• Bulletin of the Korean Chemical Society
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• 제30권8호
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• pp.1891-1892
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• 2009

• BMB Reports
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• 제39권6호
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• pp.717-721
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• 2006

In vitro analyses of type I signal peptidase activities require protein precursors as substrates. Usually, these pre-proteins are expressed in vitro and cleavage of the signal sequence is followed by SDS polyacrylamide gel electrophoresis coupled with autoradiography. Radioactive amino acids have to be incorporated in the expressed protein, since the amount of the in vitro expressed protein is usually very low and processing of the signal peptide cannot be followed by SDS polyacrylamide gel electrophoresis alone. Here we describe a rapid and simple method to express large amounts of a protein precursor in E. coli. We have analyzed the effect of ionophors as well as of azide on the accumulation of expressed protein precursors. Azide blocks the function of SecA and the ionophors dissipate the electrochemical gradient across the cytoplasmic membrane of E. coli. Addition of azide ions resulted in the formation of inclusion bodies, highly enriched with pre-apo-plastocyanine. Plastocyanine is a soluble copper protein, which can be found in the periplasmic space of cyanobacteria as well as in the thylakoid lumen of cyanobacteria and chloroplasts, and the pre-protein contains a cleavable signal sequence at its N-terminus. After purification of cyanobacterial pre-apo-plastocyanine, its signal sequence can be cleaved off by the E. coli signal peptidase, and protein processing was followed on Coomassie stained SDS polyacrylamide gels. We are optimistic that the presented method can be further developed and applied.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1070-1083
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• 2015

A gene (GT-SM3B) encoding a thermostable secreted oligoendopeptidase (GT-SM3B) was cloned from the thermophile Geobacillus thermoleovorans DSM 15325. GT-SM3B is 1,857 bp in length and encodes a single-domain protein of 618 amino acids with a 23-residue signal peptide having a calculated mass of 67.7 kDa after signal cleavage. The deduced amino acid sequence of GT-SM3B contains a conservative zinc metallopeptidase motif (His⁴⁰⁰-Glu⁴⁰¹-X-XHis⁴⁰⁴). The described oligopeptidase belongs to the M3B subfamily of metallopeptidases and displays the highest amino acid sequence identity (40.3%) to the oligopeptidase PepF_Ba from mesophilic Bacillus amyloliquefaciens 23-7A among the characterized oligopeptidases. Secretory production of GT-SM3B was used, exploiting successful oligopeptidase signal peptide recognition by Escherichia coli BL21 (DE3). The recombinant enzyme was purified from the culture fluid. Homodimerization of GT-SM3B was determined by SDS-PAGE. Both the homodimer and monomer were catalytically active within a pH range of 5.0–8.0, at pH 7.3 and 40℃, showing the K_m, V_max, and k_cat values for carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala-OH peptidolysis to be 2.17 ± 0.04 × 10^-6 M, 2.65 ± 0.03 × 10^-3 µM/min, and 5.99 ± 0.07 s^-1, respectively. Peptidase remained stable at a broad pH range of 5.0–8.0. GT-SM3B was thermoactive, demonstrating 84% and 64% of maximum activity at 50℃ and 60℃, respectively. The recombinant oligopeptidase is one of the most thermostable M3B peptidase, retaining 71% residual activity after incubation at 60℃ for 1 h. GT-SM3B was shown to hydrolyze a collagenous peptide mixture derived from various types of collagen, but less preferentially than synthetic hexapeptide. This study is the first report on an extracellular thermostable metallo-oligopeptidase.

• Molecules and Cells
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• 제26권1호
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• pp.34-40
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• 2008

To express an increased level of recombinant Mefp1 (marine mussel adhesive protein) in soluble form, we constructed expression vectors encoding truncated OmpA signal peptide-Mefp1 fusion proteins. OmpA signal peptide (OmpASP) is the 21 residue peptide fragment of the 23 residue OmpA signal sequence cleavable by signal peptidase I. We successfully produced increased levels of soluble recombinant Mefp1 (rMefp1) with various deletions of OmpASP, and found that the increased expression was caused by the increased pI of the N-terminus of the fusion proteins (${\geq}10.55$). All the OmpA signal peptide segments of 3-21 amino acids in length had the same pI value (10.55). Our results suggest that the pI value of the truncated OmpASP ($OmpASP_{tr}$) play an important role in directional signaling for the fusion protein, but we found no evidence for the presence of a secretion enhancer in OmpASP. For practical applications, we increased the expression of soluble rMefp1 with $OmpASP_{tr}$ peptides as directional signals, and obtained rMefp1 with the native amino terminus (nN-rMefp1) using an $OmpASP_{tr}$ Xa leader sequence that contains the recognition site for Xa protease.

