Reconstitution of 5-enolpyruvylshikimate-3-phosphate synthase activity by fragment complementation

摘要

The present invention relates to protein fragments of 5-enolpyruvylshikimate-3-phosphate synthase, which are selected from the protein fragment pairs of EPSPS, two such protein fragments can make up full length EPSPS and reconstitute EPSPS activities by complementation without help of any joint structure. The present invention also relates to nucleic acid molecules encoding the protein fragments, expression vectors and cells comprising such nucleic acid molecules. The present invention also relates to methods for reconstituting EPSPS activities by using the fragments or the nucleic acid molecules or the expression vectors of the present invention, as well as methods for dividing the protein fragments of the present invention.

主权项

1. A method for reconstitution of an active 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) structure from at least two inactive EPSPS fragments which constitute an EPSPS fragment pair, said method comprising, providing a first inactive EPSPS fragment and a second inactive EPSPS fragment, allowing the first and the second fragments to associate to form a structure characterized in that it, exhibits EPSPS activity; lacks a covalent bond joining the fragments; and lacks complementary non-EPSPS sequences, the fragment pair being selected from a group of E. coli EPSPS fragment pairs consisting of the following: N67/C68, N85/C86, N104/C105, N154/C155, N182/C183, N184/C185, N218/C219, N224/C225, N227/C228, N259/C260, N298/C299, N371/C372, N376/C377, N383/C384, or is selected from a group of P. putida CGMCC 0739 EPSPS fragment pairs consisting of the following: N208/C209, N214/C215, N219/C220, N222/C223, N224/C225.