E. coli에서 발현된 human HtrA1 단백질의 정제와 HtrA1의 serine protease 활성 조건에 관한 연구

김경희,김상수,김구영,임향숙,Kim, Kyung-Hee,Kim, Sang-Soo,Kim, Goo-Young,Rhim, Hyang-Shuk 한국생명과학회 2006 생명과학회지 Vol.16 No.7

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. Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (ICFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low $460{\mu}g$ for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was $42^{\circ}C$ instead of $37^{\circ}C$. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200nM of HtrA1 that cleaves half of $5{\mu}M\;of\;{\beta}-casein$ during 3 hr incubation at $37^{\circ}C$. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.

Molecular Cloning and Characterization of a Serine Protease-Like Protein from Silkworm (Bombyx Mori)

김소연,정은정,송기준,박광숙 한국유전학회 2009 Genes & Genomics Vol.31 No.5

The Bombyx mori ( B. mori) serine protease-like protein (BmSp) coding region (946 bp, GenBank accession number of mRNA, DQ118520; protein, AAZ40503) was generated from two separate and overlapping cDNA fragments using sequence homology with Trichoplusia ni azurocidin in a Bombyx EST database (Silkbase; http://www.ab.a.u-tokyo.ac.jp/silkbase/). The deduced amino acid sequence of BmSp, which encodes 303 amino acids, shows 44% amino acid identity to A. gambiae serine protease (CAA89967), 43% amino acid identity to Sarcophagi peregrina 26-kDa protease, an antibacterial protein and 31% identity to B. mori serine protease-2 (BmSP-2), a potential antiviral protein. Typical features of the BmSp included the serine protease active site triad His / Asp / Ser, three pairs of cysteine residues for disulfide bridges, and three residues, Asp / Gly / Gly, that help to confer trypsin-like specificity to the enzymes. Based on the result of sequence comparison and characterization, our results suggest that the BmSp probably the new subfamily of trypsin-like serine protease. Using RT-PCR and enzyme digestion, the full encoding sequence for BmSp was cloned into the E. coli expression vector pGEX-5X-1. The fusion protein GST-BmSp was effectively expressed in E. coli BL21(DE3) pLysS as inclusion bodies, and a denaturation and refolding procedure were performed to obtain soluble GST-BmSp. The purified protein was tested for antibacterial activity against Gram-positive and Gram-negative bacteria, but it did not show antibacterial activity in the agar well diffusion assay and liquid growth inhibition assay. The Bombyx mori ( B. mori) serine protease-like protein (BmSp) coding region (946 bp, GenBank accession number of mRNA, DQ118520; protein, AAZ40503) was generated from two separate and overlapping cDNA fragments using sequence homology with Trichoplusia ni azurocidin in a Bombyx EST database (Silkbase; http://www.ab.a.u-tokyo.ac.jp/silkbase/). The deduced amino acid sequence of BmSp, which encodes 303 amino acids, shows 44% amino acid identity to A. gambiae serine protease (CAA89967), 43% amino acid identity to Sarcophagi peregrina 26-kDa protease, an antibacterial protein and 31% identity to B. mori serine protease-2 (BmSP-2), a potential antiviral protein. Typical features of the BmSp included the serine protease active site triad His / Asp / Ser, three pairs of cysteine residues for disulfide bridges, and three residues, Asp / Gly / Gly, that help to confer trypsin-like specificity to the enzymes. Based on the result of sequence comparison and characterization, our results suggest that the BmSp probably the new subfamily of trypsin-like serine protease. Using RT-PCR and enzyme digestion, the full encoding sequence for BmSp was cloned into the E. coli expression vector pGEX-5X-1. The fusion protein GST-BmSp was effectively expressed in E. coli BL21(DE3) pLysS as inclusion bodies, and a denaturation and refolding procedure were performed to obtain soluble GST-BmSp. The purified protein was tested for antibacterial activity against Gram-positive and Gram-negative bacteria, but it did not show antibacterial activity in the agar well diffusion assay and liquid growth inhibition assay.

