Expression and purification of human keratinocyte growth factor 2 by fusion with SUMO.
Expression and purification of human keratinocyte growth factor 2 by fusion with SUMO.
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与 SUMO 融合表达和纯化人角质形成细胞生长因子 2
DOI：
10.1007/s12033-008-9135-7
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发表时间：
2009-05
影响因子：
2.6
通讯作者：
Li X
中科院分区：
文献类型：
--
作者：
Wu X;Nie C;Huang Z;Nie Y;Yan Q;Xiao Y;Su Z;Huang Y;Xiao J;Zeng Y;Tan Y;Feng W;Li X
小泛素相关修饰子（Small ubiquitin-related modifier， SUMO）融合系统已被证明可以有效地增强目标蛋白的表达并防止其降解。我们发现SUMO与人角质细胞生长因子2 （hKGF-2）基因融合是可行的，并且显著提高了蛋白的表达和效率。将融合至六组氨酸标签的SUMO基因与hKGF-2基因组成的融合DNA片段通过PCR扩增，插入表达载体pET28a，构建重组质粒pET28a-SUMO-hKGF-2。将质粒转入大肠杆菌rosettatm2 （DE3）中，在30℃条件下表达重组融合蛋白SUMO-hKGF-2，最终浓度为0.4 mM的IPTG诱导，融合蛋白的表达量可达细胞总蛋白的30%。融合蛋白采用Ni-NTA亲和层析纯化。经Sephadex G-25层析脱盐后，六组氨酸-SUMO- hkgf -2被SUMO蛋白酶消化。重组hKGF-2经Ni-NTA柱再次纯化，纯度约为95%，总产量为13.9 mg/l。有丝分裂性实验结果表明，重组hKGF-2能显著促进正常大鼠肾上皮细胞（NRK-52E）的增殖。
Small ubiquitin-related modifier (SUMO) fusion system has been shown to be efficient for enhancing expression and preventing degradation of the target protein. We showed herein that SUMO fusion to human keratinocyte growth factor 2 (hKGF-2) gene was feasible and it significantly enhanced protein expression and its efficiency. The fusion DNA fragment composed of SUMO gene, which was fused to hexahistidine tag, and hKGF-2 gene was amplified by PCR and inserted into the expression vector pET28a to construct the recombinant plasmid, pET28a-SUMO-hKGF-2. The plasmid was then transformed intoEscherichia coliRosettaTM2(DE3), and the recombinant fusion protein SUMO-hKGF-2 was expressed at 30°C for 6 h, with the induction of IPTG at the final concentration of 0.4 mM. The expression level of the fusion protein was up to 30% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography. After desalting by Sephadex G-25 size exclusion chromatography, the hexahistidine-SUMO-hKGF-2 was digested by SUMO proteases. The recombinant hKGF-2 was purified again with Ni-NTA column and the purity was about 95% with a total yield of 13.9 mg/l culture. The result of mitogenicity assay suggests that the recombinant hKGF-2 can significantly promote the proliferation of normal rat kidney epithelial (NRK-52E) cells.
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影响因子：
1.6
作者：
Zuo X;Mattern MR;Tan R;Li S;Hall J;Sterner DE;Shoo J;Tran H;Lim P;Sarafianos SG;Kazi L;Navas-Martin S;Weiss SR;Butt TR
通讯作者：
Butt TR
影响因子：
4.1
作者：
Wang Yanchun;Yuan Shengling;Zhang Zhaoshan
通讯作者：
Zhang Zhaoshan
影响因子：
1.6
作者：
Luo, YD;Cho, HH;McKeehan, WL
通讯作者：
McKeehan, WL
DOI：
10.1016/j.bbrc.2004.07.189
发表时间：
2004-09-24
影响因子：
3.1
作者：
De Marco, V;Stier, G;de Marco, A
通讯作者：
de Marco, A
影响因子：
2.2
作者：
Jimenez, PA;Rampy, MA
通讯作者：
Rampy, MA