Efficient display of two enzymes on filamentous phage using an improved signal sequence.
Directed protein-evolution strategies generally make use of a link between a protein and the encoding DNA. In phage-display technology, this link is provided by fusion of the protein with a coat protein that is incorporated into the phage particle containing the DNA. Optimization of this link can be achieved by adjusting the signal sequence of the fusion. In a previous study, directed evolution of signal sequences for optimal display of the Taq DNA polymerase I Stoffel fragment on phage yielded signal peptides with a 50- fold higher incorporation of fusion proteins in phage particles. In this article, we show that for one of the selected signal sequences, improved display on phage can be generalized to other proteins, such as adenylate cyclases from Escherichia coli and Bordetella pertussis, and that this is highly dependent on short sequences at the C-terminus of the signal peptide. Further, the display of two enzymes on phage has been achieved and may provide a strategy for directing coevolution of the two proteins. These findings should be useful for display of large and cytoplasmic proteins on filamentous phage.
Strobel
Heike
H
Unité de Chimie Organique, CNRS URA 2185, Département de Biologie Structurale et Chimie, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris, France.
Ladant
Daniel
D
Jestin
Jean-Luc
JL
D000262:Adenylyl Cyclases / Q000235:genetics* / Q000378:metabolism*; D001886:Bordetella pertussis / Q000201:enzymology* / Q000235:genetics*; D019020:Directed Molecular Evolution / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D017912:Inovirus / Q000235:genetics* / Q000378:metabolism*; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods; D021382:Protein Sorting Signals / Q000235:genetics
D019151:Peptide Library; D021382:Protein Sorting Signals; D000262:Adenylyl Cyclases