Efficient Protein Refolding Screening in a 96-well Format
Peter A. Leland, James L. Rane, Jennifer G. Reich, Tsetska Takova, and Anthony Grabski
Novagen, a brand of EMD Chemicals Inc. 441 Charmany Drive, Madison WI, USA 53719
Abstract Proteomics requires large amounts of highly pure and correctly folded protein. Most commonly, this need is met using heterologous expression systems such as Escherichia coli. Production of foreign proteins in E. coli commonly results in the formation of inclusion bodies- dense, insoluble, aggregates of mis-folded protein. Inclusion bodies are typically viewed as undesirable, however, they do have positive attributes as they are easily purified, resistant to proteolysis and can be solubilized with chaotropic agents. Unfortunately, defining conditions that promote refolding of a chemically solubilized target protein into its native conformation is both empirical and difficult. The chances of identifying an optimal refolding condition are increased by simultaneously and systematically evaluating a large number of refolding conditions. To enable this type of experiment, we have developed two 96-well plate-based protein refolding screen systems. The systems differ in the chemistry used to denature the inclusion bodies and in the refolding additives included in the 96-well plate. One system uses N-lauroylsarcosine, a chaotropic anionic detergent, to denature the inclusion bodies while the second system uses either guanidine hydrochloride or urea. Protein refolding is accomplished by rapid dilution of the denatured protein into the 96-well plate that contains an array of buffers, salts, redox couples, cyclodextrin, nondetergent sulfobetains (NDSBs), as well as other refolding additives. With these refolding screening systems, we have successfully identified optimal conditions for refolding several recombinant proteins, including a green fluorescent protein fusion, phosphatase, mammalian endopeptidase, matrix metallo-protease, and viral protease. Analysis of the various target protein activities following refolding revealed clear requirements for successful refolding, both in terms of the inclusion body denaturant and in the constituents of the refolding solutions. High-yielding refolding conditions identified by the screen have successfully been scaled 10,000-fold from a 50 µg refolding reaction to 500 mg. Significantly, all steps of the refolding screens are equally compatible with manual use and high-throughput automated liquid handling. | |
