GFP Expression Overview aug2010€¦ · Green Fluorescent Protein • Normally found in the rim of...

GFP Expression

Green Fluorescent Protein

• Normally found in the rim of Jellyfish

jellyfish produces green bioluminescencefrom small photoorgans located on its

umbrella

• When the rings of twentyto thirty jellyfish aresqueezed through arayon gauze, a faintlyluminescent liquid called“squeezate” is obtained

• GFP Green Fluorescentprotein can extractedfrom “Squeezate”

Our system

• GFP gene is on a plasmid (small DNA molecule)with the selectable marker of Amp and under aninducible Arabinose promoter.

What is a plasmid?• A circular piece of

autonomouslyreplicating DNA

• Originally evolved bybacteria

• May express antibioticresistance gene

or be modified toexpress proteins ofinterest

The Many Faces of Plasmids

Transmission electron micrograph

Agarose gelGraphic representation

BLA= beta lactamase• BLA encodes for an enzyme Beta

lactamase• The function is to cleave lactam group off

Beta lactams ( penicillin , ampicillin andrelated molecules) to render it in active

• Confers antibiotic resistance• Used as a means of selecting for

tranformed cells

Transcriptional Regulation

• pGLO plasmid• Arabinose

operon• BLA

Ampicillinase

Transcriptional Regulation

B A DaraC

B A DaraC

RNA Polymerase

Effector (Arabinose)

araC B A D

ara Operon

RNA Polymerase

Z Y A

Z Y ALacI

Effector (Lactose)

Z Y ALacI

lac Operon

Arabinose gene control is similar to Lac operon

Gene Regulation

RNA Polymerase

araC

ara GFP Operon

GFP Gene

araC GFP Gene

araC GFP Gene

Effector (Arabinose)

B A DaraC

B A DaraC

RNA Polymerase

Effector (Arabinose)

araC B A D

ara Operon

Example of an induction

Expression of GFP can be linkedwith another gene

E.Coli cell is like a bag containing allsorts of proteins

Strategy for GFP purification

• Harvest cells• Lyse cells• Precipitate protein to reduce volume• Apply to chromatography columns;

selectively desorb

Lysozyme cuts bonds betweenSugars on the cell walls

Cell Wall weakens and the cellbursts open

Lysozymefacts:

• Lysoszyme is an enzyme that fightsinfections (kills certain types ofbacteria)

• Found in Tears, Saliva and NasalDrippings

• Also found in Chicken egg whites

• Used to prevent Bacterialcontamination in Beer making

• Cows secrete lysozyme into theirstomaches as a digestive enzyme

Lysozyme in action

Why Use Chromatography?

• To purify a singlerecombinantprotein of interestfrom over 4,000naturally occuringE. coli geneproducts.

After Lysis A mixture of proteins is released

Hydrophobic InteractionChromatography:

Steps 1–3Add bacterial lysate to column matrix inhigh salt buffer

2.Wash less hydrophobic proteins fromcolumn in

low salt buffer

3.Elute GFP from column with no salt buffer

Step 1:Hydrophobic Interaction

Chromatography

• Add bacteriallysate tocolumn matrixin high saltbuffer– Hydrophobic

proteinsinteract withcolumn

Step 2:Hydrophobic Interaction

Chromatography• Wash less

hydrophobic fromcolumn with lowsalt buffer– Less

hydrophobicE. coliproteins fallfrom column

– GFP remainsbound to thecolumn

Step 3:Hydrophobic Interaction

Chromatography• Elute GFP from

column byadding no saltbuffer

GFP– Released

from columnmatrix

– Flows throughthe column