Anti-fibrinolytic and anti-microbial activities of a serine protease inhibitor from honeybee (<i>Apis cerana</i>) venom

Yang, Jie,Lee, Kwang Sik,Kim, Bo Yeon,Choi, Yong Soo,Yoon, Hyung Joo,Jia, Jingming,Jin, Byung Rae PERGAMON PRESS 2017 COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C Vol.201 No.-

<P><B>Abstract</B></P> <P>Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (<I>Apis mellifera</I>) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (<I>A</I>. <I>cerana</I>) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Apis cerana</I> venom serine protease inhibitor (AcVSPI) inhibits trypsin, but not chymotrypsin. </LI> <LI> AcVSPI is a low-molecular-weight serine protease inhibitor Api m 6-like peptide. </LI> <LI> AcVSPI inhibits plasmin and microbial serine proteases. </LI> <LI> AcVSPI functions as an anti-fibrinolytic factor. </LI> <LI> AcVSPI functions as an anti-microbial agent. </LI> </UL> </P>

Serine pretease 억제제인 4-(2-aminoethyl) benzensulfonylfluoride (AEBSF)에 의한 호중구의 자연 세포사멸의 지연과 수지상 세포로의 전이분화 연구

박해영,곽종영,Park, Hae-Young,Kwak, Jong-Young 한국생명과학회 2007 생명과학회지 Vol.17 No.7

생체 면역반응에 중요한 역할을 하는 호중구의 세포사멸은 자연적으로 일어나거나 여러 외부자극에 의한 신호의 전달에 의해 증가하거나 지연된다. 또한 사이토카인과 같은 분화제에 의해 세포사멸이 지연되고 항원 제시 기능을 가진 수지상 세포로 분화되기도 한다. 본 연구에서는 세포사멸 억제제와 사이토카인을 이용한 시스템에서 호중구가 수지상 세포로 분화되는가를 조사하였다. Pancaspase와 serine protease의 억제제인 zVAD-fmk와 AEBSF를 처리하였을 때 호중구의 세포사멸은 현저히 감소되며 AEBSF는 caspase-3와 serine protease활성을 모두 억제하였다. 호중구의 세포사멸을 효과적으로 억제하는 AEBSF와 함께 분화제로 널리 쓰이는 CM-CSF를 같이 처리하여 3일 동안 배양하면 수지상 세포에서 높이 발현되는 CD8O, CD83 및 MHC class ll의 세포표면 마커의 발현이 증가하였다. AEBSF와 CM-CSF를 처리한 호중구를 T-세포와 함께 배양하였을 때 SEB가 존재할 경우 T-세포가 증식되었으며 SEB가 없이도 $IFN{\gamma}$는 생성되었다. 이들 결과들로 부터 serine protease 억제제인 AEBSF를 호중구에 처리하여 세포사멸을 효과적으로 억제하는 것과 수지상 세포로의 분화를 촉진하는 사이토카인인 CM-CSF의작용을 나타내게 하는 조건과는 서로 상호적으로 연관되어 작용할 수 있다는 것을 제시하고 있다. Neutrophils play a key role as a first line of defense and are known to acquire the characteristics of dendritic cells (DCs) under the appropriate conditions. The spontaneous apoptosis of neutrophils was delayed by treatment with 4-(2-aminoethyl) benzensulfonylfluoride (AEBSF), a serine protease inhibitor. AEBSF inhibited both caspase-3 and serine protease activities, whereas ZVAD-fmk, a pancaspase inhibitor, inhibited only caspase-3 activity. The life span of neutrophils was prolonged up to 5 days by AEBSF in the presence or absence of granulocyte macrophage colony stimulating factor(CM-CSF). DC surface markers, such as CD80, CD83, and MHC class ll were not expressed on neutrophils treated with AEBSF alone. CM-CSF failed to prolong the survival time of neutrophils up to3 days but increased the expression levels of DC markers on neutrophils in the presence of AEBSF. Expression levels of DC markers were the highest on neutrophils treated with CM-CSF and AEBSF for 3 days. AEBSF and CM-CSF-treated neutrophils stimulated proliferation of T cells in the presence of a superantigen, Staphylococcal enterotoxin B (SEB) but produced $interferon-{\gamma}$ ($IFN{\gamma}$) in the absence of SEB. These results suggest that the inhibition of serine protease activity prolonged the life span of human neutrophils and combined treatment of neukophils with CM-CSF and serine protease inhibitor induced differentiation of neutrophils into DC-like cells.

