title: One-step purification of a recombinant protein from a whole cell extract by reversed-phase high-performance liquid chromatography.
abstract: We have developed a one-step facile, flexible and readily scalable purification method for a recombinant protein, TM 1-99 (113 amino acid residues; 12,837 Da) based on reversed-phase high-performance liquid chromatography (RP-HPLC) from an E. coli cell lysate. Following cell lysis, the cell contents were extracted with 0.1% aqueous trifluoroacetic acid (TFA), applied directly under conditions of high sample load to a narrow bore RP-HPLC C(8) column (150 mm x 2.1 mm I.D.) and eluted by a shallow gradient of acetonitrile (0.1%/min). Loads of 23 and 48 mg of lyophilized crude cell extract produced 2.4 and 4.2mg of purified product (>94% pure), respectively, at >94% recovery. Our results show the excellent potential of one-step RP-HPLC for purification of recombinant proteins from cell lysates, where high yields of purified product and greater purity are achieved compared to affinity chromatography. Such an approach was also successful in purifying just trace levels (<0.1% of total contents of crude sample) of TM 1-99 from a cell lysate.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA.
authors.forename: Janine B
authors.initials: JB
authors.lastname: Mills
authors.forename: Colin T
authors.initials: CT
authors.lastname: Mant
authors.forename: Robert S
authors.initials: RS
authors.lastname: Hodges
mesh_terms: D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification*; D015203:Reproducibility of Results
chemical_list: D011994:Recombinant Proteins