strategy allowed an integration of the cell separation step with ion exchange adsorption of the gene product with simultaneous volume reduction, which resulted in a highly condensed but still efficient recovery process (55). The two-step purification process, ion exchange chromatography in an expanded-bed format followed by IgG affinity chroma-tography for polishing, demonstrated an overall yield of more than 90%.
An alternative strategy to simplify downstream processing was investigated by Kohler and coworkers (92), where recombinant technology was applied to alter the partitioning properties of a ZZ-fusion protein, which thereby could be efficiently recovered by aqueous two-phase extraction as the primary purification step. An attractive approach to simplify recovery of products, which may be produced with modified primary sequences, e.g., industrial enzymes, would be to alter the partitioning properties by substitution of one or more surface amino acid residues, which are not involved in the biological activity, for tryptophans, which are known to influence partitioning characteristics. This type of protein engineering will most probably be used in the future to reduce production costs for bulk proteins.
A third example of how unit operations can be integrated by careful design of the gene product and the recovery process was recently demonstrated for the production of native human insulin and its C-peptide in E. coli (96,103). Human proinsulin was genetically fused to ZZ, and recovered from inclusion bodies via solubilization and refolding of the intact fusion protein, followed by IgG affinity chromatography. Native insulin, consisting of cystein-bridged A- and B-chains, as well as the clinically relevant C-peptide (104), could be released by a single-step trypsin treatment, cleaving off the ZZ-tail simultaneously to the processing of the C-peptide (96,103). The careful genetic design of a fusion protein as an intermediate product, which could be efficiently converted to the final products, allowed the design of a pilot-scale process involving few unit operations (96) that thus might result in a cost-efficient bioprocess. A further improved bioprocess for production of the proinsulin C-peptide was presented (105) in which the C-peptide-encoding gene fragment was hepta-merized to increase yields. A fusion protein containing seven copies of the C-peptide was thus produced, and after affinity purification, enzymatic digestion was employed to release native C-peptide (105).
Improved Renaturation Schemes for Recombinant Gene Products
The recovery of biologically active or native proteins by in vitro refolding from insoluble inclusion bodies can in some cases be hampered by the aggregation of the product during the procedure, leading to low overall yields. To make the procedure more efficient, several improved protocols have been developed, including the addition of different "folding enhancers" (4). Alternatively, the protein itself can be engineered to facilitate the refolding. The presence of hydrophilic peptide extensions during the refolding can dramatically improve the folding yield, probably by conferring a higher overall solubility of the protein (90). Sam-
uelsson and coworkers (42) showed that the reshuffling of misfolded disulfides in recombinant insulin-like growth factor I (IGF-I) was greatly facilitated by fusion to the highly soluble ZZ fusion partner (42). Compared to unfused IGF-I, the fusion ZZ-IGF-I could be successfully refolded at a 100-fold higher concentration (1-2 mg/mL), without forming precipitates (43).
A completely new concept to improve the fraction of correctly folded recombinant IGF-I was recently presented (106). It was demonstrated that coexpression of a specific binding protein, IGF binding protein 1 (IGFBP-1), significantly increased the relative yield of IGF-I having native disulfide bridges when expressed in a secreted form in E. coli. In addition, a glutathione redox buffer was added to the growth medium to enhance formation and breakage of disulfide bonds in the periplasm of the bacteria. In the presented example, both IGF-I and IGFBP-1 were produced as affinity fusions, to Z and BB (Fig. 4a), respectively, which facilitated the purification of in vivo-assembled het-erodimers by alternative purification methods (106). A further development of the strategy would be to express the target protein as a nonfused gene product and the specific binding protein in an affinity-tagged configuration (Fig. 4b). Correctly folded target protein would thus be affinity captured as a heterodimer via the tagged binding protein. This would employ the benefits of high specificity for affinity chromatography without introducing requirements of proteolytic processing (see following) to recover the native target protein.
Affinity-Tagged Proteases for Site-Specific Cleavage of Fusion Proteins
When gene fusion strategies are used for the production of native proteins, efficient means for site-specific cleavage of the fusion protein and subsequent removal of the affinity fusion partner are needed. In addition, if employing enzymatic strategies for cleavage, strategies for the removal of the proteolytic enzyme itself must be developed. Special considerations must be taken if the target protein itself also has to be further processed to give the desired final product (96,103). Careful upstream design of the fusion protein construct using genetic strategies can greatly facilitate the subsequent purification of the target protein and also allow for integrated systems involving coprocessing of the protein and efficient removal of the affinity fusion partner as well as the protease used for cleavage (96,102).
Enzymes for use in biotechnological applications should preferably be highly specific proteases, easy to produce by recombinant means in large scale. Interesting candidate enzymes can be found in human picorna viruses, whose maturation relies on the site-specific cleavage of a large polyprotein precursor to yield the viral components. Some of these proteases are functionally produced at high levels in bacterial expression systems (107,108), and this could facilitate the production of variants constructed by protein engineering. Recently, a new general strategy was described where the 3C protease of rhinovirus was fused to
Figure 4. Affinity-assisted in vivo folding and its use for recovery of correctly folded gene products. (a) Schematic description of the example presented by Samuelsson and coworkers (106) where a fusion protein Z-IGF-I, with correctly folded IGF-I, could be recovered using alternative affinity chromatography strategies, taking advantage of the interaction between IGF-I and IGFBP-I. (b) The basic principle might be suitable for general use both to increase the yield of correctly folded target protein and to achieve recovery of only correctly folded target protein via the interaction of a specific binding protein (BP), which binds only to the correctly folded form of the target protein. If the binding protein is expressed as an affinity fusion (Aff), this fusion protein should be a suitable ligand for recovery of correctly folded target protein.
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