| Immunoglobulin (Ig)-binding proteins (IBPs) are kinds of binding proteins with Ig in form of non-antigen produced by several of pathogens. The three well-defined IBPs contain Protein A of Staphrylococcus aureus (Staphylococcal protein A, SpA), protein G of group C and G streptococci (Streptococcal protein G, SpG), and protein L of Finegoldia magna formerly Peptostreptococcus magnus (Peptostreptococcus magnus protein L, PpL) which are produced by bacterium. They all contain a tandem repeat of multiple highly homologous Ig-binding domains and the single binding domain as the basic unit posses the entire Ig binding activity. Their unique properties of binding a large population of antibodies make them possess potential applications in purification of antibodies, antibody detection and antibody adsorption.NEIBM AL, combination of SpA A domain and PpL B3 domain, as well as LD5 and LD3 is a kind of novel evolved Ig binding molecules (NEIBMs) yielded by in vitro molecular evolution of combinatorial phage libraries displaying randomly-rearranged molecules of various Ig binding domains of SpA, SpG and PpL. They all showed the novel Ig binding property of the double-site binding to VH3 and Vk regions of human Ig Fab, and created the high affinity for human IgM which was demonstrated to enhance the IgM detection in anti-HCV ELISA assay. However, the A domain of NEIBMs AL binds to the VH3 chain with low affinity, the A domain only binds to the VH3 chain with low affinity. We have constructed several combinatorial phage libraries displaying the AL mutants which were randomly mutated at different positions at the A domain of AL. In vitro molecular evolution of these libraries directed by human IgM generated AL (VV) mutant could improve the anti-HCV detection effect. Whether the insertion of some peptides between helix Ⅱ and helix Ⅲ could produce the new bmding sites with the VH3 remains interesting.Object To obtain the novel evolved Immunoglobulin (Ig)-binding moleculars with enhanced human IgG and IgM binding activities, several the combinatorial phage libraries displaying insert-mutants of NEIBM AL. or AL(VV) were constructed, affinity selection with human IgM as bait of these phage libraries were conducted.Methods1. Construction of combinatorial phage libraries displaying insert-mutants of NEIBM AL or AL (VV). The mutants libraries of NEIBM AL or AL(VV) with inserted random peptide of 3,5,7,9 amino acids (aa) at positions between AA 38 and 39 of SpA A domain (library A3, library A5, library A7, library A9, library V3) were generated by PCR-based technology respectively.2. The in vitro molecular evolution of combinatorial phage libraries displaying insert-mutants of NEIBM AL or AL (VV) directed by human IgM molecule. The control phages disappeared through several rounds of evolutional selection cycles of coating, incubating, washing and amplifying demonstrated the successful evolution.3. Prokaryotic expression, purification and binding analysis of the inserted mutants:to further characterize the IgM and IgG binding properties of these AL mutants, we expressed these AL inserted mutants from the phages with improved binding activity, as well as NEIBM AL and AL(VV). The encoding DNA fragments were cloned into the prokaryotic expression vector pET32a(+) to construct the recombinant vectors and expressed in E.coli BL21. The IgM and IgG binding activity of these insert-mutants of NEIBM AL and AL (VV) were detected by binding ELISA, competitive inhibition ELISA and SPR assays.4. To investigate the potential application of these insert-mutants of NEIBM AL, as well as NEIBM AL, AL(VV), they were all labeled with horseradish peroxidase (HRP) respectively, and verified by binding ELISA assay. Then we established the anti-HIV ELISA assays using HRP-AL mutants as conjugated antibodies respectively to compare their detection effect in a panel comprising of forty anti-HIV positive serum specimens from HIV patients and forty serum specimens from healthy blood donors.Results:1. The size of these four libraries, A3, A5, A7, V3 were 3.2×106,2.04×l06,1.8×106, 0.9×106 and 3.1×106 members respectively, and titers were 2.3×1012,2.1×1012,1.9×1012 and 2.2x101 transformation unit (TU/mL). These results showed that the capacity, variety and randomicity of these four libraries met the requirement for the in vitro molecular evolution.2. Each 90 monoclonal phages were randomly picked up and prepared from post-selection library A3, A5, A7 and V3 respectively, and evaluated the binding activity with human IgM by phage ELISA. The phage clones from post-selection library A3, A7 and V3 exhibited the higher binding activity with human IgM than NEIBM AL phages were picked up to analyze the sequence. Six insert-mutants of NEIBM AL and AL (VV), AL137-KTS, AL137-TNQ, AL137-NNN, AL (VV) 137-NTQ, AL (VV) 137-NDD and AL (VV) 137-KNQ were obtained.3. The encoding DNA fragments of insert-mutants of NEIBM AL and AL(W) were cloned to the prokaryotic expression vector pET32a(+) to construct the recombinant vectors, named as pET32a-ALI37-KTS, pET32a-ALI37-TNQ, pET32a-ALI37-NNN, pET32a-AL(VV)I37-KNQ, pET32a-AL(VV)I37-NDD, pET32a-AL(VV)I37-NTQ and pET32a-AL as well as pET32a-AL(VV). The highly purified (purity>90%) of insert-mutants of NEIBM AL and AL(VV), AL137-KTS, AL137-TNQ, AL137-NNN, AL(VV)I37-KNQ, AL(VV)I37-NDD, AL(W)I37-NTQ and AL(VV) as well as NEIBM AL were obtained by Ni-NTA affinity chromatograph. The result showed that all of six insert-mutants of NEIBM AL and AL(VV) retained the comparable IgM and IgG binding activity as to NEIBM AL and AL(VV) through ELIS A, inhibition ELIS A and SPR assays.4. AL, AL(W), AL137-KTS, ALI37-TNQ, ALI37-NNN, AL(W)I37-KNQ, AL(W)I37-NDD and AL(VV)I37-NTQ were labeled with horseradish peroxides (HRP) to produce HRP-AL, HRP-AL(VV), HRP-AL137-KTS, HRP-ALI37-TNQ, HRP-ALI37-NNN, HRP-AL(VV)I37-KNQ, HRP-AL(VV)I37-NDD and HRP-AL(VV)I37-NTQ. The ELISA results showed all of these assays presented the same detection effects for negative serum specimens, while ALI37-NNN, AL(VV)I37-NTQ,and AL(VV)I37-KNQ based assays showed significantly improved detection effect than HRP-AL for HIV positive serum specimens (P<0.001).Summary:In this study, the in vitro molecular evolution of insert-mutants of NEIBM AL and AL(VV) were conducted successfully. And insert-mutants of NEIBM AL and AL(VV) with improved IgM and IgG binding activity were obtained. The insert-mutant of NEIBM AL and AL(VV), AL137-NNN, AL(VV)I37-NTQ, and AL(VV)I37-KNQ improved the detection effect of anti-HIV assay, presenting the application potential. This study provides an example of the successful protein engineering by in vitro molecular evolution. |
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