• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2003년도 제46회 춘계 학술연구 발표회
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• pp.82-83
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• 2003

In previous experiment, we reported when the heterologous protein is expressed by using baculovirus expression vector system (BEVS), although the amount of intracellular protein is abundant, the amount of extracellular Protein is poor. As the link in the chain of the research, we investigated the secretory pathway, important in case of the secretory protein, of the protein expressed in insect cells using BEVS. (omitted)

• 생명과학회지
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• 제11권5호
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• pp.415-422
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• 2001

Many secreted proteins have disulfide bonds that are important for their structure and function. Protein disulfide isomerase (PDI, EC 5.3.1.4.), an enzyme that catalyzes the formation and rearrangement of thiol/disulfide exchange reactions, is a resident of the endoplasmic reticulum (ER). The subcellular localization and its function as catalyst of disulfide bond formation in the biosynthesis of secretory and cell membrane proteins suggest that PDI plays a key role in the secretory pathway. We have isolated a cDNA encoding protein disulfide isomerase from Bombyx mori(bPDI). It has been characterized under ER stress conditions (dominantly induced by calcium ionophore A23187, tunicamycin and DTT), which is known to cause an accumulation of unfolded proteins in the ER. Furthermore, It has also been examined for tissue distribution(pronounced at the fat body), hormonal regulation (juvenile hormone, insulin and juvenile +transferrin; however, it is not effected by transferrin alone), and the effect of exogenous bacteria (peak at 16 h after infection) on the bPDI mRNA expression. The results suggest that bPDI is a member of the ER stress protein group, and it may play an important role in exogenous bacterial infection in fat body, and that homones regulate its expression.

• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.319-328
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• 2023

Malassezia and Staphylococcus are the most dominant genera in human skin microbiome. To explore the inter-kingdom interactions between the two genera, we examined the transcriptional changes in Malassezia and Staphylococcus species induced upon co-culturing. RNA-seq analyses revealed that genes encoding ribosomal proteins were upregulated, while those encoding aspartyl proteases were downregulated in M. restricta after co-culturing with Staphylococcus species. We identified MRET_3770 as a major secretory aspartyl protease coding gene in M. restricta through pepstatin-A affinity chromatography followed by mass spectrometry and found that the expression of MRET_3770 was significantly repressed upon co-culturing with Staphylococcus species or by incubation in media with reduced pH. Moreover, biofilm formation by Staphylococcus aureus was inhibited in the spent medium of M. restricta, suggesting that biomolecules secreted by M. restricta such as secretory aspartyl proteases may degrade the biofilm structure. We also examined the transcriptional changes in S. aureus co-cultured with M. restricta and found co-cultured S. aureus showed increased expression of genes encoding ribosomal proteins and downregulation of those involved in riboflavin metabolism. These transcriptome data of co-cultured fungal and bacterial species demonstrate a dynamic interplay between the two co-existing genera.

• 한국미생물·생명공학회지
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• 제41권1호
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• pp.17-25
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• 2013

