title: Direct production and purification of T7 phage display cloned proteins selected and analyzed on microarrays.
abstract: Phage display technology has emerged into a powerful tool for identifying proteins with specific binding properties. This technology adds amino acid sequences to the carboxy terminus of a phage capsid protein, thus generating a fusion protein displayed on the surface of the phage. Here, we have developed a high-throughput strategy to synthesize purified protein that solves many of the problems associated with crude phage lysates. Phage DNA was used as a template for a nested PCR that added the T7 promoter, ribosome binding site, and a His6-tag. The PCR product was then used as a template for in vitro transcription/translation. The resulting His6-tagged recombinant protein was then purified by nickel affinity chromatography. The functionality of the purified protein was verified using protein microarray analysis.
authors.affiliation: Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors.forename: James E
authors.initials: JE
authors.lastname: Nowak
authors.forename: Madhumita
authors.initials: M
authors.lastname: Chatterjee
authors.forename: Saroj
authors.initials: S
authors.lastname: Mohapatra
authors.forename: Sylvia C
authors.initials: SC
authors.lastname: Dryden
authors.forename: Michael A
authors.initials: MA
authors.lastname: Tainsky
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D006639:Histidine / Q000737:chemistry; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D040081:Protein Array Analysis / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016622:Silver Staining; D014764:Viral Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D006639:Histidine