Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein.
The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction. Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes. The RecA protein has been overproduced in E. coli and purified using a variety of purification schemes requiring multiple, time-intensive steps. The purification schemes share a dependence on appropriate RecA structure and/or function at one or more steps. In this report, we used a modified protein splicing element (intein) and a chitin-binding domain, fused to the C-terminus of RecA, to facilitate a one-step affinity purification of RecA protein without modification of the native protein sequence. Following the single chromatographic step, RecA protein that is greater than 95% physical purity at a concentration of greater than microM was obtained. The protein displays in vitro activities that are identical to those of protein isolated using classical procedures. The purification strategy described here promises to yield mutant RecA proteins in sufficient quantity for rigorous biophysical characterization without dependence on intrinsic RecA function.
Singleton
Scott F
SF
Department of Chemistry, Rice University, P.O. Box 1892 MS 65, Houston, TX 77251-1892, USA. sfs@rice.edu
Simonette
Rebecca A
RA
Sharma
Neil C
NC
Roca
Alberto I
AI
D002686:Chitin / Q000378:metabolism; D002793:Cholic Acids; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D019154:Protein Splicing; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D002793:Cholic Acids; D011993:Recombinant Fusion Proteins; D002686:Chitin; D011938:Rec A Recombinases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate