• KSBB Journal
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• 제13권1호
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• pp.83-89
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• 1998

Cellulose로 만들어진 다공성 미립담체를 이용하여 HeLa cell을 working volume 100mL의 spinner flask에서 배양하였으며, 세포가 완전히 자란 미립담제를 계대배양하기 위하여 담체간 세포 전이 배양 방법을 시도하였다. 부유세포의 농도는 다공성 미립담체-HeLa 시스템의 경우에 담체간 세포 전이 배양에 영향을 미치는 중요한 인자로 작용하였으며, 낮은 칼슘농도의 배지인 RPMI-1640과 빠른 교반 속도를 이용하여 활성을 유지한 많은 세포가 떨어지도록 유도하였으며, 담체간 세포 전이 배양을 효과적으로 3회 이상 실시할 수 있었다. 이렇게 배양한 세포에 재 조합 Vaccinia virus를 감염하여 그 수율을 비교한 결과 T-flask에서 떼어낸 세포로 접종한 미립담체 배양과 거의 비슷한 재조합 단백질($\beta$-galactosidase) 수율을 나타내었다. Trypsin 처리 방법에 의한 미립담체 계대 배양도 경우에 따라서는 유용한 미립담체 계대 배양 방법이 될 수 있지만 실제 생산 규모에의 적용에는 공정이 복잡해지고 정확한 제어가 필요하다는 등의 문제가 있다. 따라서, 추가적인 비용이나 공정이 필요 없는 간편한 방법인 담체간 세포 전이 배양은 동물세포 배양을 이용한 유용 단백질 및 바이러스 생산 공정의 규모 증대에 매우 유용한 수단이다.

• Asian-Australasian Journal of Animal Sciences
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• 제24권2호
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• pp.230-238
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• 2011

Effects of cooling and supplemental recombinant bovine somatotropin (rbST) on hemato-biochemical characteristics were studied at different stages of lactation of crossbred Holstein Friesian cows in a tropical environment. Ten primiparous cows were divided into two groups of five animals each. The first group was housed as the non-cooled animals in an open-sided barn with a tiled roof in a normal shaded house (NS), while the second group was housed as cooled cows in an open-sided barn with a tiled roof under misty fan cooling (MFC). Three injections with rbST (500 mg per dose) at each stage of lactation (early, mid and late lactation) significantly increased total milk yield as compared with pretreatment in both cooled and non-cooled cows. Milk fat was significantly increased, while total solids, solid not fat, milk protein and lactose were not affected by the rbST treatment. Hematological parameters, plasma proteins, albumin, glucose, triglyceride, cholesterol, creatinine, alkaline phosphatase (ALP), plasma inorganic phosphate and the activities of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were not affected by supplemental rbST in cooled and non-cooled cows. Supplementation of rbST caused a significant decrease in plasma urea concentration, while plasma FFA concentrations significantly increased in both cooled and non-cooled cows. The results of the present study suggest that exogenous rbST is efficacious in increasing milk yield without adverse effects on lactating crossbred Holstein cows in a tropical environment.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.542-549
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• 2010

In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin $\beta$-subunit gene (ApCpnB, 1,665 bp ORF) from the hyperthermophilic archaeon A. pernix K1 was amplified by PCR and subcloned into vector pET21a. The constructed pET21a-ApCpnB (6.9 kb) was transformed into E. coli BL21 Codonplus (DE3). The transformant cell successfully expressed ApCpnB, and the expression of ApCpnB (61.2 kDa) was identified through analysis of the fractions by SDS-PAGE (14% gel). The recombinant ApCpnB was purified to higher than 94% by using heat-shock treatment at $90^{\circ}C$ for 20 min and fast protein liquid chromatography on a HiTrap Q column step. The purified ApCpnB showed ATPase activity and its activity was dependent on temperature. In the presence of ATP, ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}$ and $50^{\circ}$, respectively. Specifically, the activity of malate dehydrogenase (MDH) at $85^{\circ}$ was greatly stabilized by the addition of ApCpnB and ATP. Coexpression of pro-carboxypeptidase B (pro-CPB) and ApCpnB in E. coli BL21 Codonplus (DE3) had a marked effect on the yield of pro-CPB as a soluble and active form, speculating that ApCpnB facilitates the correct folding of pro-CPB. These results suggest that ApCpnB has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1726-1736
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• 2013

