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作 者:曹赛[1,2] 戴红梅[1] 孙旭[1,3] 颛孙丹丹[1,2] 和金周[1,3] 司信喜[1] 李树龙[1] 方宏清[1] 陈惠鹏[1] 谢达平[2] 周长林[3]
机构地区:[1]军事医学科学院生物工程研究所,北京100071 [2]湖南农业大学生物安全科学技术学院,长沙410128 [3]中国药科大学生命科学与技术学院,南京210009
出 处:《中国科学:生命科学》2011年第10期1008-1015,共8页Scientia Sinica(Vitae)
基 金:国家自然科学基金(批准号:30780049);国家重点基础研究发展计划(批准号:2011CBA00801)资助项目
摘 要:胸腺肽β4(Tβ4)是N-末端乙酰化的43肽,具有多种重要生物学功能.其生物合成存在两大难点,即乙酰化修饰和小分子肽的表达.本研究发现来自古菌Sulfolobus solfataricus的乙酰化酶ssArd1可以催化Tβ4的N-末端乙酰化修饰.利用Red同源重组技术将ssArd1基因表达盒整合至E.coli BL21(DE3)染色体的lpxM位点上,构建了可以实现Tβ4N-末端乙酰化修饰的新型宿主E.coli BDA.将Tβ4编码基因融合在改造的微型Spl DnaX Intein的N端,并在Intein的C端添加His标签,构建了表达载体pET-Tβ4-Intein.在E.coli BDA中表达的融合蛋白,经镍亲和层析纯化后用β-巯基乙醇诱导融合蛋白切割释放小分子多肽,获得了具有N-末端乙酰化的Tβ4.Thymosin β4(Tβ4),a 43-amino acid Nα-acetylated peptide,has multiple significant biological functions.There are two bottlenecks to its biosynthesis:the difficulties in obtaining Nα-acetylation and expressing small peptides.In this study we found that ssArd1,an archaeal acetylase from Sulfolobus solfataricus can acetylate the N-terminal residue Ser of Tβ4.A modified E.coli BL21(DE3) with ssArd1-expressing cassette integrated in the locus of lpxM of chromosome by Red recombination was constructed and named as E.coli BDA for Nα-acetylation of proteins expressed in it.To obtain Nα-acetylated Tβ4 efficiently,a fusion protein,Tβ4-Intein,was constructed,in which Tβ4 and a His tag were fused respectively to the N-terminus and the C-terminus of a smallest mini-intein,136-amino acid Spl DnaX.After expression in E.coli BDA and purification by Ni-Sepharose affinity chromatography,the fusion protein was induced by β-mercaptoethanol to release Nα-acetylated Tβ4 through intein-mediated N-terminal cleavage.Three mutants of Tβ4 were prepared in the same way.All of Tβ4 and its three mutants have the ability to bind actin.This study laid a foundation for further investigation of the function and application of Tβ4.
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