采用DEAE-FF离子交换和Sephdax G-15葡聚糖凝胶层析方法,从玉米肽中分离、纯化,获得具有抑制HepG2细胞增殖活性的短肽,采用高效液相色谱串联质谱电喷雾法(liquid chromatography-electrospray ionization tandem mass spectrometry,LC-E...采用DEAE-FF离子交换和Sephdax G-15葡聚糖凝胶层析方法,从玉米肽中分离、纯化,获得具有抑制HepG2细胞增殖活性的短肽,采用高效液相色谱串联质谱电喷雾法(liquid chromatography-electrospray ionization tandem mass spectrometry,LC-ESI-MS/MS)对其活性片段进行结构鉴定。结果表明:其短肽的一级结构为LPPYLP[命名为玉米六肽(corn peptides-6,CPs-6)],通过四唑盐[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]比色法试验发现,CPs-6具有抑制HepG2细胞增殖的效果,且对L-02细胞的毒性低。展开更多
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
Three angiotensin I converting enzyme(ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chrom...Three angiotensin I converting enzyme(ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chromatography and reverse-phase high performance liquid chromatography(RP-HPLC), were used to isolate the ACE inhibition peptides. The amino acid sequences of the peptides were identified as Ile-Asn-Asp, Val-Glu-Pro-Gly and Leu-Ala-Asp-Glu-Phe. The IC_(50) values of the purified peptides for ACE inhibition activity were 34.72 μmol L^(-1), 20.55 μmol L^(-1) and 22.77 μmol L^(-1), respectively. These results suggested that S. nudus proteins contain specific peptides that can be released by enzymatic hydrolysis. This study may provide an experimental basis for further systematic research, rational development and clinical utilization of sandworm resources.展开更多
The preliminary purification and antimicrobial mechanism of antimicrobial peptide from Antarctic Krill were studied in this paper. The results showed that the molecular weight range of antimicrobial polypeptide （CMC...The preliminary purification and antimicrobial mechanism of antimicrobial peptide from Antarctic Krill were studied in this paper. The results showed that the molecular weight range of antimicrobial polypeptide （CMCC-1） obtained by cation exchange chromatography was between 245-709D as detected by molecular sieve chromatography, and the minimum inhibition concentration （MIC） of CMCC-1 against Staphylococcus aureus was 5.0mgmL^-1. The antimicrobial mechanism of CMCC-1 was studied with S. aureus as indicator bacterium. Compared with control group, the results of the experimental group in which S. aureus was treated with CMCC-1 were as follows： l） CMCC-1 could inhibit cell division at logarithmic phase. 2） The protein and reducing sugar con- tent, and the conductivity of culture medium increased, and the activity of alkaline phosphatase and [3-galactosidase could be detected in the culture medium. 3） Observation under scanning electron microscope revealed that somatic morphology became irregular, and then somatic surface became coarse. The cell became much smaller, and most somatic ceils gathered. The boundary between cells became dim and finally fused as a whole. 4） Observation under transmission electron microscope showed that the surface of S. aureus became rough and the reproducing ability was restrained. The cell wall became thin and the cytoplasm shrunk. Substances inside cell leaked out, which caused cells death. 5） SDS-PAGE analysis showed that some bands disappeared, and the residual bands became vague. 6） The genomic DNA electrophoresis results showed that the genomic DNA bands ofS. aureus were not degraded but the brightness significantly reduced. Thus, it is supposed that CMCC-1 could destroy the cell wall and membrane of S. aureu, increase the cell membrane permeability and the leaking-out of intracellular substances, and thus cause the death ofS. aureu.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
Recombinant ancylostoma caninum anticoagulant peptide-5 （rAcAP5） has been reported to inhibit thrombin-activatable fibrinolysis inhibitor （TAFIa） activity and have thrombolytic effect. The present study was to scr...Recombinant ancylostoma caninum anticoagulant peptide-5 （rAcAP5） has been reported to inhibit thrombin-activatable fibrinolysis inhibitor （TAFIa） activity and have thrombolytic effect. The present study was to screen a strain expressing high-yield of rAcAP5 and to assess its thrombolytic effect on embolic middle cerebral artery occlusion （MCAO） model in rats. Codons encoding for AcAP5 were optimized. Six expression plasmids and eleven E. coli strains with different characteristics were used, a total of 66 recombinant expression strains were generated and the one with the highest yield was selected to express rAcAP5, which was purified through anion- and cation-exchange chromatography. The purity of rAcAP5 and its molecular weight were determined by HPLC and mass spectrometry, respectively. The thrombolytic effect of rAcAP5 was evaluated on embolic MCAO model in rats; regional cerebral blood flow （rCBF） was monitored with a Laser-Doppler flowmetry to test the occlusion and recanalization of MCA. The highest yield recombinant strain was C2566H/pTYB 1-rAcAP5. AcAP5 （28 mg） with 90% of purity was obtained from 1 L of cell culture. In rat embolic MCAO model, vehicle （normal saline） treatment did not change the rCBF, while treatment with rAcAP5 （50-200 μg/kg, i.v.） increased the rCBF in a dose-dependent manner. In conclusion, we prepared and characterized the rAcAP5 peptide and revealed its thrombolytic effect in embolic MCAO model and our results suggested that this peptide had the potential to be used as a thrombolytic agent.展开更多
文摘采用DEAE-FF离子交换和Sephdax G-15葡聚糖凝胶层析方法,从玉米肽中分离、纯化,获得具有抑制HepG2细胞增殖活性的短肽,采用高效液相色谱串联质谱电喷雾法(liquid chromatography-electrospray ionization tandem mass spectrometry,LC-ESI-MS/MS)对其活性片段进行结构鉴定。结果表明:其短肽的一级结构为LPPYLP[命名为玉米六肽(corn peptides-6,CPs-6)],通过四唑盐[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]比色法试验发现,CPs-6具有抑制HepG2细胞增殖的效果,且对L-02细胞的毒性低。
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.
