趋化因子SDF-1β在大肠杆菌中的表达及其纯化

Expression and purification of recombinant human chemokine SDF-1β in E.coli

目的 重组人SDF 1 β在大肠杆菌中表达并获得纯化的有生物学活性的SDF 1 β蛋白。 方法 以pET32a( + ) SDF 1为人SDF 1 β克隆基因的表达载体 ,以大肠杆菌AD494(DE3)pLysS为表达菌 ,在IPTG诱导下表达出一个由 2 30个氨基酸残基组成的硫氧还蛋白 (thioredoxin) SDF 1 β的融合蛋白 ( 2 6× 1 0 3)。经细菌裂解、金属离子螯合亲和层析、肠激酶消化以游离SDF 1 β、阳离子交换层析和逆向高效液相层析等步骤纯化目的蛋白。以蛋白印迹、纯化产物的N端氨基酸测序及配体结合试验、微生理监测术鉴定纯化产物的生化性质及生物学活性。结果 在经诱导的发酵菌中融合蛋白占总菌体蛋白的 1 0 %～ 1 5 %。从 1L发酵菌液中可获得高纯度目的蛋白约 40 0 μg。蛋白印迹实验及N端氨基酸测序皆证实纯化终产物为SDF 1 β(由 71个氨基酸残基组成 ,7.8× 1 0 3)。配体结合试验及Cytosensor表明 ,该产物能与CXCR4结合 [Kd =( 1 2 .2 0± 2 .99)nmol L]并引起CXCR4表达细胞的信号传导。结论 采用本方法可获得高表达的 ,高纯度的具有与天然SDF 1 β相似活性的重组人SDF 1 β。

Objective To obtain recombinant human SDF 1β expressed in E.coli and purify SDF 1β with biological activity from the bacterium. Methods A thioredoxin SDF 1β fusion protein(26×10 3) composed of 230 amino acid residues was expressed in E.coli AD494(DE3)pLysS under the induction of IPTG when pET32a(+) SDF 1β was used as an expression vector. Purified SDF 1β was produced through following procedure: bacteria lysis, metal chelated affinity chromatography(MAC),enterokinase digestion to free SDF 1β from fusion protein, cation exchange chromatography (CEC) and reverse phase high performance liquid chromatography(RP HPLC). Western blotting with anti SDF 1β monoclonal antibody(mAb), N terminal amino acid sequencing, ligand binding assay and Cytosensor/microphysiometry were used to investigate the biochemical characters and biological activities of the purified SDF 1β. Results About 10%-15% of total bacterium protein was expressed as fusion protein. Approximately 400 μg purified SDF 1β(7.8×10 3) consisting of 71 amino acid residues were produced from 1 liter of fermented bacteria. Western blotting showed that anti SDF 1β mAb could bind with the purified SDF 1β specifically. N terminal amino acid sequencing indicated that N terminus of purified SDF 1β possessed as the same amino acid sequence as nature one. Purified SDF 1β not only had the binding activity with CXCR4 expressing cells[Kd=(12.20±2.99) nmol/L], but also could activate CXCR4 expressing cell signaling specifically in a dose dependence manner. Conclusion The purified recombinant human SDF 1β produced with this method possesses biochemical characters and biological activities as same as those nature human SDF 1β.