title: Overexpression and purification of tagged Escherichia coli proteins using a chromosomal knock-in strategy.
abstract: The purification of recombinant proteins from Escherichia coli (E. coli) has become a standard procedure both for research purposes and in biotechnology. One common way by which this is accomplished is by subcloning the gene of interest into a suitable expression vector and purifying the overexpressed protein using an affinity tag. In some cases, however, subcloning into plasmid vectors can be problematic. An alternative method could be to overexpress the gene of interest from the chromosome. Here, I describe a strategy to juxtapose strong transcriptional and translational sequences in front of any E. coli gene by recombination, which allows the gene product to be expressed in large quantities in the cell, and purified as a tagged protein. An application of this method to create a recombinant strain overexpressing the HrpA protein is described.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA. cjain@med.miami.edu
authors.forename: Chaitanya
authors.initials: C
authors.lastname: Jain
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics*; D053487:DEAD-box RNA Helicases; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods; D020365:RNA Helicases / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C092662:hrpA protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases