烟曲霉几丁质酶基因的克隆与表达

Cloning and Expression of Chitinase Gene from Aspergillus fumigatus YJ-407

Chi4 4是烟曲霉 (Aspergillusfumigatus)YJ 4 0 7产生的一种胞外几丁质酶。通过用真菌几丁质酶保守氨基酸序列与Chi4 4的N 端序列检索烟曲霉部分基因组序列数据库 ,获得一个编号为contig5 5 5的烟曲霉基因组序列 ,可能包含烟曲霉几丁质酶的基因。根据检索结果用RT PCR方法从烟曲霉YJ 4 0 7中克隆到 1 4kb的cDNA片段 ,该cDNA的ORF编码一个 395个氨基酸的蛋白 ,分子量为 4 3 6kD。对其推导氨基酸序列分析表明该蛋白与其它真菌来源的几丁质酶同源 ,而且活性中心与人巨噬细胞几丁质酶高度同源。该cDNA已在E .coli与PichiapastorisGS115中获得表达 ,分别获得 4 3kD和 4 4kD的重组蛋白 ,两种重组蛋白均有几丁质酶活性。与野生酶相比 ,大肠杆菌表达的 4 3kD重组酶及Pichia酵母表达的 4 4kD重组酶稳定性下降 ,说明Chi4

Chi44 is an extracellular chitinase produced by Aspergillus fumigatus YJ-407. By blast-searching with the conserved amino acid sequences that were found in fungal chitinases and N-terminal sequence obtained from purified chitinase, contig555 was retrieved from unfinished A. fumigatus genome database. Based on the result of blast search, a 1.4-kb cDNA was isolated from A. fumigatus YJ-407 by reverse transcriptase-PCR. The open reading frame encodes a protein of 395 amino acids with a calculated molecular mass of 43.6kD. The predicted protein exhibited a high homology with the chitinases from other fungi at the amino acid sequence level. Also, the active site region was found to be highly homologous to human macrophage chitotriosidase. When the cDNA was expressed in E. coli and Pichia pastoris GS115, respectively, both 43kD protein induced in E. coli and 44kD protein induced in Pichia GS115 were active. However, these two recombinant proteins displayed decreases in stabilities, suggesting that the glycosylation played an important role in stabilization.