메탄올 자화효모 Hansenula polymorpha에서 분비 발현이 잘 된다고 보고된 인체 혈청 알부민(human serum albumin, HSA)을 융합단편으로 사용하여 외래 재조합 단백질을 효과적으로 분비 발현할 수 있는 발현시스템을 개발하고자 하였다. 이때 조작의 용이성 및 발현 효율 제고를 위하여 전장의 HSA 뿐만 아니라 세 종류의 각기 다른 크기의 HSA 단편을 설계하여 융합단편으로 사용하였다. 즉 HSA의 N-말단으로 부터 각기 137, 172, 320, 608개 아미노산을 갖는 융합단편 HSAft (1-4)를 제작하였다. 아울러 발현되는 HSA 단편의 검출 및 분리정제를 위한 His8-tag, HSA 융합단편과 외래 단백질간의 유연성을 부여하기 위한 2조의 $Gly_4Ser_1$ linker, 융합 발현된 타겟 단백질을 HSA 단편으로부터 용이하게 분리하기 위한 담배식각바이러스 단백질분해효소(tobacco etch virus protease, Tev) 인지 서열, 타겟 단백질 유전자를 클로닝하기 위한 멀티 클로닝 사이트(multiple cloning site, MCS)서열, 그리고 타겟 재조합 단백질의 발현 검출 및 정제를 위한 Strep-tag을 포함하는 작용기 도메인을 발현카세트 기본 골격에 포함시켰다. 이렇게 구축된 4종의 HSA 융합단편 분비발현 벡터를 H. polymorpha에 형질전환한 후 각 융합단편의 발현을 조사한 결과 HSAft 단편 3, 4의 발현을 확인할 수 있었다. 녹색형광단백질 유전자 ($GFP_{uv}$)를 상기 벡터에 클로닝한 후 H. polymorpha에 도입한 결과 형질 전환체 모두에서 녹색형광단백질의 발현을 관찰 할 수 있었다. 해당 세포로부터 분비되거나 세포내에 발현되는 HSA 단편 융합 형광단백질의 발현양을 비교한 결과 HSAft 단편 4에 융합된 경우를 제외하고 나머지 경우 모두에서 세포 파쇄액과 세포 배양액 양쪽에서 해당 HSA 단편 융합 형광단백질의 발현을 확인 할 수 있었다. 한편 HSA 융합단편의 크기에 따라 자체 혹은 타겟 단백질과 융합된 형태의 단백질 분비 발현 정도가 달라지는 것은 해당 단백질의 접힘이나 단백질 분해효소에 대한 민감성 등 여러 변수에 의한 것으로 사료되며 따라서 본 연구에서 개발한 H. polymorpha용 HSA 융합단편 분비발현 시스템은 특정 외래 재조합 단백질의 효율적인 분비발현 융합단편의 선별 및 과발현 시스템 구축에 유용하게 활용될 수 있을 것으로 기대된다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권4호
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• pp.242-246
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• 2000

Human lipocortin-I was expressed as a secretory product by Saccharomyces cerevisiae harboring an expression system consisting of GAL10 promoter, inulinase signal sequence and lipocortin-I terminator. Fed-batch fermentation was carried out to overproduce recombinant human lipocortin-I. The culture medium was desalted and concentrated by ultrafiltration, and then subjected to hydroxyapatite column chromatography. The lipocortin-I was purified to >98% purity by single-step hydroxyapatite column chromato-graphy. However, it was found that the purified lipocortin-I was a proteolytically-cleaved form which was cleaved immediately after the basic amino acid Lys26.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.892-896
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• 2003

High-level expression of the lipase B26 gene from Bacillus pumilus was achieved using Bacillus subtilis Chungkookjang isolated from the Korean traditional fermented bean paste, Chungkookjang. For the secretory production of recombinant lipase B26 in a Bacillus host system, pLipB26 was constructed by ligating the lipase B26 gene into the recently designed Escherichia coli-Bacillus shuttle vector, pLipSM, and that was then transformed into B. subtilis Chungkookjang. Among the various vector, medium, and host combinations, B. subtilis Chungkookjang harboring the pLipB26 exhibited the highest lipase activity in PY medium, and B. subtilis Chungkookjang secreted two times more enzymes than B. subtilis DB 104 under the same condition. When B. subtilis Chungkookjang harboring the pLipB26 was cultured in a 5-1 jar-fermentor containing 21 of a PY medium, the maximum lipase activity (140 U/ml) and production yield (0.68 g/l) were obtained during the late exponential phase from a cell-free culture broth. Although B. subtilis Chungkookjang also secreted extracellular proteases at the late exponential phase, these results suggested the potential of B. subtilis Chungkookjang as a host for the secretory production of foreign proteins.

