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.

Figure 1. Proportion of the phage clones with different sizes of inserted fragments from the 22 phage clones after each round of selection with eight IgG molecules (A–H).

, phage clones with no inserted fragment; , phage clones displaying one domain of the combinatorial Ig-binding molecules; , phage clones displaying two domains of the combinatorial Ig-binding molecules; , phage clones displaying three domains of the combinatorial Ig-binding molecules.

Figure 2. The prokaryotic expression of SpA, SpG, D-C, D-B and D-C-G3.

M, protein marker, indicating 116.0 kDa, 66.2 kDa, 45.0 kDa, 35.0 kDa, 25.0 kDa, 18.4 kDa and 14.4 kDa; lane 1, D-C-G3; lane 2, D-C; lane 3, SpA; lane 4, SpG; lane 5, PET-32A.

Figure 3. Binding activities of D-C-G3 to hIgG, rIgG, bIgG, gIgG and four subclasses of monoclonal mIgG compared to D-C, D-B, SpA and SpG according to ELISA analysis.

, D-C-G3;, D-C; , SpA; , SpG; -, PET-32A control protein.

Figure 4. Binding activities of D-C-G3 to hIgG, rIgG, bIgG, gIgG and four subclasses of monoclonal mIgG compared to D-C, D-B, SpA and SpG according to western blot (A) and dot blot analyses (B).

Western blot results (A) are representative cropped images and every set have been processed under similar conditions as detailed in the methods section.

Figure 5. Comparison of the inhibitory potentials of D-C-G3, SpA and SpG on the binding of hIgG, rIgG, bIgG, gIgG and four subclasses of monoclonal mIgG to D-C-G3, SpA and SpG according to ELISA analysis.

, D-C-G3;, D-C; , SpA; , SpG; -, PET-32A control protein. The inhibition of the binding of SpA to bIgG, gIgG and mIgG2a was not shown, as the binding activities of SpA were too weak to be inhibited.

Figure 6. Sequence comparison of the SpG binding sites in the CH1 γ chain of these eight IgG molecules.

The sequences of bIgG CH1 (GenBank: ABE68619.1), hIgG CH1 (GenBank: AAA02914.1), rIgG CH1 (GenBank: AAB59265.1), gIgG CH1 (GenBank: AAX45026.1), mIgG1 CH1 (GenBank: CAD32497.1), mIgG2a CH1 (GenBank: BAA11361.1), mIgG2b CH1 (GenBank: BAA11359.1) and mIgG3 CH1 (GenBank: AAB59697.1) were aligned. The shaded sequences represent the binding sites to SpG. , No. 142 amino acid residue in mouse immunoglobulin gamma 1 heavy chain.

Figure 7. Comparison of IgG purification from mouse monoclonal ascites, human serum and rabbit serum with D-C-G3, SpA and SpG affinity chromatography.

(A) SDS/PAGE analysis of different IgG purified by D-C-G3, SpA and SpG affinity chromatography. M, protein molecular weight marker; 1–3, mIgG2a purified by DCG3, SpA, SpG affinity chromatography, respectively; 4–6, mIgG3; 7–9, mIgG2b; 10–12, mIgG1; 13–15 human IgG; 16–18, goat IgG purified by DCG3, SpA, SpG affinity chromatography, respectively. (B) Recovery of IgG from monoclonal antibodies, human serum and rabbit serum purified by different affinity chromatography. Black bars, purified IgG with DCG3 affinity chromatography; grey bars, purified IgG with SpA affinity chromatography; white bars, purified IgG with SpG affinity chromatography.