CAJ | 학술논문

将来源于Alcaligenes A-6的D-氨基酰化酶基因用大肠杆菌中的丰沛密码子替换,利用化学和基于聚合酶链反应(PCR)技术的酶促方法进行基因全合成,利用pET-32a构建重组表达载体pET-dan,转化进E. coil BL21(DE3)中进行融合表达.经SDS-PAGE电泳、Western-blot检测和活性测定发现, D-ANase可在大肠杆菌中高效表达,目的蛋白可达到菌体总蛋白的69.2%,密码子优化后基因构建的工程菌发酵活性为96 U/mL,重组蛋白经超声细胞破碎及Ni2+柱亲和层析纯化,比活可达1692.3 U/mg,纯度可达95%以上.
장래원우Alcaligenes A-6적D-안기선화매기인용대장간균중적봉패밀마자체환,이용화학화기우취합매련반응(PCR)기술적매촉방법진행기인전합성,이용pET-32a구건중조표체재체pET-dan,전화진E. coil BL21(DE3)중진행융합표체.경SDS-PAGE전영、Western-blot검측화활성측정발현, D-ANase가재대장간균중고효표체,목적단백가체도균체총단백적69.2%,밀마자우화후기인구건적공정균발효활성위96 U/mL,중조단백경초성세포파쇄급Ni2+주친화층석순화,비활가체1692.3 U/mg,순도가체95%이상.
The codons of D-ANase gene from Alcaligenes A-6 were substituted by the codons abundant in E. coli. , then the D-ANase gene was synthesized by the two-step method based on PCR technology. Synthetic gene and pET-32a vector were digested with BglII and XhoⅠ, ligated by T4 DNA ligase. The ligation mix-ture transformed into E. coli BL21(DE3) competent cell. Recombinant protein was detected by SDS-PAGE, Western-blot and activity assay. D-ANase can be expressed efficiently in E. coli and the expressed protein content can reach to 69.2% of the total bacterial protein content. In addition, the fermentation activity can achieve 96 U/mL. After the ultrasonic cell disruption, the recombinant protein was purified by Ni2+ affinity chromatography column. The specific activity of the purified recombinant enzyme was 1692.3 U/mg and the purity could be up to 95%. Furthermore a firm foundation was laid for the industrial use of the D-ANase.