Synonymous codons have different frequencies of usage in many species.Based on the frequency of usage,the codons can be divided into two groups,rare codons and abundant codons.Rare codons are found to be enriched at t...Synonymous codons have different frequencies of usage in many species.Based on the frequency of usage,the codons can be divided into two groups,rare codons and abundant codons.Rare codons are found to be enriched at the start regions of genes,and it is assumed that these codons can reduce elongation speed of genes.However,the rare codon usage in different genomic regions of mollusks and their relationship with selective pressure has not been systematically investigated.In this study,the patterns of rare codon usage are characterized at whole genome level,and their relationship with selective pressures is investigated in Crassostrea gigas.The rare codons are enriched at the start regions of genes with high and medium expression levels,and their proportion is higher than those in the genes with low expression level.The genes with longer coding sequences and more exon numbers have lower fraction of rare codons at start regions.Rare codons have lower level of nucleotide diversity and higher frequency of rare mutations at start regions.This work is the first comprehensive investigation of the relationships between rare codon usage and some intrinsic genetic factors in mollusca species.The results suggest that the selective pressures play an important role in shaping the rare codon usage in the C.gigas genome.展开更多
【目的】为提高重组人ADAM(A Disintegrin And Metalloproteinase)15去整合素结构域蛋白(rhADAM15)的表达水平。【方法】在详尽分析rhADAM15的cDNA和GST(谷胱甘肽-S-转移酶)-ADAM15结构的基础上,选择表达宿主菌并对表达质粒进行改造。...【目的】为提高重组人ADAM(A Disintegrin And Metalloproteinase)15去整合素结构域蛋白(rhADAM15)的表达水平。【方法】在详尽分析rhADAM15的cDNA和GST(谷胱甘肽-S-转移酶)-ADAM15结构的基础上,选择表达宿主菌并对表达质粒进行改造。【结果】(1)选择能为大肠杆菌稀有密码子提供额外tRNA的Escherichia coli.Rosetta(DE3)作为宿主菌,将质粒pGEX-ADAM15转化于其中在最佳诱导表达条件下获得298mg/L融合蛋白GST-ADAM15;(2)采用PCR体外定点突变技术将目标蛋白编码区稀有密码子GGA(Gly425)替换为GGC,使融合蛋白表达水平提高9.4%;(3)通过消除凝血酶识别序列附近的Pro-Glu-Phe残基,提高凝血酶酶切效率,使rhADAM15产量提高了35.7%;(4)在GGA替换为GGC基础上切除"Pro-Glu-Phe"残基,使rhADAM15产量提高到68mg/L,比分别切除"Pro-Glu-Phe"残基、GGA替换为GGC和野生型提高了19.2%、51.1%和61.9%。【结论】这一结果表明,在充分认识目标蛋白特性的基础上定向选择表达宿主并改造表达质粒能实现外源蛋白高水平表达。展开更多
基金supported by the National Natural Science Foundation of China(No.11701546).
文摘Synonymous codons have different frequencies of usage in many species.Based on the frequency of usage,the codons can be divided into two groups,rare codons and abundant codons.Rare codons are found to be enriched at the start regions of genes,and it is assumed that these codons can reduce elongation speed of genes.However,the rare codon usage in different genomic regions of mollusks and their relationship with selective pressure has not been systematically investigated.In this study,the patterns of rare codon usage are characterized at whole genome level,and their relationship with selective pressures is investigated in Crassostrea gigas.The rare codons are enriched at the start regions of genes with high and medium expression levels,and their proportion is higher than those in the genes with low expression level.The genes with longer coding sequences and more exon numbers have lower fraction of rare codons at start regions.Rare codons have lower level of nucleotide diversity and higher frequency of rare mutations at start regions.This work is the first comprehensive investigation of the relationships between rare codon usage and some intrinsic genetic factors in mollusca species.The results suggest that the selective pressures play an important role in shaping the rare codon usage in the C.gigas genome.
文摘【目的】为提高重组人ADAM(A Disintegrin And Metalloproteinase)15去整合素结构域蛋白(rhADAM15)的表达水平。【方法】在详尽分析rhADAM15的cDNA和GST(谷胱甘肽-S-转移酶)-ADAM15结构的基础上,选择表达宿主菌并对表达质粒进行改造。【结果】(1)选择能为大肠杆菌稀有密码子提供额外tRNA的Escherichia coli.Rosetta(DE3)作为宿主菌,将质粒pGEX-ADAM15转化于其中在最佳诱导表达条件下获得298mg/L融合蛋白GST-ADAM15;(2)采用PCR体外定点突变技术将目标蛋白编码区稀有密码子GGA(Gly425)替换为GGC,使融合蛋白表达水平提高9.4%;(3)通过消除凝血酶识别序列附近的Pro-Glu-Phe残基,提高凝血酶酶切效率,使rhADAM15产量提高了35.7%;(4)在GGA替换为GGC基础上切除"Pro-Glu-Phe"残基,使rhADAM15产量提高到68mg/L,比分别切除"Pro-Glu-Phe"残基、GGA替换为GGC和野生型提高了19.2%、51.1%和61.9%。【结论】这一结果表明,在充分认识目标蛋白特性的基础上定向选择表达宿主并改造表达质粒能实现外源蛋白高水平表达。
