Abstract: An isolated DNA is provided which encodes a protein that is capable of binding to 5?GGTACC-3?, the isolated DNA being capable of hybridizing to SEQ ID NO:3 under stringent hybridization conditions. The isolated DNA may be alternatively characterized as coding for a protein having an amino acid sequence comprising SEQ ID NO:5 or by an amino acid sequence with an expectation value of less than E=e?02 in a BLAST search using SEQ ID NO:5. Vectors containing the isolated DNA and host cells expressing the vectors as well as a method for making recombinant Acc65I having the above properties are also provided.
Type:
Grant
Filed:
June 14, 2007
Date of Patent:
September 14, 2010
Assignee:
New England Biolabs, Inc.
Inventors:
Keith Lunnen, John Greci, Geoffrey Wilson
Abstract: A method for the ligation of expressed proteins which utilizes inteins, for example the RIR1 intein from Methanobacterium thermotrophicum, is provided. Constructs of the Mth RIR1 intein in which either the C-terminal asparagine or N-terminal cysteine of the intein are replaced with alanine enable the facile isolation of a protein with a specified N-terminal, for example, cysteine for use in the fusion of two or more expressed proteins. The method involves the steps of generating a C-terminal thioester-tagged target protein and a second target protein having a specified N-terminal via inteins, such as the modified Mth RIR1 intein, and ligating these proteins. A similar method for producing a cyclic or polymerized protein is provided. Modified inteins engineered to cleave at their C-terminus or N-terminus, respectively, and DNA and plasmids encoding these modified inteins are also provided.
Type:
Grant
Filed:
February 12, 1999
Date of Patent:
February 1, 2005
Assignee:
New England Biolabs, Inc.
Inventors:
Thomas C. Evans, Ming-Qun Xu, Shaorong Chong