Refolding of the Fusion Protein of Recombinant Enterokinase Light Chain rEKL

The fusion protein of enterokinase light chain, DsbA-rEKL, 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 EKL molecule was released after the addition of 2 mmol/L of 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 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.