Yu Rong-Jie, Hong An, Dai Yun, Gao Yuan
Bio-engineering Institute of Jinan University, Guangzhou 510632, China.
Acta Biochim Biophys Sin (Shanghai). 2004 Nov;36(11):759-66. doi: 10.1093/abbs/36.11.759.
In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the PACAP-intein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by DTT and the rhPACAP was released from the chitin-bound intein tag. The activity of the rhPACAP to stimulate cyclic AMP accumulation was detected using the human pancreas carcinoma cells SW1990. Twenty-two milligrams of rhPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. The preliminary biological assay indicated that the rhPACAP, which has an extra Met at its N-terminus compared with the native human PACAP, had the similar activity of stimulating cAMP accumulation with the standard PACAP38 in the SW1990 cells. A new efficient production procedure of the active recombinant human PACAP was established.
为了通过内含肽介导的单柱纯化高效获得重组人垂体腺苷酸环化酶激活肽（PACAP），合成了编码人PACAP的基因并将其克隆到大肠杆菌表达载体pKYB中。将重组载体pKY-PAC转入大肠杆菌ER2566细胞，目标蛋白作为与可自我切割亲和标签N端融合的蛋白过量表达。通过几丁质亲和层析纯化PACAP-内含肽-CBD融合蛋白后，用二硫苏糖醇（DTT）诱导内含肽的自我切割活性，rhPACAP从几丁质结合的内含肽标签上释放出来。使用人胰腺癌细胞SW1990检测rhPACAP刺激环磷酸腺苷（cAMP）积累的活性。通过单柱纯化从1升诱导培养物中获得了22毫克纯度超过98%的rhPACAP。初步生物学检测表明，与天然人PACAP相比，N端有一个额外甲硫氨酸（Met）的rhPACAP在SW1990细胞中具有与标准PACAP38相似的刺激cAMP积累的活性。建立了一种活性重组人PACAP的高效生产新方法。
