Vitro mutagenesis

A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin.

5p zingzing09 24-04-2013 60 5   Download

Phosphorylationof the transit peptideof several chloroplast-targeted proteins enables the binding of 14-3-3 proteins. The complex that forms, together with Hsp70, has been demonstrated to be an intermediate in the chloroplast pro-tein import pathwayin vitro[May, T. &Soll, J. (2000)Plant Cell12, 53–63]. In this paper we report that mutagenesis (in order to remove the phosphorylation site) of the transit peptide of the small subunit of ribulose bisphosphate carb-oxylase/oxygenase did not affect its ability to target green fluorescent protein to chloroplastsin vivo....

8p dell39 03-04-2013 51 4   Download

We have investigated mutants of phytochrome Cph1 from the cyanobacter-ium SynechocystisPCC6803 in order to study chromophore–protein inter-actions. Cph1D2, the 514-residue N-terminal sensor module produced as a recombinant His6-tagged apoprotein in Escherichia coli, autoassembles in vitro to form a holoprotein photochemically indistinguishable from the full-length product.

15p inspiron33 26-03-2013 52 6   Download