• Journal of Microbiology and Biotechnology
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• 제24권2호
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• pp.197-208
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• 2014

A novel protease gene from Bacillus gibsonii, aprBG, was cloned, expressed in B. subtilis, and characterized. High-level expression of aprBG was achieved in the recombinant strain when a junction was present between the promoter and the target gene. The purified recombinant enzyme exhibited similar N-terminal sequences and catalytic properties to the native enzyme, including high affinity and hydrolytic efficiency toward various substrates and a superior performance when exposed to various metal ions, surfactants, oxidants, and commercial detergents. AprBG was remarkably stable in 50% organic solvents and retained 100% activity and stability in 0-4 M NaCl, which is better than the characteristics of previously reported proteases. AprBG was most closely related to the high-alkaline proteases of the subtilisin family with a 57-68% identity. The secretion and maturation mechanism of AprBG was dependent on the enzyme activity, as analyzed by site-directed mutagenesis. Thus, when taken together, the results revealed that the halo-solvent-tolerant protease AprBG displays significant activity and stability under various extreme conditions, indicating its potential for use in many biotechnology applications.

• Journal of Microbiology and Biotechnology
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• 제23권8호
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• pp.1116-1122
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• 2013

Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERT-S132A-based secretion engineering could be an effective strategy for enhancing recombinant t-PA production in CHO cells.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.463-468
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• 2011

This study investigated the changes of plasminogen activators (PAs) activity, expression and localization of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) during the estrous cycle in pigs. Estrous cycle was sorted into three group by pre-ovulation (Pre-Ov), post-ovulation (Post-Ov) and early to mid-luteal stages (Early to mid-L). Analysis for immunohistochemistry was confirmed by location of tPA and uPA. Porcine uterus tissue was cut into $1{\times}1$ cm squares, and were incubated in DMEM/F-12 medium for 1 h at $38^{\circ}C$, 5% $CO_2$ for measurement of PA activity. Western blotting was implemented for measurement of PA quantity. In results, the blood vessels and secretory glands were increased in Post-Ov stage than Pre-Ov and Early to mid-L stages. The tPA and uPA was located mainly within lumen of blood vessels and secretory glands. The PA activity in Post-Ov ($0.99{\pm}0.03$) stage were significantly (p<0.01) higher than Pre-Ov stage ($0.51{\pm}0.03$) and Early to mid-L stage ($0.21{\pm}0.04$). Expression of PAs were significantly (p<0.05) higher in Early to mid-L stage than other stages. These results indicate that PAs activity and expression may change in uterus tissue during the estrous cycle in pigs.

• Clinical and Experimental Reproductive Medicine
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• 제45권1호
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• pp.1-9
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• 2018

Objective: To determine the localization, expression, and function of Toll-like receptors (TLRs) in fallopian tube epithelial cells. Methods: The localization of TLRs in fallopian tube epithelial cells was investigated by immunostaining. Surprisingly, the intensity of staining was not equal in the secretory and ciliated cells. After primary cell culture of fallopian tube epithelial cells, ring cloning was used to isolate colonies of ciliated epithelial cells, distinct from non-ciliated epithelial cells. The expression of TLRs 1-10 was examined by quantitative real-time polymerase chain reaction, and protein localization was confirmed by immunostaining. The function of the TLRs was determined by interleukin (IL)-6 and IL-8 production in response to TLR2, TLR3, TLR5, TLR7, and TLR9 ligands. Results: Fallopian tube epithelial cells expressed TLRs 1-10 in a cell-type-specific manner. Exposing fallopian tube epithelial cells to TLR2, TLR3, TLR5, TLR7, and TLR9 agonists induced the secretion of proinflammatory cytokines such as IL-6 and IL-8. Conclusion: Our findings suggest that TLR expression in the fallopian tubes is cell-type-specific. According to our results, ciliated cells may play more effective role than non-ciliated cells in the innate immune defense of the fallopian tubes, and in interactions with gametes and embryos.