Lecture 4.3 (reporter gene GFP) Flashcards
What organism produces luminescent protein called GFP?
Aequorea Victoria
What wavelength excites GFP?
395nm
emission maximum at 509nm
Apart from GFP, what fluorescent proteins have mutations led to?
enhanced GFP
yellow GFP
cyan GFP
What is the advantage of using GFP-based reporter assays over other reporter gene assays?
they do not require cell permeabilization or the addition of exogenous substrates.
What is the downside of GFP-based reporter assays?
they do not take advantage of an enzymatic amplification step they are generally less sensitive than other reporter assays
Why are GFP-based reporter assays less sensitive?
they do not take advantage of an enzymatic amplification step
In luciferase assay, what reaction does firefly luciferase catalyse?
the oxidation of firefly luciferin
–> produce a short- lived flash of light that decays within a few seconds
State the 2 steps through which luciferase oxidises firefly luciferin?
- luciferin + ATP → luciferyl adenylate + PPi
- luciferyl adenylate + O2 → oxyluciferin + AMP + light
Are luciferase assays more sensitive that CAT assays?
Yes, 100-fold more sensitive
can detect as little as 10-20 moles of enzyme
What is the downside of luciferase assay? How is it overcome?
short- lived flash of light that decays within a few seconds
–>Reagents are available that prolong the half-life of the flash to several hours
What is a common alternative to firefly luciferase?
sea pansy Renilla reniformis enzyme (sea pansy luciferase)
–> catalyzes the oxidation of coelenterazine to produce light
What reaction does Renilla reniformis enzyme catalyse?
the oxidation of coelenterazine to produce light.
What are the main constituents of the ‘dual-luciferase reporter assay’?
-firefly luciferase
-sea pansy luciferase
–>reactions to be
monitored independently
–>two independent assays to be performed simultaneously
Thus, higher speed of screening
What is the full form of SPAP?
Secreted Placental Alkaline Phosphatase
What is SPAP?
C-terminal truncated mutant of the membrane anchored placental
alkaline phosphatase
What kind of an enzyme is SPAP?
SPAP is hydrolase enzyme responsible for removing phosphate groups from nucleotides, proteins and alkaloids
–> effective in alkaline environment
What is the advantage of using SPAP reporter gene assay?
repeated sampling
–> Removal of the 30 amino acid anchoring domain ensures that the enzyme is secreted into the culture medium
What process leads to SPAP being secreted into the culture medium?
Removal of the 30 amino acid anchoring domain ensures
How is the expression of SPAP gene monitored in SPAP gene based assay?
- Using colorimetry, after incubation with substrate p-nitrophenol phosphate
- or chemiluminecently with the CSPD
Name 2 substrates of SPAP and state how they are monitored/measured?
- p-nitrophenol phosphate
- CSPD
How to differentiate between the activity of placental from of alkaline phosphatase and that of endogenous alkaline phosphatases?
- placental form is very heat stable –> thus activity can be detected by heating to 65C and destroying endogenous phosphatases
or
2. endogenous forms of the enzyme can be inhibited by L-homoarginine
What can SPAP assay be coupled to?
coupled to luciferase reaction with substrate firefly D-luciferin-O-phosphate
What substrate is to be used if SPAP assay is to be coupled to luciferase assay?What is the resulting product?
firefly D-luciferin-O-phosphate.
product of 1st rxn is firefly luciferin, which is substrate for firefly luciferase
What is the advantage of SPAP assay couple to luciferase reaction?
The two-step reporter gene assay improves the detection limit to the zetamole range
Why is the SPAP assay + luciferase assay a versatile reporter gene system?
- higher sensitivity
- can repeatedly sample culture medium
What are beta-lactamases?
a family of bacterial enzymes that cleave penicillins and cephalosporins
What is the E.coli form of beta-lactamase called?
TEM-1 beta-lactamase
For beta lactamase assays, what forms is the E.coli form of beta-lactamase engineered into?
cytosolic, membrane associated or secreted forms
What makes beta-lactamase an excellent choice for eukaryotic reporter gene assay?
it has no endogenous counterparts
What is used to analyse beta-lactamase activity in beta-lactamase based assays?
colorimetric
Name some substrates of beta-lactamase.
membrane-permeable fluorogenic substrate, CCF2/AM
–> enables determination of
the expression of as few as 50 beta-Lactamase molecules within a cell.
The most commonly used hit/lead identification strategy nowadays is a
combination of two extremes.
What are the 2 extremes?
- knowledge-based approach
- serendipity-based approach
What does the knowledge-based approach for hit/lead identification
strategy use?
literature/patent derived molecular entities, endogenous ligands, and
biostructural information
What does the serendipity-based approach for hit/lead identification
strategy use?
combinatorial chemistry, high-throughput screening of compound libraries
