Refolding process

In the development of new products, optimization of the fermentation medium for titer only often ignores the consequences of the medium properties on subsequent downstream processing steps such as filtration and chromatography. It is imperative, therefore, that there be effective communication and understanding between workers on the upstream and downstream phases of the produc t development if rational trade-offs are to be made to ensure overall optimahty of the process. One example is to make the conscious decision, in collaboration with those responsible for the downstream operations, whether to produce a protein in an unfolded form or in its native folded form the purification of the aggregated unfolded proteins is simpler than that of the native protein, but the refolding process itself to obtain the product in its final form may lack scalabihty. 

FIG. 20-87 Illustration of a refolding process for a protein from inclusion body. 

Recently, SPE was determined to be useful in a protein refolding process. Goto, Hatton, and coworkers demonstrated that SPE was the most efficient method for solubilizing denatured ribonuclease into AOT-based w/o-MEs [59]. The w/o-ME encapsulated... 

P. fluorescens has well-developed mechanisms for the secretion of proteins into the periplasm which facilitates S—S bond formation and proper N-terminal processing. It also allows one to reduce the formation of inclusion bodies and, thus, the additional costs caused by refolding processes. Proteolytic degradation of the expressed protein is also low, and very high... 

Proper Protein Refolding The final protein structure of the recombinant protein is often obtained by the promotion of proper refolding. When the production of recombinant proteins results in inclusion bodies, an ELISA can be used to assure that the final protein product has been properly refolded and to monitor the refolding process. To do this requires antibodies that can only recognize and bind to a particular conformation that is only present upon proper refolding. 

Small amounts of surfactants may be used to prevent aggregation of proteins and may enhance the refolding process when the dried protein dissolves. Buffers may also help to prevent aggregation of the dissolved drug. Similarly, polymers may be used as aggregation inhibitors or to form matrices. Chan et al. [86] prepared crystalline powders of recombinant human deoxyribonuclease with high fractions of sodium chloride. These powders were formulated as adhesive mixtures on lactose and mannitol and showed improved aerosolization behaviour compared to the pure protein. 

Compare the efflux time of the neutral DNA (15) to that of the denatured DNA 100 min. after neutralization (11). What does the difference between these two efflux times tell you regarding the refolding process ... 

Additional evidence of difference in the refolding process is that the time taken for reoxidation of a-lactalbumin is longer than 25 hr, whereas that for lysozyme is only 90 min (Tamburro et al., 1972). Iyer and Klee (1973) found differences in the rates of S-S bond reduction in these proteins. Reduced a-lactalbumin retained hydrodynamic and optical properties characteristic of folded globular proteins, although its conformation was clearly distinguishable from that of the native protein. 

Fig. 4 Schematic depiction of the protein refolding process. (View this art in color at www.dekker.com.)...

The most important step of the inclusion body protein recovery scheme is the refolding of the denatured protein to form a biologically active product. Refolding can be relatively simple, for small monomeric proteins, or quite complicated when the protein consists of more than one polypeptide chains or contains several disulfide bonds. Difficult refolding processes can result in overall low recovery yields of active protein. This limits the use of inclusion body processes for the production of some recombinant products. 

The key variables affecting protein stability in the refolding process are temperature, pH, ionic strength, denaturant concentration and protein concentration (5). In proteins stabilized by disulfide bonds in the native state, additional variables include dissolved oxygen, trace metal ion and reducing agent concentrations (6). 

Furthermore, it has been proposed that DMSO and alcohols can be utilized to stabilize reversibly unfolded states (Arakawa and Goddette, 1985 Bhattachar-jya and Balaram, 1997). For example, DMSO has been observed to stabilize a partially unfolded conformation of lysozyme (pH 3.0) (Bhattacharjyaand Balaram, 1997). Lysozyme in 10% DMSO showed little change in structure by nuclear magnetic resonance (NMR) when compared with aqueous conditions. The lysozyme structure in 50% DMSO resembled an early kinetic intermediate observed in the refolding process. Conversely, a highly unfolded structure in 100% DMSO has been reported, probably due to the polar aprotic nature of the solvent (Jackson and Mantsch, 1991). 

Native FKBP12 contains seven trans prolyl peptide bonds, and the CTIs of some or all of them constitute a slow, rate-limiting event in folding. Its refolding process from a chemically denatured state constituted the first example of an autocatalytic formation of a native protein from kinetically trapped intermediates with nonnative prolyl isomers [76,77]. 

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