融合牛肠激酶轻链EK_L包涵体蛋白的复性纯化

Refolding of the Fusion Protein of Recombinant Enterokinase Light Chain rEK_L

大肠杆菌高密度发酵表达肠激酶轻链融合蛋白DsbA-rEKL,主要以包涵体形式存在。包涵体经4mol/L尿素和0.5%TritonX-100洗涤,以6mol/L盐酸胍、100mmol/LDTT溶解,在胱氨酸存在下,以脉冲加样方式复性。融合蛋白复性在6mmol/L胱氨酸存在下、脉冲加量0.03mg/mL和复性终蛋白浓度0.3mg/mL为最佳复性方案。复性的融合蛋白加2mmol/LCaCL2后快速自切。经IDA-Sepharose及Q-Sepharose纯化,rEKL纯度可达95%以上,可高效酶切重组瑞特普酶融合蛋白Trx-rPA。实现了大规模生产rEKL,每升发酵液经复性及纯化后,可得rEKL60mg/L以上,使以融合蛋白表达rPA等药用蛋白成为现实。

The fusion protein of enterokinase light chain, DsbA-rEKL, was expressed mainly in inclusion body in E. coli. The recombinant bacteria was fermented to high density, with high expression of the fusion protein. After being washed with 0.5 % Triton X-100 and 4mol/L urea, the inclusion body was dissolved in 6mol/L guanidine and 100mmol/L DTT, derivatized by cystine and refolded by pulse refolding. The strategy of pulse refolding involved the addition of 0.03mg/mL of fusion protein until its final concentration reached 0.3mg/mL. The refolded protein was autocleaved and the active EKE molecule was released after adding 2mmol/L CaCl2. Using the two-step purification processes of IDA-Sepharose chromatography and Q-Sepharose chromatography, the purity of rEKL was found to be above 95 %, with a high activity to cleave the recombinant reteplase fusion protein Trx-rPA. The yield of purified rEKL was more than 60mg/L of cultures. As a result, the therapeutic proteins like rPA could be produced on a large-scale in a way such as expressed in the form of fusion proteins.