Abstract
BackgroundProtein A, protein G and protein L are three well-defined immunoglobulin (Ig)-binding proteins (IBPs), which show affinity for specific sites on Ig of mammalian hosts. Although the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. The single domains of protein A, protein G and protein L were all demonstrated to have function to bind to Ig. Whether combinations of Ig-binding domains of various IBPs could exhibit useful novel binding is interesting.ResultsWe used a combinatorial phage library which displayed randomly-rearranged various-peptide-linked molecules of D and A domains of protein A, designated PA(D) and PA(A) respectively, B2 domain of protein G (PG) and B3 domain of protein L (PL) for affinity selection with human IgG (hIgG), human IgM (hIgM), human IgA (hIgA) and recombinant hIgG1-Fc as bait respectively. Two kinds of novel combinatorial molecules with characteristic structure of PA(A)-PG and PA(A)-PL were obtained in hIgG (hIgG1-Fc) and hIgM (hIgA) post-selection populations respectively. In addition, the linking peptides among all PA(A)-PG and PA(A)-PL structures was strongly selected, and showed interestingly divergent and convergent distribution. The phage binding assays and competitive inhibition experiments demonstrated that PA(A)-PG and PA(A)-PL combinations possess comparable binding advantages with hIgG/hIgG1-Fc and hIgM/hIgA respectively.ConclusionIn this work, a combinatorial phage library displaying Ig-binding domains of protein A, protein G, or protein L joined by various random linking peptides was used to conducted evolutional selection in vitro with four kinds of Ig molecules. Two kinds of novel combinations of Ig-binding domains, PA(A)-PG and PA(A)-PL, were obtained, and demonstrate the novel Ig binding properties.
Highlights
Protein A, protein G and protein L are three well-defined immunoglobulin (Ig)-binding proteins (IBPs), which show affinity for specific sites on Ig of mammalian hosts
The precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria
Distribution of various fragment sizes displayed by phage library and post-selection populations To evaluate the Ig affinity selection efficacy, some markers including phage library binding capacity, output/input ratio of phages, distribution of various fragment size etc. were measured
Summary
Protein A, protein G and protein L are three well-defined immunoglobulin (Ig)-binding proteins (IBPs), which show affinity for specific sites on Ig of mammalian hosts. The precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. Protein A of Staphylococcus aureus (SpA), protein G of group C and G streptococci (SpG), and protein L of Finegoldia magna formerly Peptostreptococcus magnus are three well-defined IBPs. the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. The extracellular part of SpA contains a tandem repeat of five highly homologous IgG-binding domains designated (from the N terminus) E, D, A, B and C, each of which contains about 58 amino acid residues. SpG (about 63 KD) is composed of 594 amino acid residues, containing 3 highly homologous Ig-binding domains identified as B1, B2 and B3 [5]. The fold of the Ig-binding domains of protein L is similar to that of the domains of SpG [8,9]
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