• Applied Biological Chemistry
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• 제49권4호
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• pp.293-297
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• 2006

포유류의 조직에 널리 분포되어 있으며 혈압 조절에 중요한 역할을 하는 Angiotensin-converting enzyme(ACE)은 아연을 함유하는 dipeptidase로서 angiotensin I을 가수분해하여 강력한 혈압상승제인 angiotensin II를 생성하는 효소이다. 최근에 돼지의 난소에서 ACE 활성이 측정되었으며, 돼지의 신장에서 ACE 단백질이 분리되어 그 특성이 알려졌다. 그러나 돼지의 어떠한 ACE DNA 염기서열도 아직까지 보고 된 바는 없다. 그러므로 본 연구에서 reverse transcriptase-polymerase chain reaction(RT-PCR)을 이용하여 돼지의 신장 ACE cDNA를 클로닝하고 그 염기서열을 분석하였다. ACE cDNA는 1309개의 아미노산으로 구성되어 있으며 그 분자량은 150kDa이다. 염기서열로부터 유추한 아미노산의 서열을 분석한 결과, N 말단의 33개 아미노산이 signal peptide 역할을 하는 것으로 보이며, C 말단 근처의 짧은 transmembrane 영역은 세포막에 anchor역할을 하는 것으로 보인다. 돼지 신장의 ACE에서 두 개의 매우 유사한 amino acid peptidase domain은 tandem duplication 되어 있으며, 각각의 domain은 다른 포유류의 체세포 ACE들과 마찬가지로 putative metal-binding site(His-Glu-Met-Gly-His)를 하나씩 가지고 있는 것으로 나타났다. 돼지 신장 ACE 서열과 인간, 토끼, 쥐 등과 같은 포유류의 ACE 아미노산 서열들과의 상동성 비교는 진화과정 중 두 domain이 매우 잘 보전되어 왔음을 보여주고 있다.

• 한국정보과학회논문지:소프트웨어및응용
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• 제34권7호
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• pp.595-602
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• 2007

본 논문에서는 미지의 아미노산 서열이 신호 펩티다제 I에 의해 절단되는 분비성 단백질인지를 판별하고, 분비성 단백질일 경우에는 절단 위치를 예측하는 방법을 제안한다. 아미노산의 소수성을 이용한 전처리를 수행하여 분비성 단백질의 선도서열인 신호서열의 존재와 절단 위치를 추정한다. 전처리를 통해서 신호서열 아닌 서열을 초기에 제외함으로써 신호서열 예측의 정확도를 높인다. 지지벡터기계를 신호서열의 예측에 효과적으로 적용하기 위해서, 생물학적 정보와 관련된 아미노산 서열간의 거리를 제안한다. 아미노산의 세포내 위치를 예측할 수 있는 소수성 척도와 아미노산의 진화적인 관계를 나타낼 수 있는 치환행렬을 이용하여 아미노산 서열간의 거리를 정의한다. Swiss-Prot release 50 단백질 자료에 대하여 교차타당성 기법을 사용하여 실험한 결과 제안한 방법은 신호서열중에 98.9%를 신호서열로 판별하였고, 88%의 절단위치 예측정확도를 보였다. 기존의 방법과의 비교실험을 통해서 제안한 방법이 신호서열의 예측에 더욱 효과적임을 확인하였다.

• 한국미생물·생명공학회지
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• 제49권1호
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• pp.65-74
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• 2021

Serine proteases are the most versatile proteolytic enzymes with tremendous applications in various industrial processes. This study was designed to investigate the biochemical properties, critical residues, and the catalytic potential of alkaline serine protease using in-silico approaches. The primary sequence was analyzed using ProtParam, SignalP, and Phyre2 tools to investigate biochemical properties, signal peptide, and secondary structure, respectively. The three-dimensional structure of the enzyme was modeled using the MODELLER program present in Discovery Studio followed by Molecular Dynamics simulation using GROMACS 5.0.7 package with CHARMM36m force field. The proteolytic potential was measured by performing docking with casein- and keratin-enriched residues, while the effect of the inhibitor was studied using phenylmethylsulfonyl fluoride, (PMSF) applying GOLDv5.2.2. Molecular weight, instability index, aliphatic index, and isoelectric point for serine protease were 39.53 kDa, 27.79, 82.20 and 8.91, respectively. The best model was selected based on the lowest MOLPDF score (1382.82) and DOPE score (-29984.07). The analysis using ProSA-web revealed a Z-score of -9.7, whereas 88.86% of the residues occupied the most favored region in the Ramachandran plot. Ser327, Asp138, Asn261, and Thr326 were found as critical residues involved in ligand binding and execution of biocatalysis. Our findings suggest that bioengineering of these critical residues may enhance the catalytic potential of this enzyme.