Protein engineering based

Protein engineering based on sequence-activity relationships is attractive because of the small numbers of variants that need to be tested in order to obtain improvement. In principle this frees the investigator from the tyranny of high throughput screening. It also allows one to design and create the most informative set of variants, rather than relying upon a stochastically constructed library (i-6). 

In 1989, two enzymes based on genetic engineering techniques were introduced, ie, a cloned alkaline protease (IBIS) and a protein engineered Subtihsin Novo (Genencor, California). Lipase and ceUulase types of detergent enzymes have also begun to appear. 

Recently Alan Fersht, Cambridge University, has developed a protein engineering procedure for such studies. The technique is based on investigation of the effects on the energetics of folding of single-site mutations in a protein of known structure. For example, if minimal mutations such as Ala to Gly in the solvent-exposed face of an a helix, destabilize both an intermediate state and the native state, as well as the transition state between them, it is likely that the helix is already fully formed in the intermediate state. If on the other hand the mutations destabilize the native state but do not affect the energy of the intermediate or transition states at all, it is likely that the helix is not formed until after the transition state. 

Cyproase 1 is in effect a new enzyme, produced by systematic protein engineering firmly based on sound chemical principles. Its status as an enzyme mimic may be debatable its efficiency is not. It was shown to hydrolyze 25% of bonds to proline in a (denatured) peptide toxin in two hours at pH 70, with some 400 turnovers. It is one of the two most efficient enzyme mimics we will encounter in this article1131... 

Separation from culture media or broth is the primary step in collecting the product found either in cells (sohd) or medium (liquid). This initial separation step is engineered based on cell size and density differences between solid and liquid (Table 4.10). In the case where the recombinant product is localized in the intracellular content such as the cytoplasm or inclusion bodies, which are highly insoluble particles found in bacteria, the cells are hrst isolated from the medium and then disrupted to collect the recombinant protein fraction. A number of cell disruption techniques have been developed to facilitate this step, and some are listed in Table 4.11. 

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