Acholeplasma lysate를 처리한 Tenebrio molitor larva 에서 동정된 serine protease 유전자에 관한 연구

정지은,황희주,조용훈,한연수,방인석,이용석 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

Serine protease는 병원체의 표면 melanization, hemolymph coagulation, antimicrobial peptide synthesis 등을 통해 여러 무척추동물의 방어기작을 조절하는것으로 알려 져 있다. 곤충의 경우 Tribolium을 대상으로 이와 같은 연구가 이루어져 왔지만, 혈 액의 량이 그리 많지 않아 연구자들은 최근 갈색거저리(Tenebrio)를 이용하기 시작 하였다. 하지만 아직 유전체(자) 서열정보가 충분하지 않은 상황이다. 본 연구에서 는 이러한 갈색거저리 유충을 이용하여 세포벽이 없으며 사람에서 pneumonia나 다른 호흡기 질환을 일으키는 mycoplasma 와 유사한 acholeplasma lysate를 처리 한 후 접종 전과 후의 전사체의 비교를 통하여 무척추 동물에서의 선천성 면역 관련 유전자들을 동정하고자 하였다. Acholeplasma lysate를 처리하기 전과 후의 각 샘 플들로부터 cDNA library를 구축한 후 random sequencing 을 통해 염기서열을 분 석하였고, 얻어진 서열들로부터 NCBI nr 데이터베이스에 Blastx 분석을 하여 획득 한 서열들을 comparative transcriptomic 방법을 이용하여 분석한 결과, 여러 종류 의 Serine protease 관련 유전자들이 동정되었다. Serine protease (XP_970766.1)의 경우에는 acholeplasma를 처리한 샘플에서 2배 정도 발현이 증가하였고, serine protease P66 (EFA09207.1)의 경우 증감의 변화는 보이지 않았다. 또한 serine protease P146 (EFA04636.1), serine protease H1 (EEZ99180.1) 등의 유전자들도 동정되어 연구하고 있다.

잉어(Cyprinus carpio)로부터 분리된 Aeromonas hydrophila의 extracelluar proteases 연구

이종규,김종필,최태진,송영환,Lee, Jong-Kyu,Kim, Jong-Pil,Choi, Tae-Jin,Song, Young-Hwan 한국어병학회 1997 한국어병학회지 Vol.10 No.1

Aeromonas hydrophila isolated from the intestine of carp produced several kinds of proteases into the medium. Inhibitor assay with the culture supernatant of A. hydrophila showed that there were major metalloproteases and minor serine proteases. Gelatin SDS-PAGE showed two proteolytic bands. One broad protease band was inhibited by metalloprotease specific inhibitor, EDTA, indicating a metalloprotease. The other was inhibited by serine protease specific inhibitor, PMSF, suggesting a serine protease. The proteolytic activities of both extracellular proteases remained on Gelatin SDS-PAGE after heating at $70^{\circ}C$ for 30 min. However, the major metalloprotease was separated into two proteolytic bands on Gelatin PAGE by gel filtration chromatography on Sephadex G-75. 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.

