通过染色体整合表达古菌乙酰化酶合成N-末端乙酰化胸腺肽β4

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.

Biosynthesis of Nα-acetylated Thymosin β4 by Co-expressing an Archaeal Acetylase Integrated in Chromosome of Escherichia coli

Thymosin β4 (Tβ4), a 43-amino acid N^α-acetylated peptide, has multiple significant biological functions. There are two bottlenecks to its biosynthesis: the difficulties in obtaining N^α-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 N^α-acetylation of proteins expressed in it. To obtain N^α-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 N^α-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.