• BMB Reports
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• 제43권12호
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• pp.801-806
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• 2010

The existence of glycine residues in long-chain scorpion toxins has been well documented. However, their role as analgesics has not been evaluated. To address this issue, we investigated the functional role of glycines in the C-terminal end of Chinese-scorpion toxin from Buthus martensii Karsch (BmK AGP-SYPU2) using site-directed mutagenesis and analgesic activity assays. Recombinant BmK AGP-SYPU2 and its mutants were efficiently expressed in E. coli and purified to homogeneity using immobilized metal ion affinity chromatography (IMAC) and cation exchange chromatography. The mouse-twisting test was used to detect the analgesic activity of BmK AGP-SYPU2 and its mutants. As a result, we identified glycines at the C-terminal end that, when altered, significantly affected analgesic activity. Also, Mut6566 was significantly decreased compared to BmK AGP-SYPU2. These data indicate that the glycines at the C-terminal end are important for the analgesic activity of BmK AGP-SYPU2.

• 공업화학
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• 제31권1호
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• pp.67-72
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• 2020

Biomaterials are frequently functionalized with Arg-Gly-Asp (RGD) peptides to provide cell adhesion sites. In this study, RGD peptides were enzymatically coupled on to the surface of fibroin microspheres. Papain exhibited a strong preference for dansyl phenylalanine for the peptide formation with fibroin microspheres. Thus, RGD1 peptide was designed to carry cysteine to both sides of the sequence, glycine as a spacer and two residues of phenylalanine at the C-terminal (CRGDCGFF). The enzymatic modification facilitated by an increasing amount of substrate and by the presence of organic solvent, dimethylsulfoxide at 25% (v/v). Microspheres coupled with RGD1, showed a significantly different precipitation property and an increased apparent volume, possibly due to the steric hindrance of RGD peptides on the surface. Transmission electron microscopy also confirmed the presence of cysteine residues in RGD1 coupled on the surface of microspheres stained with gold nanoparticles. RGD1-microspheres significantly facilitated the growth of murine fibroblast 3T3 cells even under non-adhesion culture conditions.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.169-177
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• 1992

토양으로부터 alkaline protease 생성능이 강한 Streptomyces griseus HC-1141을 분리하였으며, 효소생산의 최적 배양조건은 0.5 casein, 0.05 ammonium chloride, 0.1 ferrous sulfate, 2.0의 lactose, pH 8.0에서 84시간 배양했을 때이다. 효소의 정제는 ammonium sulfate 침전, DEAE-cellulose ion exchange chromatography, Sephadex G-150 gel filtration, crystallization으로 하여 53.23배 정제할 수 있었으며 polyacrylamide gel 전기영동상 단일밴드를 나타내었다.

• Journal of Plant Biotechnology
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• 제36권4호
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• pp.407-414
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• 2009

A cDNA clone encoding phytocystatin was isolated from Brassica rapa seedlings, through rapid amplification of cDNA ends (RACE). This gene (name as Brcpi1; GenBank accession no.: EF079953) had a total length of 881 bp with an open reading frame of 609 bp, and encoded predicted polypeptide of 203 amino acid (aa) residues including a putative N-terminal signal peptide. Other relevant regions found its sequence included the G and PW conserved aa motifs, and the consensus LARFAV sequence for phytocystatins and the reactive site QVVAG. The BrCPI1 protein shared 95, 94, 81, 80 and 78% identity with other CPI proterins isolated from Brassica oleracea (BoCPI-1), Arabidopsis thaliana (AtCY SB), Glycine max (GmCPI), Oryza sativa (OsCYS-2) and Zea may (ZmCPI) at amino acid level, respectively. Southern blot analysis showed that Brcpi1 was a low copy gene. Expression pattern analysis revealed that Brcpi1 was a tissue-specific expressing gene during reproductive growth and strongly expressed at mature seedling stages. Furthermore, overexpression of Brcpi1 in transgenic Arabidopsis was enhanced tolerance to salt and cold stresses. Meanwhile the juvenile seedling of Brcpi1 transgenic plants was not affected by various concentrations ABA in MS medium. Taken together, the results showed that Brcpi1 functioned as a cysteine protease inhibitor and it exhibited a protective agent against diverse types of abiotic stress, which induced this gene in a tissue- and stress-specific manner.

• 한국식품과학회지
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• 제31권6호
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• pp.1635-1641
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• 1999

재래식 메주로부터 분리한 Bacillus subtilis PCA203이 생산하는 protease를 분리 정제하였다. 먼저 효소 생산용 배지$(0.2%\;soytone,\;2%\;soluble\;starch,\;0.1%\;(NH_4)_2SO_4,\;0.1%\;CaCl_2,\;0.01%\;yeast\;extract,\;0.1%\;K_2HPO_4,\;0.1%\;KH_2PO_4)$를 이용하여 $30^{\circ}C$에서 20시간 배양한 다음, 원심분리하여 상징액을 분획한 후, 80% 포화 황산암모늄에 의한 염석과 CM Sephdex C-50 및 Sephadex G-100을 이용하여 비활성도 76.0 unit/mg, 수율 2.7%, 정제배수 7.6배로 효소를 정제하였다. 정제 단백질의 YMC-pack protein-RP column chromatography에 의한 순도검증에서 순도가 95% 이상인 것으로 나타났다. SDS-PAGE 분석에서 주 밴드의 분자량은 약 31.5 kDa이었고 아미노산 조성은 alanine, glycine, serine, valine의 함량이 많았으며 분자량 31,500 Da를 기준으로 하였을 경우 본 protease의 잔기수는 321잔기였다. RP-HPLC로 분획한 main peak의 N-terminal amino acid sequence를 확인한 결과 $Val^1-Pro^2-Tyr^3-Gly^4-Val^5-Ser^6-Gln^7-Gly^8-Lys^9-Ala^{10}$인 것으로 밝혀졌다.