• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.212-213
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• 2005

• BMB Reports
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• 제31권5호
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• pp.432-443
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• 1998

The poliovirus Sabin 1 strain has features that make it a particularly attractive live recombinant mucosal vaccine vehicle. Sabin 1 cDNA was manipulated to have multiple cloning sites and a viral specific 3C-protease cutting site at the N-terminal end of the polyprotein. The gene for the N-terminal 169 amino acids of the HIV-1 p24 was cloned into the multiple cloning site of the manipulated Sabin cDNA. A recombinant progeny virus was produced from HeLa cells when it was transfected with the RNA synthesized from the p24-Sabin chimeric cDNA. The recombinant progeny virus expresses substantial amounts of the HIV-1 p24 protein, which was clearly detected in the infected cell lysates and culture supernatants in Western blot experiments with rabbit anti-p24 serum and AIDS patients' sera. Differing from the Mahoney strain, the recombinant Sabin 1 poliovirus maintained the foreign gene stably during the subsequent passages. Replication capacity was about 1 to 1.5 log lower than that of the wild-type Sabin 1. Other physicochemical stability characteristics of the recombinant virus were similar to that of the wild-type Sabin 1. These results suggest that the manipulated Sabin 1 poliovirus can be used as a live viral vaccine vector for the development of mucosal vaccines.

• 생명과학회지
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• 제22권4호
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• pp.552-558
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• 2012

사스(Severe acute respiratory syndrome, SARS)는 사람의 신종 폐렴인 중증 급성 호흡기 질환으로 신종 코로나바이러스, SARS-CoV에 의해 유발된다. 3CL protease는 SARS-CoV의 복제, 전사 및 단백질 합성을 조절하는 복제효소 복합단백질의 프로세싱에 결정적인 역할을 담당하는 중요한 효소이다. 따라서, 이 효소를 저해함으로써 SARS-CoV의 증식을 억제하고 사스의 증폭 및 확산을 막을 수 있다. SARS-3CL protease의 활성 저해물질의 탐색은 사스의 치료제 개발에 중요한 목표 중의 하나로 인식되고 있으며 이를 위해서는 활성형 SARS-3CL protease의 대량 생산이 필요하다. 본 연구에서는 활성형 SARS-3CL protease를 대량 생산하기 위하여 여러 가지 발현 벡터 및 단백질 발현 방법 등을 검토하였다. 그 결과, pET29a/3CLP 발현 벡터를 이용한 무세포 단백질 합성법이 SARS-3CL protease 생산에 최적 조건인 것으로 확인되었다. 또한 발현된 효소를 완전히 정제하여 그 특성을 분석한 결과, 본 효소는 무세포 단백질 합성계에서 전구체로 합성됨과 동시에 자가분해됨으로써 모든 단백질이 활성형인 성숙체 단백질로 전환되어 간단히 활성형 SARS-3CL protease 효소를 생산할 수 있음을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제19권11호
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• pp.1295-1300
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• 2009

A 2,172-bp full-length gene (aga-F78), encoding a protease-resistant $\alpha$-galactosidase, was cloned from Rhizopus sp. F78 and expressed in Escherichia coli. The deduced amino acid sequence shared highest identity (45.0%) with an $\alpha$-galactosidase of glycoside hydrolase family 36 from Absidia corymbifera. After one-step purification with a Ni-NTA chelating column, the recombinant Aga-F78 migrated as a single band of ~82 and ~210 kDa on SDS-PAGE and nondenaturing gradient PAGE, respectively, indicating that the native structure of the recombinant Aga-F78 was a trimer. Exhibiting the similar properties as the authentic protein, purified recombinant Aga-F78 was optimally active at $50^{\circ}C$ and pH 4.8, highly pH stable over the pH range 5.0-10.0, more resistant to some cations and proteases, and had wide substrate specificity (pNPG, melidiose, raffinose, and stachyose). The recombinant enzyme also showed good hydrolytic ability to soybean meal, releasing galactose of $415.58\;{\mu}g/g$ soybean meal. When combined with trypsin, the enzyme retained over 90% degradability to soybean meal. These favorable properties make Aga-F78 a potential candidate for applications in the food and feed industries.

• BMB Reports
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• 제36권4호
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• pp.390-398
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• 2003

The CDP/Cux transcription factor was previously shown to be proteolytically processed at the G1/S transition. In view of characterizing and eventually identifying the protease responsible for CDP/Cux processing, we have established an in vitro proteolytic processing assay. CDP/Cux recombinant proteins expressed in mammalian or bacterial cells were efficiently processed in vitro using as a source of protease either whole cell extracts, the nuclear or the cytoplasmic fraction. Processing was found to take place optimally at a lower pH, to be insensitive to variations in salt concentration, and to be inhibited by the protease inhibitors MG132 and E64D. Interestingly, the bacterially-produced substrate was more efficiently processed than the substrate purified from mammalian cells. Moreover, processing in vitro was more efficient when CDP/Cux substrates were purified from populations of cells enriched in the S phase than in the G1 phase of the cell cycle. Altogether, these results suggest that post-translational modifications of CDP/Cux in mammalian cells inhibits processing and contributes to the cell cycle-dependent regulation of processing. The in vitro processing assay described in this study will provide a useful tool for the purification and identification of the protease responsible for the processing of CDP/Cux.

