What are abzymes?
- Catalytic antibodies
- monoclonal Abs w/catalytic activity
4 reasons to study catalytic Abs?
- Insights into enzyme catalysis
- Insights into the evolution of enzymes
- Create designer enzymes
- An alternate approach to “protein engineering”
“…binding specificity and catalytic activity may be inextricably linked in enzymes,…” Linus Pauling - Insights into autoimmune diseases
Describe the steps in producing monoclonal Abs
A means of obtaining large amounts of pure antibody (exactly the same amino acid sequence)
- Inject antigen (TS-analog) into mouse.
- Take B cells from spleen (which will make Ab but do not grow well in culture) and myeloma cells (cancerous mouse B cells) (which will grow well in culture but do not make Ab) and add polyethylene glycol (PEG) (which is used to fuse B cells and myeloma cells)
- Hybrid cells (hybridoma cells) grow in culture and make Ab. Only fused hybridoma cells will survive
- Cells put in media (called HAT) - unfused myeloma cells die (they lack an enzyme only available from B cells)
- Separate hybridoma cells - 1 cell per well (Fas separation). Screen for the presence of Ab w/the desired properties
- Purify identical Mab.
Draw a plot showing the energy involved in catalyzing a chemical rxn
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Describe the 4 steps to find (select for) catalytic antibodies with hydrolase activity
- Use a transition state analog as an antigen
- Make monoclonal antibodies to the antigen
- Look for activity in antibodies (selection with an assay)
- Purify Fab fragment from antibody
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The amino acid ester hydrolysis reaction catalyzed by antibody ___
17E8
For the 17E8 Fab fragment with bound Ts-analog, the binding site mostly formed by ____
CDR3 (Complementarity-Determining Region)
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