title: Production of a thermostable DNA polymerase by site-specific cleavage of a heat-eluted affinity fusion protein.
abstract: A novel strategy is described for bacterial expression and affinity purification of a recombinant truncated version of the heat-stable DNA polymerase I from Thermus aquaticus. The DNA polymerase (deltaTaq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the deltaTaq DNA polymerase, affinity-purified ABP-deltaTaq could be heat-eluted from HSA columns by incubation at 85 degrees C. To produce free deltaTaq DNA polymerase, efficient site-specific cleavage of the affinity tag was performed using a recombinant coxsackievirus 3C protease (3Cpro), also produced as an ABP affinity fusion. Thus, an integrated strategy could be devised where both the cleaved ABP affinity tag and the protease fusion could be recovered after site-specific cleavage using HSA-affinity chromatography. The flow-through fraction contained essentially pure deltaTaq DNA polymerase with full enzymatic activity.
authors.affiliation: Department of Biochemistry and Biotechnology, Kungliga Tekniska Högskolan, Stockholm, Sweden.
authors.forename: T
authors.initials: T
authors.lastname: Gräslund
authors.forename: J
authors.initials: J
authors.lastname: Nilsson
authors.forename: A M
authors.initials: AM
authors.lastname: Lindberg
authors.forename: M
authors.initials: M
authors.lastname: Uhlén
authors.forename: P A
authors.initials: PA
authors.lastname: Nygren
mesh_terms: D000345:Affinity Labels; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D003546:Cysteine Endopeptidases / Q000235:genetics; D004256:DNA Polymerase I / Q000235:genetics / Q000302:isolation & purification*; D004770:Enterovirus / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D006358:Hot Temperature; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012709:Serum Albumin; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
chemical_list: D000345:Affinity Labels; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D004256:DNA Polymerase I; D003546:Cysteine Endopeptidases