带跨膜片段的α-PEC的分子构建

Molecular Designing of Phycoerythrocyanin α-subunit with Transmembrane Motif

通过分子设计构建一段来自人红细胞膜上的血型糖蛋白A(glycophorin A,GPA)中的跨膜片段基因,并连接到藻红蓝蛋白基因片段(pecA)的N端,将融合基因插入到表达载体pET30a进行表达。表达产物以包涵体形式存在,通过优化条件使其溶解,并与藻蓝胆素PCB在裂合异构酶PecE/PecF催化下进行体外重组,通过可逆光致变色活性大小确定包涵体溶解的最佳条件。结果表明,超声后的细胞加入终浓度8 mol/L尿素,0.2%Triton X-100和一定量的PecE和PecF,透析时采用含0.2%Triton X-100的尿素梯度透析可使重组活性最高。研究为可逆光致变色生物材料在生物光电材料中的应用打下了基础。

Glycophorin A existing in human erythrocyte membrane has a transmembrane motif （TS）, which can locate protein at membrane. In order to bind phycoerythrocyanin α-subunit（ α-PEC, i.e. PecA-PVB. PVB was abbreviation of phyeoviolobilin, and PecA was apo-protein of α-PEC） at membrane, TS was designed to fuse with PecA. By means of molecular biological methods, the DNA fragment of TS （ ts ） was amplified with PCR and cloned with vector pBluescript, and then ligated with PecA gene （pecA） to give ts-pecA DNA fragment. After cloning and .sequencing, ts-pec A was subeloned in expression vector pET30. When pET30-ts-pecA was expressed in E. coli, the expressed product TS-PecA existed in inclusion body. In order to renature TS-PecA, detergents （Tween 20, Tween 80 and Triton X-100） were tested. It was shown that Triton X-100 was best suitable for TS-PecA to be renatured. In the presence of 0.2% Triton X-100 and phyeoerythrocyanin lyase/isomerase （PeeE/PecF）, the denatured TS-PecA with urea Of 8 mol/L was well renatured by gradually dialyzing away urea. After its renaturation, TS-PecA could be well reconstituted with phyeocyanobilin （PCB） to produce TS-PecA-PVB under catalysis of PecE/PecF. α-PEC （i. e. PeeA-PVB） has reversible photochromic properties, and the designed TS-PeeA-PVB also has the properties, which was being tested to apply in photosensitive protein materials. Therefore, the design of TSyPecA and the further reconstitution of TSyPecA-PVB were important for researches that photochromic a-PEC proteins are applied as biological opto-electrie materials.