Assay shuffling

Fig. 5. Activities of 654 active clones from the shuffled subtilisin library compared to twenty-six parents. Relative activities of each clone in five screens are plotted as concentric circles. Each color represents one of the five screening conditions pH5.5 (orange), pH7.5 (blue), pHIO (dark red), thermostability (yellow), and activity in 35% DMF at pH 7.5 (green). The area of the circle is proportional to the activity in the five assays relative to the best parent in each assay.

E. coli ProFARI shuffling growth assay evolved PRAI activity from ProFARI 29... 

Staphylococcus aureus lipase epPCR/ shuffling chromogenic assay 12-fold increase in phospholipase activity 38... 

E. coli aldolase epPCR/ shuffling UV assay acceptance of phosphate free substrates 41... 

Low solubility or improper folding may sometimes hamper the use of enzymes, particularly when expressed in a non-native host. A method of expressing proteins with a C -terminal GFP fusion to use fluorescence as a measure of the amount of correctly folded protein has been introduced (44). DNA shuffling produced variants of ferritin that showed increased solubility, even when they were recloned without the GFP fusion. This assay has been used to produce proteins for X-ray crystallography structure determination (45). The protein nucleoside diphosphate kinase from Pyrobaculum aerophilum is insoluble when expressed in E. coli, but after DNA shuffling, a functional variant with six mutations was found to have 90% solubility, which enabled its crystallization, and its structure was determined. 

As these techniques have been improved, reports have started to appear describing the evolution of enzymes with medical applications. A recent paper has reported the gene shuffling of over 20 human interferon-a genes and the use of phage display, which was used to select mutant cytokines that demonstrated superior antiviral and antiprohferation activities in a mouse ceU assay [ 13 ]. In an other recent work, a butyrylchoUnesterase (BChE) enzyme has been evolved using a mammalian ceU expression system to... 

---