Question

lab question

1. What is the basis of the different purification methods?

2. What are some of the factors the might have interfered with your results?
3. How might you improve the process to increase the yield and purity?

lab process

E. coli BL21 (DE3) cells were transformed with the pET Topo-1521 vector containing a reading frame encoding the green fluorescent protein (GFP). Cells were cultured in M9ZB media at 37°C until the absorbance at 600 nm reached 0.7, at which point expression was induced by the addition of IPTG and cultures were transferred to 26°C and incubated overnight. The cells were harvested by centrifugation (10 min and 13000 g) and resuspended in 2 vol. of extraction buffer (50 mM NaH2PO4, 100 mM NaCl, 50 mM Imidazole, pH 8.0). After lysis by sonication, cell debris was pelleted by centrifugation at 13000 g for 10 min. Solutions provided: Crude lysate – a mixture of lysed cells containing GFP Protein standard – 4 mg/ml BSA Biuret reagent – Coomassie solution that reacts with proteins to produce a purple colour Resuspension buffer - 50 mM NaH2PO4, 100 mM NaCl, 30 mM Imidazole, pH 8.0 IMAC elution buffer - 50 mM NaH2PO4, 100 mM NaCl, 300 mM Imidazole, pH 8.0 IEX elution buffer - 50 mM NaH2PO4, 500 mM NaCl, 30 mM Imidazole, pH 8.0 Saturated ammonium sulphate Why has each of the components of the different buffers been included? Measuring the protein concentration: Make a protein standard curve, and test the protein concentration of the crude lysate (dilutions may be required). Making the standard curve– each person should prepare a set of cuvettes a) Prepare 6 cuvettes, and add the appropriate amount of protein standard, buffer, and Biuret reagent to each cuvette (see table next page) and mix by inverting immediately. Wait for 5 minutes while the colours develop – the colour is then stable for about an hour. b) The Novaspec spectrophotometer should be set to read absorbance at 530 nm. Using the sample from cuvette 1 as your blank, set the reference to 0.000 AU c) Read the absorbance of your tubes and record the readings in the table below. For both sets of cuvettes, graph the absorbance against the concentration. This is your calibration curve.Make up 0.5 mL of a diluted protein sample (suggest starting with a 1/10 dilution, dilute using resuspension buffer) and test as above. If the reading is too low, try a higher concentration. If it is too high, try a lower concentration. It needs to be on the linear part of the curve. Measuring the GFP concentration: Test the crude lysate for GFP concentration by measuring the absorbance at 488 nm (the extinction coefficient of GFP is 53,000 M-1 cm-1 and the molecular weight of GFP is 27,000 g/mol). You may need to dilute your sample using resuspension buffer so that the absorbance is between 0-1. Use resuspension buffer to blank the spectrophotometer. 3 different purification procedures will be carried out. 1) Affinity chromatography (IMAC) – 1 mL of crude lysate will be applied to a 1 mL His-Trap column (GE Health Science). The column will be washed with 5 column volumes (5 mL) of the resuspension buffer, giving you a final volume of 6 mL, and then the protein eluted into a separate tube with 6 mL IMAC elution buffer. 2) Selective precipitation – 300 µL of crude lysate will be added to 700 µL of saturated ammonium sulphate solution and then centrifuged (5 min, 8,000 rpm). The pellet will be separated from the supernatant by gently decanting the superantent into another tube, and resuspended in 1 mL of resuspension buffer. Samples will be collected for the supernatant and precipitate. 3) Ion Exchange – 1 mL of crude lysate will be applied to a 1 mL Q-sepharose column (GE Health Science). The column will be washed with 5 column volumes (5 mL) of the resuspension buffer, giving you a final volume of 6 mL, and then the protein eluted into a separate tube with 6 mL IEX elution buffer. Samples will be run by SDS-PAGE (4-12% Bis-Tris) as per manufacturer’s instructions, and stained with simply blue stain. The marker used contained bands at 260, 160, 110, 80, 60, 50, 40, 30, 20, 15, 10, 3.5 kDa)

Answer 1

Answer 1:-

1) AFFINITY CHROMATOGRAPHY:- This method works on the basis of specific binding affinity of a protein molecules towards a ligand that has been immobilized onto a solid matrix.

In this case, an agarose based solid column is prepared onto which a ligand is bound which will interact with the protein of interest.

The interaction can be specific or non-specific. Specific interaction includes formation of covalent bonds between functional groups such as amine and carboxyl which can be eluted out after the chromatography process is finished.

2) SELECTIVE PRECIPITATION:-

This process is based on changing the ionic strength of the solution used to precipitate out the protein molecules.

Proteins show presence of ions and charged amino acids such as lysine which interacts with the water molecules to maintain the soluble state of the protein however when the ionic strength increases, this interaction is affected and the solubility is altered. At high concentration, in other words there will be reduced solubility with increase in ionic strength. Ammonium salts are mostly preferred for this method.

3) ION EXCHANGE CHROMATOGRAPHY:-

In case of ion-exchange chromatography, the proteins are separate on the basis of charged associated with them at particular pH. Proteins may have a positive or negative charge depending upon the amino acid side chain that indicates whether they are acidic or basic.

Protein when present at pI (isoelectric point) is neutral and when placed in buffered solution having pH less than pI, the net charge on proteins will be positively charged and hence will bind to a negatively charged substrate. The reverse is the case when the solution pH is more than proteins pI. Here the protein will be negatively charged and will bind to positively charged substrate.

Fhe negatively charged substrate is called Cation Exchange Resin and the positively charged substrate is called Anion Exchange Resin.