On the basis of yeast surface display, we developed a yeast surface hybrid system of which a bait is displayed on the yeast surface through the secretory pathway, where the prey is supposed to bind the bait if it could,otherwise it will be secreted out side of the cell. By virtue of immunochemical labeling of tag fused to the prey, the binding could be easily monitored and the signal correlates to the binding affinity. The system uses an all-in-one construct, including selections for both in E.coli and yeast, and bait and prey regulated by bidirectional Gal1/10 promoters. The system,as we called yeast surface two hybrid (YS2H),would add novelty and options to the diversity screening of protein-protein interactions and would greatly benefit the discovery of protein interactions, especially for antigen-antibody engineering.
The detail of YS2H is indicated in the Fig.1-3.
Figure 1. Diagram of YS2H and its verification by coiled coil interactions. (A) Schematic diagram of gene expression cassette and protein processor pathway using Gal1/10 bidirectional promoters to express base and acid coils seperately, and the interaction was intended to be monitored by immunological labeling of Myc tag exposure on the yeast surface. (B) The same as (C), except that interaction was monitored by fluorescence coming from GFP complementation. (C) Affinity of different En-Kn pairs measured by fluorescence from immunological conjugated antibodies against Myc epitope, or GFP and OPT-GFP complementation using FACS. (D) A diagram of heptad repeats of acid- base coils and their interaction strength coming from electrostatic attraction and hydrophobic interaction. (E) Correlation diagram of disassociate constant (Kd) with relative mean fluorescence intensity (MFI) of different En-Kn pairs, gained from (C).
Figure 2. Protein interactions verified by yeast surface 2 hybrid using FACS. (A) Schematic diagram of construct used for the analysis. Antigen fusing with Aga2 and Flag tag and is supposed to display on the surface, and antibody, fused with Myc tag, is expressed as secrete form. (B) YS2H shows interaction of alpha L integrin wild type (Wt) and high affinity (HA) I domain with their antibodies AL-57, TS1/22. Immunological fluorescence against Myc and Flag tag epitope was measured by FACS, with thin line indicated uninduced cells and thick lines indicates induced (the same as the figures followed ), while green indicates 10 mM MgCl2 in the presence of labeling solution, and red indicates 10 mM EDTA in the presence of labeling solution. (C) YS2H showed interaction of TS1/22 with HA I domain, as well as the TS1/22 antibody disassociated by pH2.2 HCl-Glycine rebinding to a Wt I domain expression cells (showing in red). (D) Start from a YS2H strain with AL-57 interacting with Wt I domain, maturation against Wt I domain, two higher affinity mutations, F265S and L295P were screened.
Figure 3. YS2H was used to screen new VHH binders for botulinium neurotoxin light chain B (B-LC), a comparing with phage display. (A) Using YS2H to determine affinity of botulinium neurotoxin light chain A (A-LC) or B-LC interacting with VHHs screened by phage display. (B) YS2H screened representative clones from a library of VHHs, binding affinity was shown. (C) Sequences alignment of VHHs screened by YS2H and phage display, with conserved cysteines shown in yellow, non-conserved shown in filemot and complementary determine regions (CDR) shown in grey. Different frame work (FR) and CDR were also shown in the upper of the sequence. (D) Protein resonance patterns of four VHHs screened by phage display and YS2H. VHH B8,G6,B10 and C3 were screened by phage display, others were isolated by YS2H.
Current interests:
A) Systematic methods to generate antibodies that can cross-react with multiple serotypes of bacterial toxins
Botulinum neurotoxin (BoNT), produced by bacteria strains of Clostridium botulinum, is most potent poison in the world and thus could be a biological weapon. Antibodies may offer an opportunity to release victims suffered from BoNT baiting. There are seven different serotypes of BoNTs, from BoNT-A to BoNT G. BoNT-A and BoNT-B are most likely to be introduced to food and cause human disease. So making antibodies against both light chain of BoNT-A and BoNT-B will be most favorable for practical treatment of botulism disease. And it will also open the possibility to use one therapeutic protein to anchor different targets, with the advantage of less management and side-effects. We will use YS2H system to engineer a variable heavy chain (VHH) antibody B8, which shows high affinity to A-LC only, to cross react with B-LC.
B) Novel strategy to develop antibodies against short epitope tags
Monoclonal antibodies (mAbs) against short peptides around 10 amino acids are very useful for protein detection and purification, as could been seen from multiple widely used commercial mAbs against epitope tags, such as mAbs against c-myc, flag and HA tag. However ,mAbs against more epitopes and binding in various conditions are needed for different purpose. Candidate scFv can be easily converted into full-length of mAbs and expressed in mammalian cells. Compared to multiple limitations of producing antibodies by hybridoma, our method provides unique advantage for mAbs discovery against short peptides. On the other hand, most of antigens for mAbs have several or a cluster of key residues for antibody binding. It indicates we can engineer specific mAbs for a special region of antigens. For those diseases-related pivotal proteins with their 3D structure availability, quick generation of mAbs for the surface epitope may provide opportunity of disease therapy.