In vivo biotinylated proteins as targets for phage-display selection experiments.
Screening phage-displayed combinatorial libraries represents an attractive method for identifying affinity reagents to target proteins. Two critical components of a successful selection experiment are having a pure target protein and its immobilization in a native conformation. To achieve both of these requirements in a single step, we have devised cytoplasmic expression vectors for expression of proteins that are tagged at the amino- or carboxy-terminus (pMCSG16 and 15) via the AviTag, which is biotinylated in vivo with concurrent expression of the BirA biotin ligase. To facilitate implementation in high-throughput applications, the engineered vectors, pMCSG15 and pMCSG16, also contain a ligase-independent cloning site (LIC), which permits up to 100% cloning efficiency. The expressed protein can be purified from bacterial cell lysates with immobilized metal affinity chromatography or streptavidin-coated magnetic beads, and the beads used directly to select phage from combinatorial libraries. From selections using the N-terminally biotinylated version of one target protein, a peptide ligand (Kd= 9 microM) was recovered that bound in a format-dependent manner. To demonstrate the utility of pMCSG16, a set of 192 open reading frames were cloned, and protein was expressed and immobilized for use in high-throughput selections of phage-display libraries.
Combinatorial Biology, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.
Michael D
MD
Scholle
Frank R
FR
Collart
Brian K
BK
Kay
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D019921:Biotinylation; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D019151:Peptide Library; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D019151:Peptide Library; D011506:Proteins; D011993:Recombinant Fusion Proteins