Phage display as a novel screening method to identify extracellular proteins.
Extracellular proteins are involved in many diverse and essential cell functions and in pathogenic bacteria, and they may also serve as virulence factors. Therefore, there is a need for methods that identify the genes encoding this group of proteins in a bacterial genome. Here, we present such a method based on the phage display technology. A novel gene III-based phagemid vector, pG3DSS, was constructed that lacks the signal sequence which normally orientates the encoded fusion protein to the Escherichia coli cell membrane, where it is assembled into the phage particle. When randomly fragmented DNA is inserted into this vector, only phagemids containing an insert encoding a signal sequence will give rise to phage particles displaying a fusion protein. These phages also display an E-tag epitope in fusion with protein III, which enables isolation of phages displaying a fusion protein, using antibodies against the epitope. From a library constructed from Staphylococcus aureus chromosomal DNA, genes encoding secreted as well as transmembrane proteins were isolated, including adhesins, enzymes and transport proteins.
Rosander
Anna
A
Department of Microbiology, SLU, Swedish University of Agricultural Sciences, P.O. Box 7025, SE-750 07 Uppsala, Sweden.
Bjerketorp
Joakim
J
Frykberg
Lars
L
Jacobsson
Karin
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D015723:Gene Library; D008969:Molecular Sequence Data; D019151:Peptide Library
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D019151:Peptide Library