Green Fluorescent Protein (GFP) Purification. Hydrophobic Interaction Chromatography

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1 Green Fluorescent Protein (GFP) Purification Hydrophobic Interaction Chromatography

2 What is the GFP gene? GFP is a green fluorescent protein that is normally found in jellyfish. It has been engineered into other organisms.

3 Mouse under UV light (left) Same mouse under normal light (right) Mouse blood vessels (red-rfp) in tumor (red-dsred). Mouse with brain tumor expressing RFP.

4 Three 60 day old kittens. Two have been genetically modified to make RFP. All three look similar under normal light, but under UV light, only the two genetically modified kittens glow red.

5 GFP has been added to : rabbits rats mice frogs flies worms and countless others S. Stevens 6/14/2006

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7 Remember what a plasmid is? A plasmid is a small circular piece of DNA (about 2,000 to 10,000 base pairs) that contains important genetic information for the growth of bacteria.

8 pglo or GFP Plasmid

9 Gene Regulation & the Arabinose Operon Every living organism has the capability to turn genes on and off at different times and under certain conditions. The process of gene regulation is similar in all organisms, increasing in complexity as you climb up the evolutionary ladder. Much of the regulation occurs during the synthesis of messenger RNA (mrna).

10 Lac Z Operon The plasmids we have been using to transform our bacteria in the last two labs have been engineered to switch on the gene of interest only when a certain substance is present in the environment that the bacteria is living in.

11 Isopropyl β-d-1- thiogalactopyranoside (IPTG) is a molecular biology reagent. This compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon and induce protein expression. Any substance, particularly certain sugars can be used as the trigger. Arabinose is also very commonly used. In this discussion, arabinose serves the same function as IPTG on this same plasmid to do exactly what IPTG does.

12 Each cell that makes up our various organs and tissues contains the exact same complement of DNA - our personal genome. In order for our cells to become a skin cell vs. a hair cell, certain genes must be turned on or kept off. This process of switching the expression of genes on and off is known as gene regulation. Some genes must be continually expressed in all cells to maintain protein production. However, many genes are active only in specific cells or tissues.

13 The LacZ operon of E.coli: This is how the bacteria, E. coli, regulates the genes involved in the breakdown of the sugar arabinose, IPTG, etc.: Arabinose, is a sugar. It serves as an energy and carbon source in any organisms that comsume it. E. coli encodes 3 genes - arab, araa, and arad - that need to be expressed breakdown of arabinose. These genes are only expressed when the sugar arabinose is present in the environment. These three ara genes are clustered together and make up the LacZ operon.

14 LacZ operon consists of the 3 genes, arab, araa and arad, regulated by the arabinose gene activator, arac.

15 The Ara regulatory sequences are upstream (they precede the 5 end) of the arab gene. The promoter region is the site that binds the RNA polymerase enzyme, which is always required for the transcription of genes.

16 Just upstream from the promoter lies the binding site for the arabinose gene activator, arac. This activator protein is bound to a regulatory sequence and changes shape when the sugar binds to it. This shape change allows RNA polymerase to bind at the promoter sequence and transcription is initiated for the genes, arab, araa, and arad.

17 The pglo plasmid has been engineered to incorporate the LacZ operon and its regulatory regions. Both the promoter and the arac gene are present. But the arab, araa, and arad region have been replaced by the single gene for GFP. This places the GFP gene under the regulation of the LacZ promoter and regulatory regions.

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19 This means that the GFP Gene can only be transcribed when Arabinose, IPTG or some other simple sugar is present. If the gene is not transcribed, no GFP can be made.

20 The pglo plasmid Beta Lactamase (Ampicillin Resistance Gene) arac regulator protein Regulates GFP transcription Green Fluorescent Protein Aequorea victoria jellyfish gene ori bla pglo arac GFP

21 Transformed Bacteria With Fluorescence

22 Bacterial Colonies

23 Transformed Bacteria

24 Why Use Chromatography? To purify a single recombinant protein of interest from over 4,000 naturally occuring E. coli gene products.

25 Column Chromatography Chromatography used for protein purification Size exclusion Ion exchange Hydrophobic interaction

26 There are 3 Basic Steps in Chromatography 1. Adding/Binding bacterial lysate to column matrix in high salt buffer. 2. Wash less hydrophobic proteins from column in low salt buffer. 3. Elute GFP from column with no salt buffer

27 4 Buffers Used in the Lab: Equilibration buffer - A medium salt buffer used to "equilibrate" or "prime" the chromatography column for the binding of GFP. Binding buffer - A high salt Buffer that is added to the bacterial lysate. This causes the hydrophobic regions of proteins to bind to the hydrophobic regions of the column. Wash buffer - A medium salt Buffer is used to wash other proteins from the column. Elution buffer - In low salt buffers, the conformation of GFP changes so that the hydrophilic parts of the molecule are more exposed. This causes the GFP to have a higher affinity for the buffer than for the column and allows the GFP to wash off the column.

28 Binding Add the bacterial lysate to the column. Hydrophobic proteins bind to the column.

29 Washing Medium salt wash buffer. Washes weakly associated proteins from the column. GFP remains bound to column.

30 Elution Low salt buffer. GFP releases from column.