Green Fluorescent Protein (GFP) Purification by Chromatography Lab

Inoculating-Growing a cell culture Observe sterile techniques throughout the experiment/lab exercise Add 3 mls of TE solution to vial containing ampicillin and arabinose, respectively on Feb 20. Add 55 mls DI water to a 250 mls Erlenmeyer flask-heat to boiling in a microwave. Add single LB tablet to the flask. Let it soak for 20 minutes. Repeat heating and swirling several times till the entire tablet is dissolved. It takes 10 minutes to rehydrate arabinose. Ampicillin is an antibiotic which inhibits growth of bacterial contaminants which may be introduced from environment. Arabinose is a sugar which induces the overexpression of GFP in cloned cells.

Inoculating-Growing a cell culture Cool the LB media and add 500 μl of arabinose and ampicillin into flask. Swirl the flask to mix the components. Aliquot 2 mls of liquid media into 16 culture tubes. Store in refrigerator till Feb 23. Per head use three culture tubes – inoculate a colony from +pGLO/LB/Amp/Ara, +pGLOLB/Amp and –pGLO/LB/Amp into three separate tubes (on Feb 23). Shake for 30 seconds. Place the tubes horizontally in incubator at 32°C till Feb 24. LB is made up of extract of yeast and an enzymatic digest of meat byproducts which provides a mixture of carbohydrates, amino acids, nucleotides, salts and vitamins all of which are nutrients for bacterial growth. Single bacterial colony originates from single bacterium, all the individual bacteria in colony are genetically identical and are called clones. Single isolated colonies are chosen for transformation to the culture media as these should be 1-2 mm away from other colonies which are not contaminated with other bacteria. Orienting the tubes horizontally – more surface area of culture media is exposed to the air in tube, allowing more oxygen to diffuse into cells. Shake periodically will enhance more growth.

Purification phases Bacterial Concentration and Lysis – Feb 25 - Rehydrate vial of lysozyme with 1 ml of TE buffer Removing bacterial debris – Feb 27 Protein chromatography – Mar 2 Place the lysozyme in refrigerator till sue

Purification phase 1 Centrifugation – Transfer from culture tube into microtube. Results in pellet of bacteria found at bottom of tube and liquid supernatant above the pellet. Pour off the liquid supernatant Observe the pellet with UV light The pellet will fluoresce bright green upon exposure to UV light because green protein is being expressed within the bacteria.

Purification phase 1 Add 250 μl of TE buffer to pellet of bacteria – resuspend rapidly by pipetting up and down or vortex gently Keep on resuspending till no bacterial chunks are seen - Lysozyme – break cell wall Lysozyme is an enzyme that functions to degrade or lyse the cell wall by cleaving polysaccharide sugars. The subsequent freeze-thaw step aids in complete disruption of wall and internal membranes. Cytoplasm expands and lyses the cells and complete disruption will release soluble components including GFP into medium.

Purification phase 2 Removing bacterial debris: Centrifugation step to separate large particles of lysed bacteria from smaller proteins like GFP Supernatant will fluoresce – UV exposure Remove supernatant into new 2 mls microtube-done immediately Separate large particles of lysed bacteria such as cell membrane and walls from small proteins like GFP. Transfer of supernatnat should be done immediately to prevent pellet debris from leaching and contaminating supernatant and might also clog chromatography column.

Purification phase 3 Hydrophobic interaction chromatography: Thousands of endogenous protein + GFP separation GFP has several stretches of hydrophobic regions GFP sticks to column Less hydrophobic and more hydrophillic will elute out Column has bed of microscopic beads. These beads form matrix through which proteins must pass before being collected. The matrix has affinity for molecule of interest (GFP) and not other bacterial proteins. GFP sticks to column allowing other contaminants to separate out.

Four buffers used are: Equilibration buffer – medium salt buffer (2 M (NH4)2SO4) – used to equilibrate or prime the column for binding of GFP Binding buffer- high salt binding buffer (4M (NH4)2SO4) + bacterial lysate Wash buffer – medium salt buffer (1.3M (NH4)2SO4 – washes weak proteins (GFP starts penetrating into column) Elution buffer – low salt buffer – 10mM TrisEDTA – wash GFP from column Euilibration buffer- prepares the column for application of GFP. It raises salt concentration of column to match that of bacterial GFP lysate. Binding buffer raises salt concentration of GFP which causes a conformational change in GFP, exposing hydrophobic regions which are further able to interact with and bind the hydrophobic regions of column and hydrophillic regions are shielded Wash buffer functions to wash away less hydrophobic and contaminating proteins from column. TE buffer has lowest salt concentration because it functions to remove GFP from column. The hydrophobic patches of proteins re-orient to the interior and hydrophillic regions are exposed in low salt buffer. The buffer has high concentration of water molecules which will change conformation of GFP so that GFP are more exposed to surface causing GFP to have higher affinity for buffer than for the column.

Successful chromatography Place column gently into collection tubes. Create a paper crutch – size of match stick and wedge between column and collection tube – impossible for air tight seal to form and insures column will flow Cap the tubes in elution steps – creates air pressure which pushes on column bed causing sample to flow faster The GFP flow out – UV light If column is disturbed – no elution as distinct ring – elute out as irregular and distorted shape. Jamming the column tightly into collection tubes will create an air tight seal and sample will not flow through.

This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

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