• 생명과학회지
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• 제16권7호
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• pp.1133-1140
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• 2006

E. coli HtrA (High temperature requirement protein A)의 human homologue 중 하나인 HtrA1은 IGFBP를 절단하여 IGF의 활동을 조절하는 serine protease으로 알려졌다. HtrA1의 serine protease 활성이 여러 질병의 발병 mechanism과 연관성을 가진 것으로 예상되고 있지만, 이런 상관관계를 밝히기 위해서 기본적으로 필요한 다량의 HtrA1 단백질의 발현 및 정제조건과 HtrA1 serine protease의 최적 활성조건이 확립되어 있지 않은 상황이다. 따라서 본 연구에서는 pGEX 시스템을 이용하여 E. coli에서 mature HtrA1인 ${\Delta}149(WT)$와 catalytic site mutant인 ${\Delta}149(S328A)$를 85%의 순도로 1 liter 배양 시, 정제된 단백질을 각각 $400{\mu}g,\;520{\mu}g$ 얻을 수 있는 발현조건을 정립하였다. 또한 HtrA1 serine protease 활성은 protease의 농도와 substrate와의 반응시간에 dependent하며, substrate와의 반응온도가 $42^{\circ}C$일 때 최적의 serine protease활성을 나타내는 것을 알 수 있었다. 특히 $200{\mu}M$의 HtrA1 serine protease를 $37^{\circ}C$에서 3시간 반응 시켰을 때, substrate로 사용한 ${\beta}-casein$의 약 50%가 절단되는 것을 관찰하였다. 따라서 이 반응조건에 사용한 HtrA1의 양을 1 unit으로 하여 HtrA1의 serine protease활성을 여러 조건에서 비교 분석할 수 있다 본 연구에서 정립한 mature HtrA1을 다량으로 얻을 수 있는 발헌 및 정제조건과 serine protease 최적 활성조건은 HtrA1의 serine protease 활성과 생물학적 기능의 상관관계를 이해하는데 활용될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제28권2호
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• pp.275-283
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• 2018

Ischemic stroke can result from blockage of blood vessels, forming fibrin clots in the body and causing irreparable brain damage. Remedial thrombolytic agents or anticoagulants have been studied; however, because the FDA-approved tissue plasminogen activator has low efficacy and side effects, it is necessary to develop safer and more effective treatment candidates. This study aimed at assessing the fibrinolytic and anticoagulation features of a novel serine protease extracted and purified from Diopatra sugokai, a polychaeta that inhabits tidal flats. The purified serine protease was obtained through ammonium sulfate precipitation, affinity chromatography, and ion-exchange chromatography. Its molecular size was identified via SDS-PAGE. To characterize its enzymatic activities, the protease activity at various pH and temperatures, and in the presence of various inhibitors, was measured via azocasein assay. Its fibrinolytic activity and anticoagulant effect were assessed by fibrin zymography, fibrin plate assay, and fibrinogenolytic activity assays. The novel 38 kDa serine protease had strong indirect thrombolytic activity rather than direct activity over broad pH (4-10) and temperature ($37^{\circ}C-70^{\circ}C$) ranges. In addition, the novel serine protease exhibited anticoagulant activity by degrading the ${\alpha}$-, ${\beta}$-, and ${\gamma}$-chains of fibrinogen. In addition, it did not produce cytotoxicity in endothelial cells. Therefore, this newly isolated serine protease is worthy of further investigation as a novel alkaline serine protease for thrombolytic therapy against brain ischemia.

• 한국어병학회지
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• 제10권1호
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• pp.31-38
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• 1997

잉어로부터 분리한 Aeromonas hydrophila는 세포 밖으로 여러 종류의 proteases를 생산한다. A. hydrophila의 배양 상층액을 이용한 inhibitor assay를 통하여, 주된 활성을 나타내는 metallopretease와 약한 활성을 나타내는 serine protease가 있음을 알게 되었다. Gelatin SDS-PAGE를 통하여 두 개의 활성 band가 관찰되었으며, 이 들 중 넓게 퍼진 band는 metalloprotease에 특이하게 작용하는 inhibitor인 EDTA에 의해 활성이 상실되었고 따라서 metalloprotease임을 알 수 있었다. 다른 하나는 serine protease에 특이하게 반응하는 inhibitor인 PMSF에 의해 저해되어 serine protease임을 알 수 있었다. 이러한 두 extracellular protease의 활성은 $75^{\circ}C$에서 30 분간 열을 가한 후에도 Gelatin SDS-PAGE상에 남아있었다. 그런데, 주된 metalloprotease는 Sephadex G-75를 이용한 column chromatography를 거친 후 Gelatin Gel상에서 두 개의 band로 분리되었다.

