黑曲霉脂肪酶:基因克隆、表达及体外复性

Aspergillus niger F044 Lipase:Gene Cloning,Overexpression and in vitro Refolding

根据黑曲霉F044脂肪酶N-端氨基酸序列,运用生物信息学方法,找到与黑曲霉脂肪酶基因同源的候选基因A84689。根据该基因序列,设计引物直接PCR扩增得到黑曲霉脂肪酶全长基因anl。anl全长1044bp,含3个内含子,编码297个氨基酸(含信号肽27个氨基酸),与其它脂肪酶基因没有明显同源性。将编码成熟脂肪酶的anl连接到pET28a载体上得到重组表达质粒,转化大肠杆菌BL21(De3),诱导表达并纯化出目的蛋白。通过大量稀释和DEAESepharoseFastFlow层析相结合的方法,变性后的纯化蛋白在体外实现再折叠复性。

From the N-termlnal amino acid sequence of the Aspergillus niger F044 lipase, a potential homologous gene A84689 to the anl（the gene encoding the Aspergillus niger lipase） was found by means of bioinformatics. Based on the nucleotide sequence of the A84689, primers were designed to amplify anl. Nucleotide sequencing of the genomic anl gene revealed an open reading frame of 1 044 nueleotides, containing three introns（54, 45 and 51 nucleotides）. The deduced amino acid sequence of the anl gene corresponds to 297 amino acid residues including a signal sequence of 27 amino acid residues. The cloned cDNA coding for mature Anl（the protein of the Aspergillus niger lipase） was overexpressed in Escherichia coli BL21 （De3）, and the recombinant Anl was purified. The denatured recombinant Anl by 8mol/L urea was refolded in vitro by dilution and DEAE Sepharose Fast Flow chromatography.