CONSTRUCTION AND EXPRESSION OF SYNTHETIC WILD-TYPE AND MUTANT-GENES ENCODING PORCINE PANCREATIC COLIPASE - TRYPTOPHAN FLUORESCENCE STUDIES
CONSTRUCTION AND EXPRESSION OF SYNTHETIC WILD-TYPE AND MUTANT-GENES ENCODING PORCINE PANCREATIC COLIPASE - TRYPTOPHAN FLUORESCENCE STUDIES
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DOI：
10.1016/0167-4781(91)90173-j
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发表时间：
1991-07-23
期刊：
影响因子：
--
通讯作者：
BEHNKE, WD
中科院分区：
文献类型：
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作者：
ERNST, EG;BEHNKE, WD
根据猪胰脏脂肪酶(CLP)的已知氨基酸序列(95个氨基酸残基)，设计并组装了一个297个碱基的基因。将CLP编码基因(CLP)插入到小表达载体pUC8中的LacZ基因片段中，并克隆到大肠杆菌JM109中，优化了其在细菌中的表达。该结构的表达产生了大约一个蛋白质。11 kDa，相当于中电，M(R)为10336，在N-末端加上10个额外的氨基酸。将重组CLP(ReCLP)从细菌包涵体中溶解，纯化后复性。然后通过定点突变构建了一个突变的CLP基因，将Tyr-55改变为Trp。由于猪CLP不含色氨酸，该策略为生物物理研究提供了一种带有内部荧光探针的蛋白质。荧光光谱分析证实突变蛋白中存在色氨酸。野生型和突变型CLP都能与亲和纯化的兔抗CLP抗体反应，该抗体是针对天然CLP的。Tyr-55到Trp的交换不影响reCLP的活性。对reCLP与胆盐牛磺酸脱氧胆酸盐(TDOC)相互作用的荧光研究表明，突变体reCLP疏水结合部位的Trp-55插入了胆盐胶束的内部。
Based on the known (95-residue) amino acid (aa) sequence of porcine pancreatic colipase (CLP), a cofactor of pancreatic lipase, a 297 bp gene was designed and assembled from eight synthetic, overlapping DNA fragments. Optimized for expression in bacteria, the CLP-encoding gene (CLP) was inserted into the lacZ gene fragment contained in the small expression vector, pUC8, and cloned in Escherichia coli JM109. Expression of this construct yielded a protein approx. 11 kDa in size, equivalent to CLP, with an M(r) of 10336, plus ten additional amino acids at the N-terminus. The recombinant CLP (reCLP) was solubilized from bacterial inclusion bodies and then purified and refolded. A mutant CLP gene, changing Tyr-55 to Trp, was then constructed by site-directed mutagenesis. Since porcine CLP contains no Trp, this strategy provided a protein with an internal fluorescent probe for biophysical studies. The presence of Trp in the mutant protein was confirmed using fluorescence spectroscopy. Both wild-type (wt) and mutant reCLP reacted on Western blots with an affinity-purified rabbit anti-CLP antibody, raised against native CLP. The Tyr-55 to Trp exchange did not affect the activity of reCLP. Fluorescence studies of the interaction between reCLP and the bile salt, taurodeoxycholate (TDOC), showed that Trp-55 in the hydrophobic binding site of mutant reCLP inserted into the interior of the bile salt micelle.