Lecture 11 Flashcards
1
Q
What are abzymes?
A
- Catalytic antibodies
- monoclonal Abs w/catalytic activity
2
Q
4 reasons to study catalytic Abs?
A
- 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
3
Q
Describe the steps in producing monoclonal Abs
A
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.
4
Q
Draw a plot showing the energy involved in catalyzing a chemical rxn
A
Slide 5
5
Q
Describe the 4 steps to find (select for) catalytic antibodies with hydrolase activity
A
- 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
6
Q
Slide 7
A
N/A
7
Q
Slide 8
A
N/A
8
Q
Slide 9
A
N/A
9
Q
Slide 10
A
N/A
10
Q
Slide 11
A
N/A
11
Q
The amino acid ester hydrolysis reaction catalyzed by antibody ___
A
17E8
12
Q
For the 17E8 Fab fragment with bound Ts-analog, the binding site mostly formed by ____
A
CDR3 (Complementarity-Determining Region)
13
Q
Slide 14
A
N/A
14
Q
Slide 15
A
N/A
15
Q
Slide 16
A
N/A
