Refolding of the Fusion Protein of Recombinant Enterokinase Light Chain rEK L

Abstract

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