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1.
Annu Rev Virol ; 2(1): 25-40, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26958905

ABSTRACT

Cre-lox of bacteriophage P1 has become one of the most widely used tools for genetic engineering in eukaryotes. The origins of this tool date to more than 30 years ago when Nat L. Sternberg discovered the recombinase, Cre, and its specific locus of crossover, lox, while studying the maintenance of bacteriophage P1 as a stable plasmid. Recombinations mediated by Cre assist in cyclization of the DNA of infecting phage and in resolution of prophage multimers created by generalized recombination. Early in vitro work demonstrated that, although it shares similarities with the well-characterized bacteriophage λ integration, Cre-lox is in many ways far simpler in its requirements for carrying out recombination. These features would prove critical for its development as a powerful and versatile tool in genetic engineering. We review the history of the discovery and characterization of Cre-lox and touch upon the present direction of Cre-lox research.


Subject(s)
Bacteriophage P1/enzymology , Genetic Engineering/history , Integrases/metabolism , Viral Proteins/metabolism , Virology/history , Bacteriophage P1/genetics , Bacteriophage P1/physiology , History, 20th Century , Integrases/genetics , Recombination, Genetic , Viral Proteins/genetics , Virus Integration
2.
Protein Eng ; 16(1): 65-72, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12646694

ABSTRACT

The specific binding of RGD-containing proteins to integrin is a function of both the conformation of and the local sequence surrounding the RGD motif. To study the effect of these factors on integrin binding affinity and specificity, we obtained RGD-containing ligands specific for different integrins presented on the same protein scaffold. The beta-turn region between two anti-parallel beta-strands on the loop I of tendamistat, an inhibitor of alpha-amylase, was extended by two residues and randomized in a phagemid library. This library and two subsequently constructed RGD-containing loop I libraries were biopanned with purified integrins alphaIIbbeta3, alphaVbeta3 and alphaVbeta5 individually. The sequence analysis of selected tendamistat variants and characterization by phage ELISA revealed that phage adhesion is mediated exclusively by an RGD motif located at only two out of four possible positions on loop I. Further, sequences flanking the RGD motif were specific for different integrin targets. Interestingly, selected tendamistat variants mimic natural integrin ligands, both in sequence similarity and in integrin binding specificity, indicating that various ligand specificity patterns can be generated by driving towards maximum affinity in the integrin-ligand complexes.


Subject(s)
Bacteriophages/genetics , Integrins/genetics , Integrins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Blotting, Western , Enzyme-Linked Immunosorbent Assay/methods , Models, Molecular , Peptide Library , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Protein Binding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , alpha-Amylases/antagonists & inhibitors
3.
Curr Pharm Biotechnol ; 3(1): 23-8, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11883504

ABSTRACT

Bacteriophage lambda has emerged as an alternative vehicle for the surface display of peptides and proteins to the commonly used filamentous phage. There are a number of unique features that make lambda an attractive display vehicle including the ability to display multimeric proteins, no requirement for secretion of the displayed fusion protein and the means to vary the valency of the displayed fusion protein. With its increasing use for cDNA encoded display, the lambda display systems will be an important tool for functional genomics.


Subject(s)
Bacteriophage lambda/genetics , Peptide Library , Peptides/genetics , Proteins/genetics , Genotype , Phenotype , Viral Fusion Proteins/genetics
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