Clones/cloning from microbial cells

Second, the flow cytometric isolation of very rare clones from a library is technically demanding. For most protein engineering applications microorganisms are used as the expression host. Whereas the sorting of rare mammalian cells is widely practiced in cell biology research and in clinical medicine and is therefore highly advanced, the screening and isolation of microbial cells is still much more of an art (Davey and Kell, 1996). 

Fuhrman, J. A., and Ouverney, C. C. (1998). Marine microbial diversity studied via 16S rRNA sequences Cloning results from coastal waters and counting of native Archaea with fluorescent single cell probes. Aquat. Ecol. 32, 3—15. 

The recombinant technique involves transfecting cells with DNA that codes for the production of the intended protein. The process of transfection is shown in Exhibit 10.8. Once transfected, the cells (microbial or mammalian) grow and divide as clones and express the intended protein through instructions from the foreign DNA genes introduced into the cells. 

In an alternate approach, the enantioselective microbial reduction of methyl-4-(2 -acetyl-5 -fluorophenyl) butanoates 80 (Figure 16.19B) was demonstrated using strains of Candida and Pichia. Reaction yields of 40%-53% and EEs of 90%-99% were obtained for the corresponding (5)-hydroxy esters 77. The reductase that catalyzed the enantioselective reduction of ketoesters was purified to homogeneity from cell extracts of Pichia methanolica SC 13825. It was cloned and expressed in E. coli, and recombinant cultures were used for the enantioselective reduction of the keto-methyl ester 80 to the corresponding (5)-hydroxy methyl ester 77. On a preparative scale, a reaction yield of 98% with an EE of 99% was obtained [99]. 

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