Cassettes random

Hallet, B., Sherratt, D.J. and Hayes, F. (1997) Pentapeptide scanning mutagenesis random insertion of a variable five amino acid cassette in a target protein. Nucleic Acids Research, 25, 1866-1867. 

Fig. 16. Binding pocket of the lipase from P. aeruginosa for the acid part of 2-methyldecanoic acid p-nitrophenyl ester showing the geometric position of amino acids 160-163 which were randomized in cassette mutagenesis (49).

The starting point for further exploration of protein sequence space was the conjecture that Stemmer s method of Combinatorial Multiple Cassette Mutagenesis (CMCM) [74] can be applied in appropriately modified form. It is a special type of DNA-shuffling which can be used to generate mutant gene libraries in which cassettes composed of random or defined sequences and the wild-type are incorporated randomly. CMCM had been developed for use in the area of functional antibodies [74],... 

As expected the PKS for rapamycin showed a Type I organisation strongly reminiscent of the erythromycin PKS, with catalytic activities arranged in modules (Scheme 27) and with sets of modules housed in turn in three multi-modular cassettes designated RAPS 1, RAPS 2 and RAPS 3. RAPS 1 contains modules 1 to 4, RAPS 2 modules 5 to 10, and RAPS 3 modules 11 to 14. The domain structure of the rapamycin PKS may not correspond in every detail to the pattern expected from the proposed structure for the PKS product however. In modules 3 and 6, there appear to be potentially active KR and DH domains which are not required module 3 also contains a potentially active but functionally redundant ER domain. It is possible that the active sites of these extra domains have been inactivated in a way that is not apparent from the primary sequence, and that the now redundant protein residues have still to be edited out by the random processes of evolution. There is also a chance that all these domains are indeed active and that the true rapamycin PKS product is more fully reduced than that shown. Extra post-PKS reoxidations would then be required to reintroduce the oxygen functionality at the relevant sites in the final structure. 

Asp mentioned above and Lys Ser, which was introduced in order to remove a proteolytic cleavage site [39]. Thus, there were altogether four fixed amino acid replacements in addition to fhe randomized side chains. The mutagenized gene cassette was fhen inserted into an appropriate E. coli vector and a genetic library comprising 3.7x10 variants was prepared [40]. 

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