The study and understanding of the natural evolution of protein function clearly has important implications for the design of in vitro evolution strategies. Nature must explore enormously large sequence spaces in a manner that preferentially examines areas relatively high in function.
There are many methods to create new function such as point mutation, error-prone PCR and DNA shuffling. Functional salvage screen(FSS) introduces randomly fragmented genomic DNA to the target protein. Therefore, FSS creates functionally new proteins that have a variety of sequence spaces. In order to control sequence spaces, we used specific size of random nucleotides. GFP was screened as a model protein for the directed evolution with random nucleotides. Defective GFP was constructed at the two positions. N-terminal one thirds(82Asp~89Pro) and two thirds(136Ile-145Tyr) loop regions of GFP(238aa) were firstly selected. And restoration of defective GFP with random nucleotides was executed with overlapping PCR technique. We screened 8 colonies that returned to original function at the two thirds loop region of GFP.