• Journal of Microbiology and Biotechnology
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• 제17권4호
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• pp.594-599
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• 2007

It is well known that the Escherichia coli inner membrane-bound protease DegS is a periplasmic stress sensor for unfolded outer membrane proteins (OMPs). Previous studies have also shown that the outer membrane protease OmpT activates plasminogen in vitro and this may be exploited by bacteria in the course of pathogenesis. However, there has been no research on the plasminogen activation ability of the important periplasmic protein DegS. Accordingly, in this study, the whole-length and truncated degS genes were separately overexpressed in Escherichia coli, the recombinant proteins purified by affinity chromatography, and their plasminogen activator role tested in vitro. The results suggested that the whole-length DegS was able to activate plasminogen on a plasma plate. The truncated form of DegS (residues 80-345), designated ${\Delta}DegS$, also acted as a plasminogen activator, as confirmed by different assays. The serine protease property of ${\Delta}DegS$ was verified based on the complete inhibition of its enzyme activity by PMSF (phenylmethanesulfonyl fluoride). Therefore, the present results indicate that DegS is a plasminogen activator in vitro.

• BMB Reports
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• 제42권8호
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• pp.506-510
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• 2009

The Toll signalling pathway in invertebrates is responsible for defense against Gram-positive bacteria and fungi, leading to the expression of antimicrobial peptides via NF-$\kappa$B-like transcription factors. Gram-negative binding protein 3 (GNBP3) detects beta-1,3-glucan, a fungal cell wall component, and activates a three step serine protease cascade for activation of the Toll signalling pathway. Here, we showed that the recombinant N-terminal domain of Tenebrio molitor GNBP3 bound to beta-1,3-glucan, but did not activate down-stream serine protease cascade in vitro. Reversely, the N-terminal domain blocked GNBP3-mediated serine protease cascade activation in vitro and also inhibited beta-1,3-glucan-mediated antimicrobial peptide induction in Tenebrio molitor larvae. These results suggest that the N-terminal GNBP homology domain of GNBP3 functions as a beta-1,3-glucan binding domain and the C-terminal domain of GNBP3 may be required for the recruitment of immediate down-stream serine protease zymogen during Toll signalling pathway activation.

• Journal of Microbiology and Biotechnology
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• 제20권8호
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• pp.1243-1250
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• 2010

A cDNA encoding a cysteine protease inhibitor (CPI) was isolated from the cDNA library of clamworm Perinereis aibuhitensis Grube. The deduced amino acid sequence analysis showed that the protein had 51%, 48%, and 48% identity with Zgc:153129 from Danio rerio, cystatin B from Theromyzon tessulatum, and the ChainA, stefin B tetramer from Homo sapiens, respectively. The gene was cloned into the intracellular expression vector pET-15b and expressed in Escherichia coli. The recombinant CPI (PA-CPI) was purified by affinity chromatography on Ni-charged resin and ion-exchange chromatography on DEAE-Sepharose FF. The relative molecular mass of PA-CPI was 16 kDa as deduced by SDS-PAGE. Activity analysis showed that the recombinant protein could inhibit the proteolytic activity of papain. A constitutive and secretive expression vector was also constructed, and the cDNA encoding CPI was subcloned into the vector for extracellular expression. Western blotting analysis results showed that the PA-CPI was secreted into the medium. Bioassay demonstrated that E. coli DH5${\alpha}$ harboring pUC18ompAcat-CPI showed a significant difference in mortality to the Asian longhorned beetle Anoplophora glabripennis compared with untransformed E. coli DH5${\alpha}$ and control.

• Parasites, Hosts and Diseases
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• 제52권1호
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• pp.41-46
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• 2014

The mature domain of a cysteine protease of Spirometra erinacei plerocercoid larva (i.e., sparganum) was expressed in Escherichia coli, and its value as an antigen for the serodiagnosis of sparganosis was investigated. The recombinant protein (rSepCp-1) has the molecular weight of 23.4 kDa, and strongly reacted with the sparganum positive human or mice sera but not with negative sera by immunoblotting. ELISA with rSepCp-1 protein or sparganum crude antigen (SeC) was evaluated for the serodiagnosis of sparganosis using patient's sera. The sensitivity and specificity of ELISA using rSepCp-1 protein were 95.0% (19/20) and 99.1% (111/112), respectively. In contrast, the sensitivity and specificity of ELISA with SeC were 100% (20/20) and 96.4% (108/112), respectively. Moreover, in experimentally infected mice, the sensitivity and specificity of both ELISA assays were 100% for the detection of anti-sparganum IgG. It is suggested that the rSepCp-1 protein-based ELISA could provide a highly sensitive and specific assay for the diagnosis of sparganosis.

• Journal of Microbiology and Biotechnology
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• 제34권2호
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• pp.436-456
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• 2024

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80℃. In addition, the enzyme showed a half-life of 15 h at 80℃, and there was a 60% increase in its activity at 10 mM Ca²⁺ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.