Biosynthesis of indole-3-acetic acid (IAA) via the indole-3-pyruvic acid pathway involves three kinds of enzymes; aminotransferase encoded by aspC, indole-3-pyruvic acid decarboxylase encoded by ipdC, and indole-3-acetic acid dehydrogenase encoded by iad1. The ipdC from Enterobacter cloacae ATCC 13047, aspC from Escherichia coli, and iad1 from Ustilago maydis were cloned and expressed under the control of the tac and sod promoters in E. coli. According to SDS-PAGE and enzyme activity, IpdC and Iad1 showed good expression under the control of $P_{tac}$, whereas AspC was efficiently expressed by $P_{sod}$ originating from Corynebacterium glutamicum. The activities of IpdC, AspC, and Iad1 from the crude extracts of recombinant E. coli Top 10 were 215.6, 5.7, and 272.1 nmol/min/mg-protein, respectively. The recombinant E. coli $DH5{\alpha}$ expressing IpdC, AspC, and Iad1 produced about 1.1 g/l of IAA and 0.13 g/l of tryptophol (TOL) after 48 h of cultivation in LB medium with 2 g/l tryptophan. To improve IAA production, a tnaA gene mediating indole formation from tryptophan was deleted. As a result, E. coli IAA68 with expression of the three genes produced 1.8 g/l of IAA, which is a 1.6-fold increase compared with wild-type $DH5{\alpha}$ harboring the same plasmids. Moreover, the complete conversion of tryptophan to IAA was achieved by E. coli IAA68. Finally, E. coli IAA68 produced 3.0 g/l of IAA after 24 h cultivation in LB medium supplemented with 4 g/l of tryptophan.

• 생명과학회지
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• 제18권12호
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• pp.1665-1670
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• 2008

콩의 oil은 식량유지 자원으로서 매우 중요한 부분을 차지하고 있으며, 전세계 식용유의 22%를 콩 oil이 차지하고 있으며 식품에서 매우 중요한 영양학적인 요소이다. 이중 불포화지방산은 지방산 중에서 종자 구성물질들은 polygenetic 형질들로 되어있다. 본 시험은 큰올콩과${\times}$신팔달콩의 RIL 계통과 SSR marker를 이용하여 유전자지도를 작성하고, 이를 바탕으로 불포화지방산의 함량과 관련된 양적형질 유전자좌(QTLs)를 탐색하였다. Oleic acid 함량과 관련된 QTLs는 7개의 연관군에서 8개의 마커가 확인되었으며, linoleic acid는 5개의 연관군에서 7개의 마커가 확인되었다. 그리고 linolenic acid는 5개의 연관군에서 각각 하나씩의 마커가 확인되었다. 본 시험의 결과 불포화지방산에 공통적으로 나타난 QTL은 연관군 C1과 L이었다.

• 생명과학회지
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• 제27권10호
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• pp.1145-1151
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• 2017

세제에 의하여 대장균의 periplasm에서 penicillin G amidase (PGA)를 방출하는 방법을 연구하였다. 결과적으로 세제와 lysozyme의 혼합 작용이 효과적인 것으로 나타났다. 세포 투과성의 최적 조건을 알아보기 위하여 세제의 종류, 농도, pH, 반응 시간, 온도 등의 영향을 살펴보았다. 그리하여 대장균에서 재조합 PGA를 periplasm에서 방출하는 모델을 만들 수 있었고 방출된 PGA를 농축할 수 있었다. 실리카 구슬을 이용한 고정화 시스템으로 PGA 용액을 농축할 수 있었으며, 더 이상의 정제 과정 없이 순수하게 추출 할 수 있었다. 고정화된 PGA는 penicillin G 생성의 원료인 6-APA를 생산하는데 사용할 수 있었다. 이 방법은 대장균으로부터 재조합 단백질을 추출하는 간단한 방법이며 고정화 PGA를 이용하여 ${\beta}-lactam$ 항생물질의 산업적 생산 이용될 수 있을 것으로 사료된다.

