In vitro molecular evolution yields an NEIBM with a potential novel IgG binding property

Abstract

Staphylococcus aureus protein A (SpA) and protein G of groups C and G streptococci (SpG) are two well-defined bacterial immunoglobulin (Ig)-binding proteins (IBPs) with high affinity for specific sites on IgG from mammalian hosts. Both SpA and SpG contain several highly-homologous IgG-binding domains, each of which possesses similar binding characteristic of the whole corresponding proteins. Whether specific combinations of these domains could generate a molecule with novel IgG-binding properties remained unknown. We constructed a combinatorial phage library displaying randomly-rearranged A, B, C, D and E domains of SpA as well as the B2 (G2) and B3 (G3) domains of SpG. In vitro molecular evolution directed by human, rabbit, bovine, or goat polyclonal IgGs and four subclasses of mouse monoclonal IgGs generated one common combination, D-C-G3. A series of assays demonstrated that D-C-G3 exhibited a potential novel IgG binding property that was obviously different from those of both parent proteins. This study provides an example of successful protein engineering through in vitro molecular evolution and useful approaches for structure and function studies of IBPs.

A facile label-free electrochemiluminescent biosensor for specific detection of Staphylococcus aureus utilizing the binding between immunoglobulin G and protein A

“…As seen in Fig. 1, green fluorescence from FITC was observed mainly on the outer layer of bacterial cells, which was consistent with the previous claim that SPA existed in the cell wall of S. aureus [25]. The phenomenon also demonstrated the feasibility of label-free detection of S. aureus based on the binding of porcin IgG to S. aureus.…”

“…This protein has been conventionally adopted in immunoassays to oriented immobilization of antibody and expose Fab region of IgG to facilitate immunobinding between antibody and antigen [24][25][26]. Graphene, a two-dimensional carbon sheet with almost one atom thickness, has been proved to be an ideal carrier for protein immobilization due to its powerful mechanical strength, large surface area, and excellent thermal stability [27,28].…”

Binding characteristics of staphylococcal protein A and streptococcal protein G for fragment crystallizable portion of human immunoglobulin G

“…The authors believe that the results of this study have three representative implications in biotechnology. First, these intuitive and quantitative observations can be useful for biochemists and biophysicists to decide the bacterial protein to use in experiments such as immunoprecipitation techniques, double sandwich immunoassays, and affinity purification [73] , [74] . Conventionally, the experimenter knew from experience whether spA binds to human IgGs more strongly than spG but did not know how strongly they bind and what non-covalent intermolecular interactions facilitate this [73] , [75] , [76] .…”

Wireless poly(dimethylsiloxane) quartz-crystal-microbalance biosensor chip fabricated by nanoimprint lithography for micropump integration aiming at application in lab-on-a-chip

“…Qi et al60) 6.47 × 105 2.30 × 10 −5 2.81 × 10 10 result of comparison is shown in the last row of Table I. Qi et al calculated the affinity parameters using the SPR sensor.…”