Using offset recombinant polymerase chain reaction to identify functional determinants in a common family of bacterial albumin binding domains.
The 46 amino acid GA albumin binding module is a putative virulence factor that has been identified in 16 domains from four bacterial species. Aside from their possible effects on pathogenicity and host specificity, the natural genotypic and phenotypic variations that exist among members of this module offer unique opportunities for researchers to identify and explore functional determinants within the well-defined sequence space. We used a recently developed in vitro recombination technique, known as offset recombinant PCR, to shuffle seven homologues that encode a broad range of natural GA polymorphisms. Phage display and selection were applied to probe the recombinant library for members that showed simultaneous improvements to human and guinea pig serum albumin binding. Thermodynamic data for the most common phage-selected mutant suggest that domain-stabilizing mutations substantially improved GA binding for both species of albumin.
Rozak
David A
DA
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA. david.rozak@amedd.army.mil
Alexander
Patrick A
PA
He
Yanan
Y
Chen
Yihong
Y
Orban
John
J
Bryan
Philip N
PN
D000418:Albumins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D002151:Calorimetry; D002942:Circular Dichroism; D015723:Gene Library; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013696:Temperature
D000418:Albumins; D001426:Bacterial Proteins