Patent ID: 6423515
Filing Date: 2002-07-23
Classification: C12N

Abstract:
A method of constructing an isolated nucleic acid encoding a protein having an N-terminal methionine residue at position âˆ’1 and a glutamine residue at position 1, said encoded protein, after its position âˆ’1 methionine residue has been cleaved and its glutamine residue has been autocyclized, being the Ribonuclease of SEQ ID NO:1 or the Ribonuclease of SEQ ID NO:2, comprising the following steps:starting with pET11d-rOnc(Q1, M23L) recombinant plasmid DNA; identifying a specific site-directed mutation of said DNA that must be carried out to produce an isolated nucleic acid encoding the Ribonuclease of SEQ ID NO:1 or the Ribonuclease of SEQ ID NO:2; using first and second PCR reactions in an overlapping PCR protocol to generate two overlapping nucleic acid fragments, each bearing said identified site-directed mutation within their regions of overlap; and using said two overlapping nucleic acid fragments in a third PCI reaction in the overlapping PCR protocol to generate an isolated nucleic acid encoding one of said proteins.