Antimicrobial proteins and peptides had been found from a wide variety of organisms in the last few years These molecules have attracted much research interest because of their biochemical diversity, broad specificity...Antimicrobial proteins and peptides had been found from a wide variety of organisms in the last few years These molecules have attracted much research interest because of their biochemical diversity, broad specificity on anti-viral, anti-bacterial, anti-fungi, anti-protozoan parasites, anti-tumoural, and wound-healing effects. Antimicrobial proteins and peptides play key roles in innate immunity. They interact directly with bacteria and kill them. The brown-spotted grouper, Epinephelusfario, is an important marine fish cultured in southem China. Recently, bacteria and virus have caused high mortality in E. fario cultures, but its endogenous antimicrobial peptides and proteins have not been explored. An antimicrobial component was found from the skin homogenate of E. fario. After the skin homogenate was digested with trypsin, its antimicrobial activity was lost, which showed that the antimicrobial component is a protein. The antimicrobial protein （Efap） was purified from the skin homogenate of E. fario by successive ion-exchange and gel filtration chromatography. Efap was demonstrated to be single protein band by SDS-PAGE, with the apparent molecular weight of 41 kD. Efap exhibited antimicrobial activity both for the Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis, and for the Gram-negative bacteria, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio fluvialis, Pasteurella multocida, Aeromonas hydrophila, Eschrrichiu coli, and Pseudomonas aeruginosa. Except A. hydrophila, P. aeruginosa, and E. coli （MIC〉20 mol/L）, most of the tested Gram-negative bacteria were sensitive to Efap （MIC〈20 mol/L）. Interestingly, Efap showed potent antimicrobial activity against Gram-positive bacteria S. aureus （MIC 5-10 mol/L） but comparatively weak antimicrobial activity against M. luteus and B. subtilis. The broad antimicrobial activities of Efap suggest that it contributes to the innate host defence of E. fario.展开更多
目的:对重组Arg^(34)-胰高血糖素样肽-1(Arg^(34)-Glucagon-like Peptide-1[7-37],Arg^(34)-GLP-1[7-37])生产制备过程中的潜在杂质进行分离纯化和结构确证。方法:将发酵后的料液进行高效液相色谱分析检测,利用离子交换色谱和制备液相...目的:对重组Arg^(34)-胰高血糖素样肽-1(Arg^(34)-Glucagon-like Peptide-1[7-37],Arg^(34)-GLP-1[7-37])生产制备过程中的潜在杂质进行分离纯化和结构确证。方法:将发酵后的料液进行高效液相色谱分析检测,利用离子交换色谱和制备液相纯化得到Arg^(34)-GLP-1[7-37]纯品和杂质组分,使用纯品进行高温、酸碱等不同条件下的破坏实验,来探知杂质产生机理,利用MALDI-TOF质谱、蛋白质全序列测序等方式推断杂质的分子量与结构。结果:经阴离子交换色谱及反相制备纯化后,Arg^(34)-GLP-1[7-37]的纯度可达97.9%。分离出3种主要杂质并将其纯化至91.4%,99.2%和96.7%。基质辅助激光解吸电离飞行时间质谱(Matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF)测试其[M+H]+分子量分别为1033.5446,2744.2397和3112.4475,通过破坏实验及Arg^(34)-GLP-1[7-37]的氨基酸序列来推测杂质1、杂质3与杂质9可能序列分别为E-F-I-A-W-L-V-R,H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W,H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-R。将纯化前后的成分进行比对,表明上述3个主要杂质是由发酵过程产生,通过极端条件处理这些杂质,其含量会显著增加。本研究解析的3个杂质结构及机理探究均为首次发表。结论:由于重组Arg^(34)-GLP-1[7-37]在高温、过酸过碱及氧化环境中不稳定,为防止纯化过程中主产物降解,应注意避免这些条件。本研究为胰高血糖素样肽-1(glucagon-like peptide1,GLP-1)及其类似物的研究指出了一些应避免的误区。展开更多
基金Key Research Program for International Cooperation(2005DFA30610)Program for New Century Excellent Talents in University(NCET-05-0755)+2 种基金National Natural Science Foundation(30700128)Natural Science Foundation of Hainan Province(80623)Research Foundation of Education Department of Hainan Province(Hj200731)
文摘Antimicrobial proteins and peptides had been found from a wide variety of organisms in the last few years These molecules have attracted much research interest because of their biochemical diversity, broad specificity on anti-viral, anti-bacterial, anti-fungi, anti-protozoan parasites, anti-tumoural, and wound-healing effects. Antimicrobial proteins and peptides play key roles in innate immunity. They interact directly with bacteria and kill them. The brown-spotted grouper, Epinephelusfario, is an important marine fish cultured in southem China. Recently, bacteria and virus have caused high mortality in E. fario cultures, but its endogenous antimicrobial peptides and proteins have not been explored. An antimicrobial component was found from the skin homogenate of E. fario. After the skin homogenate was digested with trypsin, its antimicrobial activity was lost, which showed that the antimicrobial component is a protein. The antimicrobial protein （Efap） was purified from the skin homogenate of E. fario by successive ion-exchange and gel filtration chromatography. Efap was demonstrated to be single protein band by SDS-PAGE, with the apparent molecular weight of 41 kD. Efap exhibited antimicrobial activity both for the Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis, and for the Gram-negative bacteria, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio fluvialis, Pasteurella multocida, Aeromonas hydrophila, Eschrrichiu coli, and Pseudomonas aeruginosa. Except A. hydrophila, P. aeruginosa, and E. coli （MIC〉20 mol/L）, most of the tested Gram-negative bacteria were sensitive to Efap （MIC〈20 mol/L）. Interestingly, Efap showed potent antimicrobial activity against Gram-positive bacteria S. aureus （MIC 5-10 mol/L） but comparatively weak antimicrobial activity against M. luteus and B. subtilis. The broad antimicrobial activities of Efap suggest that it contributes to the innate host defence of E. fario.
文摘目的:对重组Arg^(34)-胰高血糖素样肽-1(Arg^(34)-Glucagon-like Peptide-1[7-37],Arg^(34)-GLP-1[7-37])生产制备过程中的潜在杂质进行分离纯化和结构确证。方法:将发酵后的料液进行高效液相色谱分析检测,利用离子交换色谱和制备液相纯化得到Arg^(34)-GLP-1[7-37]纯品和杂质组分,使用纯品进行高温、酸碱等不同条件下的破坏实验,来探知杂质产生机理,利用MALDI-TOF质谱、蛋白质全序列测序等方式推断杂质的分子量与结构。结果:经阴离子交换色谱及反相制备纯化后,Arg^(34)-GLP-1[7-37]的纯度可达97.9%。分离出3种主要杂质并将其纯化至91.4%,99.2%和96.7%。基质辅助激光解吸电离飞行时间质谱(Matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF)测试其[M+H]+分子量分别为1033.5446,2744.2397和3112.4475,通过破坏实验及Arg^(34)-GLP-1[7-37]的氨基酸序列来推测杂质1、杂质3与杂质9可能序列分别为E-F-I-A-W-L-V-R,H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W,H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-R。将纯化前后的成分进行比对,表明上述3个主要杂质是由发酵过程产生,通过极端条件处理这些杂质,其含量会显著增加。本研究解析的3个杂质结构及机理探究均为首次发表。结论:由于重组Arg^(34)-GLP-1[7-37]在高温、过酸过碱及氧化环境中不稳定,为防止纯化过程中主产物降解,应注意避免这些条件。本研究为胰高血糖素样肽-1(glucagon-like peptide1,GLP-1)及其类似物的研究指出了一些应避免的误区。
