title: Application of a new vector system for efficient protein purification in the crystallization of PA5104/ORF from Pseudomonas aeruginosa.
abstract: We have developed a new T7-based vector system for rapid purification and high-throughput capability applicable for structural studies. The system allows purification of target proteins to homogeneity in two steps with a single Ni-affinity column. The first step relies on affinity purification of the N-terminal His-tagged protein in the conventional way, eluting the protein with imidazole. Addition of a His-tagged 3C protease to cleave the His-tag permits a second pass through the nickel column, this time all impurities bind to the column while the pure protein does not. This has the major advantage of quickly removing the residual contaminating proteins that are associated with nickel affinity purification as well as the protease and His-tag. Here, we describe the application of this system to over-express and purify ORF PA5104 from Pseudomonas aeruginosa. The protein was successfully crystallized and crystals were shown to diffract to atomic resolution. Additionally preliminary X-ray diffraction analysis of two crystals forms is presented, one diffracting to 1.9 A and the other to 0.96 A resolution.
authors.affiliation: IBLS, University of Glasgow, Glasgow G12 8QQ, UK. Bernhard.Lohkamp@ki.se
authors.forename: Bernhard
authors.initials: B
authors.lastname: Lohkamp
authors.forename: Neil G
authors.initials: NG
authors.lastname: Paterson
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Lapthorn
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015870:Gene Expression; D005822:Genetic Vectors / Q000138:chemical synthesis*; D016366:Open Reading Frames; D011550:Pseudomonas aeruginosa / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins