Chromatographic refolding of rhodanese and lysozyme assisted by the GroEL apical domain, DsbA and DsbC immobilized on cellulose.

The Escherichia coli expression system remains the first choice for the production of recombinant proteins when biological activity is not compromised by posttranslational modification. Many strains of E. coli, vectors and culture conditions are available to express recombinant proteins in a soluble and correctly folded conformation. Often these strategies fail, and misfolded recombinant proteins aggregate into inclusion bodies. To recover its biological activity, a recombinant protein must be refolded, a step that has become the major limitation of the E. coli expression system. Chromatographic refolding, assisted by immobilized chaperones and foldases, is an in vitro refolding protocol that significantly improves refolding yields, yet its application at the bioprocess scale has been limited. Therefore, new cost-efficient alternatives to utilize chromatographic refolding assisted by chaperones and foldases might improve the production of biologically active proteins in E. coli. In this work, the GroEL apical domain (AD) and the oxidoreductases DsbA and DsbC fused to a carbohydrate-binding module (CBM) were purified and immobilized on microcrystalline cellulose particles. A column packed with a 60:40 (v/v) mixture of gel filtration media and cellulose particles with immobilized AD significantly improved the chromatographic refolding of rhodanese. A similar column with equimolar amounts of AD, DsbA and DsbC immobilized on cellulose particles significantly improved the oxidative chromatographic refolding of lysozyme. The assisted refolding yields were up to 80% for rhodanese and 100% for lysozyme, compared with 33% and 23%, respectively, obtained in the experiments without immobilized chaperones. In addition, AD, DsbA and DsbC immobilized on cellulose exhibited significant operational stability under the extreme denaturing conditions used in the chromatographic refolding batches. These results suggest that chromatographic refolding assisted by AD, DsbA, and DsbC immobilized on cellulose is suitable for the oxidative refolding of proteins expressed in E. coli as inclusion bodies.