A series of highly taxonomically diverse Trichoderma strains were investigated using proteomic approaches, to investigate the utility of protein profiles as taxonomic markers and to identify proteins of potential econ...A series of highly taxonomically diverse Trichoderma strains were investigated using proteomic approaches, to investigate the utility of protein profiles as taxonomic markers and to identify proteins of potential economic importance. Initial studies have focused on a comparison of single strains of T. aureoviride, T. saturnisporum, T. polysporum, T. longbrachiatum and T. spirale, along with two strains of T. harzianum. All seven strains were grown in synthetic medium supplemented with 2%(w/v) glycerol, to maximize the diversity of extracellular protein production. Samples of secreted protein were separated by 2D gel electrophoresis and will be characterized by MALDI-TOF peptide fingerprinting.展开更多
A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot (Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5’ untra...A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot (Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5’ untranslated region (UTR) of 84 bp, a 3’ UTR of 451 bp and an open reading frame (ORF) of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5’UTR of SmTCTP started with a 5’-terminal oligopyrimidine tract (5’-TOP), a typical feature for translationally controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known verte- brate TCTPs in a range of 58.8% to 64.1%. The length of fish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch (Lateolabrax japonicus) is 170 aa in length, while that of zebrafish (Danio rerio) and rohu (Labeo rohita) is 171 aa in length. North- ern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET30a-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further inves- tigation of the biological function of TCTP in fish.展开更多
为了优化肽质量指纹谱(peptide mass fingerprint,PMF)鉴定蛋白质的生物信息学分析条件。将牛碳酸酐酶2(carbonic anhydrase-2,CAH2)和人热休克蛋白70s(Hsp70s)进行2-DE分离、酶解,肽段经过MALDI-TOFMS分析得到PMF数据。选择Swissprot、...为了优化肽质量指纹谱(peptide mass fingerprint,PMF)鉴定蛋白质的生物信息学分析条件。将牛碳酸酐酶2(carbonic anhydrase-2,CAH2)和人热休克蛋白70s(Hsp70s)进行2-DE分离、酶解,肽段经过MALDI-TOFMS分析得到PMF数据。选择Swissprot、MSDB、NCBInr、Random等数据库和MASCOT与MS-Fit搜索引擎,以牛CAH2为模型优化搜索参数,结果表明:Swissprot是适合做蛋白PMF分析的数据库;主要参数最佳设置为:漏切位点数为1个,肽质量容错数为±1Da,同时肽质量类型选择平均分子质量比单同位素质量更便于候选蛋白的筛选。最后用人Hsp70s蛋白的PMF数据检验优化条件,结果表明,所选择的数据库及参数是可靠的。展开更多
文摘A series of highly taxonomically diverse Trichoderma strains were investigated using proteomic approaches, to investigate the utility of protein profiles as taxonomic markers and to identify proteins of potential economic importance. Initial studies have focused on a comparison of single strains of T. aureoviride, T. saturnisporum, T. polysporum, T. longbrachiatum and T. spirale, along with two strains of T. harzianum. All seven strains were grown in synthetic medium supplemented with 2%(w/v) glycerol, to maximize the diversity of extracellular protein production. Samples of secreted protein were separated by 2D gel electrophoresis and will be characterized by MALDI-TOF peptide fingerprinting.
文摘A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot (Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5’ untranslated region (UTR) of 84 bp, a 3’ UTR of 451 bp and an open reading frame (ORF) of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5’UTR of SmTCTP started with a 5’-terminal oligopyrimidine tract (5’-TOP), a typical feature for translationally controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known verte- brate TCTPs in a range of 58.8% to 64.1%. The length of fish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch (Lateolabrax japonicus) is 170 aa in length, while that of zebrafish (Danio rerio) and rohu (Labeo rohita) is 171 aa in length. North- ern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET30a-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further inves- tigation of the biological function of TCTP in fish.
文摘为了优化肽质量指纹谱(peptide mass fingerprint,PMF)鉴定蛋白质的生物信息学分析条件。将牛碳酸酐酶2(carbonic anhydrase-2,CAH2)和人热休克蛋白70s(Hsp70s)进行2-DE分离、酶解,肽段经过MALDI-TOFMS分析得到PMF数据。选择Swissprot、MSDB、NCBInr、Random等数据库和MASCOT与MS-Fit搜索引擎,以牛CAH2为模型优化搜索参数,结果表明:Swissprot是适合做蛋白PMF分析的数据库;主要参数最佳设置为:漏切位点数为1个,肽质量容错数为±1Da,同时肽质量类型选择平均分子质量比单同位素质量更便于候选蛋白的筛选。最后用人Hsp70s蛋白的PMF数据检验优化条件,结果表明,所选择的数据库及参数是可靠的。
