Intein-mediated protein ligation of expressed proteins

Details Intein-mediated protein ligation of expressed proteins Intein-mediated protein ligation of expressed proteins

2005-02-01

2005-02-01

Achutamurthy, Ponnathapura (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S068100, C435S252300, C435S320100, C435S471000, C530S350000

Reexamination Certificate

active

06849428

ABSTRACT:
A method for the ligation of expressed proteins which utilizes inteins, for example the RIR1 intein fromMethanobacterium 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.

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