含组氨酸标签的枯草杆菌5-氨基酮戊酸脱水酶的高效表达

High level expression of His-tagged 5-aminolevulinate dehydratase from Bacillus subtilis

以枯草杆菌基因组为模板,通过PCR扩增枯草杆菌编码5-氨基酮戊酸脱水酶基因hemB。将NdeⅠ和EcoRⅠ双酶切的纯化产物插入同样处理的pET-28 a中构建成表达载体。表达蛋白的N端含有6个组氨酸标签。构建的表达载体转入大肠杆菌BL21(DE3),在IPTG诱导下,重组蛋白获得高效表达。SDS-PAGE检测到41 kD的特异带,表达菌株的上清检测到ALAD的比活为337 mU.mg-1,表明组氨酸标签不显著影响酶活。

The hemB gene from Bacillus subtilis encoding 5-aminolevulinate dehydratase （ALAD） was amplified by PCR, using the B. subtilis genome as the template. The purified hemB gene digested with Nde Ⅰ and EcoR Ⅰ was inserted into pET-28a vector treated with the same manner to yield the recombinant vector, which encoded ALAD with His6-tag at N-terminus. The constructed expression vector was transformed into Escherchia coli BL21 （DE3）. Upon the induction with 0.5 mmol· L^-1 IPTG, a specific band at 41 kD was displayed on SDS-PAGE. About 337 mU · mg^-1 specific activity for ALAD was analyzed from the crude of the induced strains, suggesting that His6-tag fusion did not affect the enzyme activity obviously.