An integrated vector system for cellular studies of phage display-derived peptides.
Peptide phage display is a method by which large numbers of diverse peptides can be screened for binding to a target of interest. Even when successful, the rate-limiting step is usually validation of peptide bioactivity using living cells. In this paper, we describe an integrated system of vectors that expedites both the screening and the characterization processes. Library construction and screening is performed using an optimized type 3 phage display vector, mJ(1), which is shown to accept peptide libraries of at least 23 amino acids in length. Peptide coding sequences are shuttled from mJ(1) into one of three families of mammalian expression vectors for cell physiological studies. The vector pAL(1) expresses phage display-derived peptides as Gal4 DNA binding domain fusion proteins for transcriptional activation studies. The vectors pG(1), pG(1)N, and pG(1)C express phage display-derived peptides as green fluorescent protein fusions targeted to the entire cell, nucleus, or cytoplasm, respectively. The vector pAP(1) expresses phage display-derived peptides as fusions to secreted placental alkaline phosphatase. Such enzyme fusions can be used as highly sensitive affinity reagents for high-throughput assays and for cloning of peptide-binding cell surface receptors. Taken together, this system of vectors should facilitate the development of phage display-derived peptides into useful biomolecules.
Voss
Stephan D
SD
Department of Pediatric Radiology, Children's Hospital Medical Center, Boston, MA 02115, USA.
DeGrand
Alec M
AM
Romeo
Giulio R
GR
Cantley
Lewis C
LC
Frangioni
John V
JV
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008546:Melanoma, Experimental / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D000073718:Pancreatitis-Associated Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D008164:Luminescent Proteins; D000073718:Pancreatitis-Associated Proteins; D010446:Peptide Fragments; D019151:Peptide Library; C000612141:REG3A protein, human; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase