Recombinant Proteins

Question 1

Applications of GFP and derivatives

Answer 1

Recombinant starting plasmid containing GFP
1. Large amount of compatible MCS
- restrictio digest based cloning strategy
- form fusion proteins, introduced into cells as DNA constructs
2. Small amount
- PCR based cloning

Question 2

Red Fluorescence

Answer 2

Most red shifted emission in natural RFPs
Expression of eqFP611 in HeLa cells
Large stokes shift
Bright fluorescence

Question 3

Application test of eqFP611 as fusion partner for protein tracking

Answer 3

Notch family members play a role in a variety of developmental processes by controlling cell fate decisions
The notch signalling network is an evolutionarily conserved intercellular that regulates interactions between physically adjacent cells
Multidomain protein with the intracellular domain being involved in gene regulation

Question 4

Reporter assay, monitoring gene activity

Answer 4

Regulatory sequence to be studied (gene’s promoter)
Reporter gene (encoding GFP or luciferase)
DNA > mRNA > a reporter protein, amount is easily measured (fluorescence)

Question 5

Purification of recombinant proteins

Answer 5

6 histodines = histag
Purification using ion affinity chromatography
Bacterial expression, selection

Question 6

IMAC

Answer 6

Elution with acid and imidazole
Protein extract > TALON metal affinity flow through
Wash > elute > purified

Question 7

X ray crystallography

Answer 7

Solubilised protein
Vapor diffusion
X ray through crystal > x ray diffraction
Electron density map
3D structure

Question 8

Structure of GFP like proteins to study function

Answer 8

Large, polar, hydrophilic
- changes in Q structure
- recombinant production/IMAC
- functional test by gel filtration chromatography

Question 9

Gel filtration chromatography

Answer 9

Gel beads
Molecules move through and sorted by size