基金supported by research grant of Guangxi Key Laboratory Traditional Chinese Medicine Quality Standards (No. GXGZZK201501)the Open Research Fund Program of Guangxi Key Laboratory of Marine Biotechnology (No. GLMBT-201407)+1 种基金partly supported by Shanghai Fengxian District Science and Technology Project (Nos. 20141001 and 20151205)Shanghai No. 6 People’s Medical Group Project and research project of Shanghai municipal health and Family Planning Commission (No. 201540027)
文摘Three angiotensin I converting enzyme(ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chromatography and reverse-phase high performance liquid chromatography(RP-HPLC), were used to isolate the ACE inhibition peptides. The amino acid sequences of the peptides were identified as Ile-Asn-Asp, Val-Glu-Pro-Gly and Leu-Ala-Asp-Glu-Phe. The IC_(50) values of the purified peptides for ACE inhibition activity were 34.72 μmol L^(-1), 20.55 μmol L^(-1) and 22.77 μmol L^(-1), respectively. These results suggested that S. nudus proteins contain specific peptides that can be released by enzymatic hydrolysis. This study may provide an experimental basis for further systematic research, rational development and clinical utilization of sandworm resources.
基金supported by the National Science and Technology Pillar Program(2013BAD13B03)National Natural Science Foundation of China(Grant 31201311)Special Scientific Research Funds for Central Non-profit Institutes,Yellow Sea Fisheries Research Institute(20603022012001)
文摘The preliminary purification and antimicrobial mechanism of antimicrobial peptide from Antarctic Krill were studied in this paper. The results showed that the molecular weight range of antimicrobial polypeptide （CMCC-1） obtained by cation exchange chromatography was between 245-709D as detected by molecular sieve chromatography, and the minimum inhibition concentration （MIC） of CMCC-1 against Staphylococcus aureus was 5.0mgmL^-1. The antimicrobial mechanism of CMCC-1 was studied with S. aureus as indicator bacterium. Compared with control group, the results of the experimental group in which S. aureus was treated with CMCC-1 were as follows： l） CMCC-1 could inhibit cell division at logarithmic phase. 2） The protein and reducing sugar con- tent, and the conductivity of culture medium increased, and the activity of alkaline phosphatase and [3-galactosidase could be detected in the culture medium. 3） Observation under scanning electron microscope revealed that somatic morphology became irregular, and then somatic surface became coarse. The cell became much smaller, and most somatic ceils gathered. The boundary between cells became dim and finally fused as a whole. 4） Observation under transmission electron microscope showed that the surface of S. aureus became rough and the reproducing ability was restrained. The cell wall became thin and the cytoplasm shrunk. Substances inside cell leaked out, which caused cells death. 5） SDS-PAGE analysis showed that some bands disappeared, and the residual bands became vague. 6） The genomic DNA electrophoresis results showed that the genomic DNA bands ofS. aureus were not degraded but the brightness significantly reduced. Thus, it is supposed that CMCC-1 could destroy the cell wall and membrane of S. aureu, increase the cell membrane permeability and the leaking-out of intracellular substances, and thus cause the death ofS. aureu.
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。
基金National Technology Graveness Special Purpose Fund(Grant No.2009ZX09301-010)
文摘Recombinant ancylostoma caninum anticoagulant peptide-5 （rAcAP5） has been reported to inhibit thrombin-activatable fibrinolysis inhibitor （TAFIa） activity and have thrombolytic effect. The present study was to screen a strain expressing high-yield of rAcAP5 and to assess its thrombolytic effect on embolic middle cerebral artery occlusion （MCAO） model in rats. Codons encoding for AcAP5 were optimized. Six expression plasmids and eleven E. coli strains with different characteristics were used, a total of 66 recombinant expression strains were generated and the one with the highest yield was selected to express rAcAP5, which was purified through anion- and cation-exchange chromatography. The purity of rAcAP5 and its molecular weight were determined by HPLC and mass spectrometry, respectively. The thrombolytic effect of rAcAP5 was evaluated on embolic MCAO model in rats; regional cerebral blood flow （rCBF） was monitored with a Laser-Doppler flowmetry to test the occlusion and recanalization of MCA. The highest yield recombinant strain was C2566H/pTYB 1-rAcAP5. AcAP5 （28 mg） with 90% of purity was obtained from 1 L of cell culture. In rat embolic MCAO model, vehicle （normal saline） treatment did not change the rCBF, while treatment with rAcAP5 （50-200 μg/kg, i.v.） increased the rCBF in a dose-dependent manner. In conclusion, we prepared and characterized the rAcAP5 peptide and revealed its thrombolytic effect in embolic MCAO model and our results suggested that this peptide had the potential to be used as a thrombolytic agent.