• KSBB Journal
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• 제10권3호
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• pp.343-348
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• 1995

LeI은 스테로이드를 통울에 투여하였을 때 분비가 촉진되어 항염증성 효과를 나타내는 calcium 의 존성 phospholipid 결합 단백질이다. S. cerevisiae는 대장균과 통물세포의 장점을 모두 가지고 있으므로 동물세포 유래의 이종 단백질의 분비 생산에 많이 이용되고 있다. 본 연구에서는 GAL10 promoter­p ppL-LCI유전자 LCI terminator로 구성된 pYGLPT5 로 LCI을 S. cerevisiae SEY2102에서 발현 분비시키고 각 분획으로 나누어 LCI양을 비교한 결과 protoplast 68.6 %. periplasmic 24 %, culture supernatant 7.4%로 분포하였다. pYGLPT5로 형질전환된 S. cereviswe 2102를 유가 배양한 결과, 최종적인 LCI의 생산량은 약 $500mg/\ell$ 였다. LCI은 N 말단 부근에 $CA^{++}$ 결합부위가 있으므로 이를 이용하여 hydroxylapatite column chromatography로 정 제하 였다. 배지로 분비된 34kDa LCI을 ultrafiltration 과 hydroxylapatite column chromatography 의 두 단계로 순도 99% 이상으로 정제할 수 있었다.

• Journal of Pharmaceutical Investigation
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• 제32권3호
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• pp.199-207
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• 2002

In order to develop a sustained release formulation of bovine somatotropin (BST), which has been used to increase the body weight of oxen or the milk production of dairy cows, poly(D,L-lactide-co-glyceride)(PLGA) microspheres were made by W/O/W multiple emulsification method and solvent extraction method. Physical properties including particle size, drug entrapment, drug release, protein denaturation, and in vivo body weight increase in rats were characterized. The size of the microspheres was increased as the molecular weight of PLGA increased. When Span 65 and stearic acid during preparation were added, the size was decreased but the amount of surface protein was increased, resulting in a high loading efficiency, with fast release of BST from the microspheres. Aggregation or fragmentation of BST by SDS-PAGE during microsphere preparation and drug release study was not observed. Body weight of Sprague-Dawley's male rats was significantly increased after subcutaneous administrations of BST-loaded PLGA microspheres. There was a good correlation between in vivo weight gain and in vitro release rate of microspheres. PLGA microspheres with a high surface protein ratio could be a good candidate for the sustained delivery of BST.

• Journal of Microbiology and Biotechnology
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• 제22권9호
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• pp.1237-1244
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• 2012

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

• IMMUNE NETWORK
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• 제5권3호
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• pp.172-178
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• 2005

Background: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. However, it is known that the induction of immune response to CEA is very difficult because CEA is a self-antigen expressed in fetal cells and weakly expressed in normal colorectal epithelial cells. To enhance anti-tumor immunity specific for CEA, recombinant CEA protein was modified using listeriolysin O (LLO) for endosomal lysis and trans activator of transcription (Tat) domain for transducing extracellular proteins into cytoplasm. Methods: After immunization using dendritic cells pulsed with Tat-CEA, both Tat-CEA and LLO, and both Tat-CEA and Tat-LLO, antibody titer to CEA and LLO, cytotoxic T lymphocyte activity and the frequency of IFN-${\gamma}$ producing T lymphocytes were measured. Results: Immunization using DC pulsed with both Tat-CEA and Tat-LLO protein showed the increasement of production of CEA-specific antibody in serum, cytotoxic T lymphocyte activity, the frequency of IFN-${\gamma}$ secreting T cells, compared with DC pulsed with both Tat-CEA and LLO. Furthermore the ratio of CD8+T cell to $CD4^+$ cell among CEA-specific T cells was increased in group pulsed with both Tat-CEA and Tat-LLO. Conclusion: These results suggested that DC vaccine using Tat-LLO could be used for the development of effective immunotherapy for the treatment of tumor.