 와 73% 로 나타났다. 또한 분비된 endoglucanase A는 분자량 100kDa 이상에서 활성 밴드를 보여, N-linked 당쇄부가에 의해 상당한 비율의 당쇄가 부가됨을 추측할 수 있었다.

• Journal of Microbiology and Biotechnology
• /
• 제13권6호
• /
• pp.926-936
• /
• 2003

Overexpression of human Bcl-2 protein in recombinant Chinese hamster ovary (rCHO) cells producing humanized antibody (SH2-0.32) considerably suppressed sodium butyrate (NaBu)-induced apoptosis during batch culture by using commercially available serum-free medium, which extended the culture longevity. Due to the extended culture longevity provided by the anti-apoptotic effect of Bcl-2 overexpression, the final antibody concentration of 14C6-bcl-2 culture (Bcl-2 high producer, $23\;\mu\textrm{g}\;ml^{-1}$) was 2 times higher than that of the $SH2-0.32-{\Delta}bcl-2$ culture (cells transfected with bcl-2-deficient plasmid, $10.5\;\mu\textrm{g}\;ml^{-1}$) in the presence of NaBu. To determine the effect of NaBu/Bcl-2 overexpression on the molecular integrity of protein products, antibodies purified from 14C6-bcl-2 and $SH2-0.32-{\Delta}bcl-2$ cultures in the presence of NaBu were characterized by using various molecular assay systems. For comparison, antibody purified from the parental rCHO cell culture (SH2-0.32) in the absence of NaBu was also characterized. No significant changes in molecular weight of antibodies could be observed by SDS-PAGE. From GlycoSep-N column analysis, it was found that the core oligosaccharide structure ($GlcNAc_2Man_3GlcNAc_2$) was not affected by NaBu/Bcl-2 overexpression, while the microheterogeneity of N-linked oligosaccharide structure was slightly affected. Compared with the antibody produced in the absence of NaBu, the proportion of neutral oligosaccharides was increased from 10% (14C6-bcl-2) to 16% ($SH2-0.32-{\Delta}bcl-2$) in the presence of NaBu, which was accompanied by the reduced proportion of acidic oligosaccharides, especially of monosialylated and disialylated forms. The changes in microheterogeneous oligoformal structures of antibody in turn affected the mobility of antibody isoforms in isoelectric focusing (IEF), resulting in the occurrence of some more basic antibody isoforms produced in the presence of NaBu. However, the antigen-antibody binding properties were not changed by alteration of glycosylation pattern. The competitive enzyme-linked immunosorbent assay (ELISA) showed that the antibody produced by NaBu/Bcl-2 overexpression maintained its antigen-antibody binding properties with binding affinity of about $2.5{\times}10^9{\;}M^{-1}$. Taken together, no significant effects of NaBu/Bcl-2 overexpression on the molecular integrity of antibodies, produced by using serum-free medium, could be observed by the molecular assay systems.