Dual function of a bee venom serine protease: Prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals

Young Moo Choo,Kwang Sik Lee,Hyung Joo Yoon,Bo Yeon Kim,Mi Ri Sohn,Jong Yul Roh,Yeon Ho Je,Nam Jung Kim,Iksoo Kim,Soo Dong Woo,Hung Dae Sohn,Byung Rae Jin 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.10

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles forBi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

Bacillus sp. KUN-17 균주가 생산하는 균체외 Serine Protease의 정제 및 특성

황세영 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.1

토양 세균인 Bacillus sp. KUN-17의 배양액으로부터 51배 정제된 protease는 분자량 38000의 단량체이며, phenylmethyl sulfonyl fluoride(PMSF)에 의하여 선택적으로 활성이 억제되고, 반응 최적 온도 및 pH는 40℃, pH 10.5를 보이므로써 serine(-alkaline) protease(E.C 3.4.21.14)에 속하는 것으로 판단되었다. 효소 활성의 half-life는 55℃에서 1시간으로 나타났으며 SDS나 urea 등의 고농도 하에서도 활성이 유지되었다. 본 효소의 기질특이성은 사용한 chromogen기질의 대부분이 본 효소에 의하여 가수분해 되므로써, random proteolysis가 시사되었다. 이 효소의 천연단백질에 대한 친화력은 ester 기질보다 약 10배 이상인 것으로 나타났으며 이들 기질은 효소 단백질에 대하여 서로 길항저해 효과를 보였다. 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℃ and pH 10.5 for optimal condition. The half-life of the enzyme activity was 1 hr at 55℃, 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.

Molecular Cloning and Characterization of a Serine Protease-Like Protein from Silkworm (Bombyx Mori)

So Youn Kim,Eon Jeong Jeong,Ki Joon Song,Kwang Sook Park 한국유전학회 2009 Genes & Genomics Vol.31 No.5

The Bombyx mori (B. mori) serine protease-like protein (BmSp) coding region (946 bp, GenBank accession number of mRNA, DQ118520; protein, AAZ40503) was generated from two separate and overlapping cDNA fragments using sequence homology with Trichoplusia ni azurocidin in a Bombyx EST database (Silkbase; http://www.ab.a.u-tokyo.ac.jp/silkbase/). The deduced amino acid sequence of BmSp, which encodes 303 amino acids, shows 44% amino acid identity to A. gambiae serine protease (CAA89967), 43% amino acid identity to Sarcophagi peregrina 26-kDa protease, an antibacterial protein and 31% identity to B. mori serine protease-2 (BmSP-2), a potential antiviral protein. Typical features of the BmSp included the serine protease active site triad His/Asp/Ser, three pairs of cysteine residues for disulfide bridges, and three residues, Asp/Gly/Gly, that help to confer trypsin-like specificity to the enzymes. Based on the result of sequence comparison and characterization, our results suggest that the BmSp probably the new subfamily of trypsin-like serine protease. Using RT-PCR and enzyme digestion, the full encoding sequence for BmSp was cloned into the E. coli expression vector pGEX-5X-1. The fusion protein GST-BmSp was effectively expressed in E. coli BL21(DE3) pLysS as inclusion bodies, and a denaturation and refolding procedure were performed to obtain soluble GST-BmSp. The purified protein was tested for antibacterial activity against Gram-positive and Gram-negative bacteria, but it did not show antibacterial activity in the agar well diffusion assay and liquid growth inhibition assay.

Characterization, Cloning, and Heterologous Expression of a Subtilisin-Like Serine Protease Gene VlPr1 from Verticillium lecanii

Gang Yu,Jin-Liang Liu,Li-Qin Xie,Xue-Liang Wang,Shi-Hong Zhang,Hong-Yu Pan 한국미생물학회 2012 The journal of microbiology Vol.50 No.6

The entomopathogenic fungus Verticillium lecanii is a wellknown biocontrol agent. V. lecanii produces subtilisin-like serine protease (Pr1), which is important in the biological control activity of some insect pests by degrading insect cuticles. In this study, a subtilisin-like serine protease gene VlPr1 was cloned from the fungus and the VlPr1 protein was expressed in Escherichia coli. The VlPr1 gene contains an open reading frame (ORF) interrupted by three short introns, and encodes a protein of 379 amino acids. Protein sequence analysis revealed high homology with subtilisin serine proteases. The molecular mass of the protease was 38 kDa, and the serine protease exhibited its maximal activity at 40°C and pH 9.0. Protease activity was also affected by Mg2+ and Ca2+ concentration. The protease showed inhibitory activity against several plant pathogens, especially towards Fusarium moniliforme.