Zz

X'

Figure 3. The concept of dual-affinity fusions for recovery of proteolytically labile target proteins. The gene encoding the target protein X is fused between the genes encoding the ZZ and BB tags, respectively. A first passage over an IgG column results in recovery of the full-length product as well as potential degradation products. A second passage over an HSA column results in recovery of only the full-length product.

Flowthrough

Figure 3. The concept of dual-affinity fusions for recovery of proteolytically labile target proteins. The gene encoding the target protein X is fused between the genes encoding the ZZ and BB tags, respectively. A first passage over an IgG column results in recovery of the full-length product as well as potential degradation products. A second passage over an HSA column results in recovery of only the full-length product.

proteins, ZZ and BB. The concept was applied to the production of proteolytically sensitive human insulin-like growth factor II (IGF-II) in E. coli. The concept first of all allows the use of two consecutive affinity chromatography steps, but in addition, dramatic stabilization effects can be obtained as compared with more conventional expression routes. The dual affinity fusion approach was found to have a stabilizing effect on three small and labile recombinant proteins, namely human proinsulin, a domain from rat protein disulfide isomerase, and a fragment from a human T-cell receptor (88), when expressed in E. coli. The concept of dual affinity fusions has thereafter been employed also with affinity tags other than the SpA and SpG tags (99102).

Strategies for Integrated Downstream Processing

An important part of modern biotechnology is to develop simplified schemes for fermentation and downstream processing of recombinant proteins by the integration of unit operations. Genetic product design can be used in several ways to obtain such schemes, that is, by influencing the yield and localization of the product as well as to adapt the gene product by fusion technology to specific unit operations suitable for large-scale downstream processing.

The recently presented expanded-bed adsorption procedure (mentioned earlier) constitutes a nice example of process integration (55). Efficient recovery of a secreted recombinant fusion protein ZZ-M5 was achieved directly from a crude fermentor broth without prior cell removal. The fusion protein had a relatively low isoelectric point (pI), which allowed anionic exchange adsorption at pH 5.5 at which most E. coli host proteins are not adsorbed. This

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