Producing a transposase of a mauritiana subfamily mariner transposon in a prokaryotic host cell comprises transforming the host cell with vectors containing the transposase coding sequence and a kinase coding sequence

摘要

#CMT# #/CMT# Producing a transposase of a mauritiana subfamily mariner transposon in a prokaryotic host cell comprises cloning the transposase coding sequence (S1) into an expression vector, cloning a cAMP-dependent protein kinase catalytic subunit coding sequence (S2) into an expression vector and transforming the host cell with the vectors. #CMT# : #/CMT# Independent claims are also included for: (1) transposase produced as above; (2) prokaryotic host cell comprising S1 and S2; (3) expression vector comprising S1 and S2; (4) kit for insertional, sequencing or cloning mutagenesis comprising a transposase as above. #CMT#USE : #/CMT# The transposase is useful for transposing a DNA sequence of interest into a target DNA sequence in vitro or in vivo (claimed). #CMT#ADVANTAGE : #/CMT# The transposase is stabilized by kinase-induced phosphorylation. #CMT#BIOTECHNOLOGY : #/CMT# Preferred Method: The transposase is a hyperactive mutant transposase. The two coding sequences are cloned into the same vector. The two coding sequences are under the control of the same regulatory elements. The host cell is an Escherichia coli cell. The transposon is Mos1. #CMT#EXAMPLE : #/CMT# Plasmid pMalC2x-Tnp is a vector for bacterial expression of Mos1 transposase N-terminally fused to maltose binding protein. Plasmid pET-pKa is a vector for bacterial expression of cAMP-dependent protein kinase catalytic subunit under the control of the pol7 promoter. ER2566 bacteria transformed with both pMalC2x-Tnp and pET-pKa produced a transposase fusion protein that was free of autoproteolysis products.