• 미생물학회지
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• 제39권4호
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• pp.230-234
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• 2003

Bacillus amyloliquefaciens psychrotrophic strain이 분비하는 세포 외 단백질 가수분해효소를 정제하여, endopeptidase 활성에 관한 특성을 분석하였다. Protease SE910로 명명된 효소는 단백질 내부의 leucine에 연결된 peptide 결합만을 가수분해하는 endopeptidase로 작용한다. 효소를 특이한 아미노산 잔기에 작용하는 화학수식제와 반응하였을 때 효소의 활성부위에 관여하는 아미노산 잔기가 수식되었을 때는 효소활성이 저해를 받는다. 본 효소는 serine을 수식하는 PMSF에의해 endopeptidase 활성이 완전히 저해되었으며, 카르복실 기능기를 수식하는 화학수식제에 의해 저해되었고, lysine을 화학수식하는 PLP에 의해서는 큰 영향을 받지 않았다. 이것은 본 효소의 endopeptidase 활성에 serine과 aspartic acid 잔기가 관여하는 것을 의미한다. 구조적으로 leucine을 포함하는 유도체인 bestatin은 효소의 endopeptidase 활성을 경쟁적으로 저해하였다.

• BMB Reports
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• 제34권6호
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• pp.509-516
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• 2001

A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and $38^{\circ}C$, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum 19A, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.

• Parasites, Hosts and Diseases
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• 제41권4호
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• pp.189-196
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• 2003

In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, we purified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Korean keratitis patient The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequential chromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDa protease had a pH optimum of 8.5 and a temperature optimum of $55^{\circ}C$. Phenylmethylsulfonylfluoride and 4-(2-Aminoethyl)-benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activity of the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degraded various extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine protease secreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis, such as in corneal tissue invasion, immune evasion and nutrient uptake.

• Journal of Microbiology and Biotechnology
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• 제21권1호
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• pp.20-27
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• 2011

The purification and characterization of a $Mn^{2+}$-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be $60^{\circ}C$. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. $Mn^{2+}$ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants ($H_2O_2$, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial applications.

• Molecules and Cells
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• 제19권2호
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• pp.223-227
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• 2005

We have identified TMPRSS6, a novel type 2 transmembrane serine protease. TMPRSS6 possesses all the signature motifs of the family of transmembrane serine proteases (TMPRSSs), including a transmembrane domain, an LDL receptor class A (LDLRA) domain, a scavenger receptor cysteine-rich (SRCR) domain, and a serine protease domain. The substrate specificity of TMPRSS6 is slightly different from those of other TMPRSS family members. Combined with the finding that TMPRSS6 is expressed strongly in the thyroid and weakly in the trachea, this may indicate that TMPRSS6 has a specialized role.

• 한국미생물·생명공학회지
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• 제23권1호
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• pp.53-59
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• 1995

A protease isolated and purified 51 fold from the culture filtrate of a soil bacterium, Bacillus sp. KUN-17, which was appeared to be a monomeric protein with molecular weight of 38, 000 daltons, was suggested to be involved in the serine (-alkaline) protease (E.C 3.4.21.14) since its activity was selectively inhibited by phenylmethylsulfonyl fluoride (PMSF) and required 40$\circ$C and pH 10.5 for optimal condition. The half-life of the enzyme activity was 1 hr at 55$\circ$C, and the activity was maintained even under high concentrations of SDS or urea. The enzyme was indicated to perform random proteolysis from the fact that most of the chromogenic substrates employed were hydrolyzed by the enzyme. The affinity of the enzyme for natural proteins was approximately 10-times higher than ester compounds, and both substrates showed mutual inhibitory effect competitively for the enzyme activity.

• Journal of Microbiology and Biotechnology
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• 제2권4호
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• pp.231-236
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• 1992

A 5.8 kb chromosomal DNA fragment of Serratia marcescens ATCC 27117 including an extracellular serine protease gene was cloned in Escherichia coli. The cloned gene(pSMP18) caused specific excretion of the protease into the extracellular medium through the outer membrane of E. coli host cells. The protease purified from E. coli harboring pSMP18 was inactivated not by 100 mM EDTA but by 10 mM phenyl methyl sulfonyl flouride (PMSF). The molecular weight of the purified serine protease was about 66, 000 in the SDS-PAGE and the isoelectric point was approximately 5.7 in IEFㆍGel electrophoresis. The optimal pH and temperature for reaction of the purified serine protease were 9.5 and $45^\circ{C}$, respectively.