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Enzyme Purification

The document provides an overview of various techniques used to purify and characterize enzymes, including precipitation methods like ammonium sulfate precipitation, dialysis to remove small molecules, gel filtration chromatography to separate by size, ion exchange chromatography to separate by charge, hydrophobic interaction chromatography to separate based on hydrophobicity, and affinity chromatography to separate based on specific binding interactions. The key steps and principles of each technique are described at a high level.

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421 views25 pages

Enzyme Purification

The document provides an overview of various techniques used to purify and characterize enzymes, including precipitation methods like ammonium sulfate precipitation, dialysis to remove small molecules, gel filtration chromatography to separate by size, ion exchange chromatography to separate by charge, hydrophobic interaction chromatography to separate based on hydrophobicity, and affinity chromatography to separate based on specific binding interactions. The key steps and principles of each technique are described at a high level.

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ranjish_007
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© Attribution Non-Commercial (BY-NC)
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Module 4: Enzyme purification and characterization Lecture5: Enzyme purification and characterization

Protein Purification
In an organism, organism any one protein is present as a very

small percentage of the total biomolecules. In order to characterize a protein fully, it is necessary to purify it. There are many possible strategies that one could use to purify a protein. In general, the more that you know about the protein's t i ' properties, ti the th better b tt strategy t t you could ld adopt to purify it. A typical protein purification strategy will be comprised of several stages, each one taking advantage g of different characteristics of the p protein.

Downstream processing of enzymes

Modulating Solubility 1) Precipitation at the pI A protein's average net charge at its isoelectric point is 0. Above or below this pH, the protein molecules are negatively or positively charged, respectively, causing them to repel each other.

Modulating g Solubility y 2) ) Salting g in Many proteins are poorly soluble in pure water, but are much more soluble in salt-containing g solutions. Thus, lowering ionic strength can be precipitate p certain proteins. p used to p

Modulating Solubility 3) Salting out At very high concentrations (>1 M) of certain salts, proteins solubility is reduced due to a competition with the protein for interaction with water molecules.

Ammonium sulfate precipitation Precipitation with ammonium sulfate is a common first step in protein purification. Because proteins precipitate at different concentrations of salt, one can perform a first "cut" with a concentration that will leave the desired protein soluble, remove the precipitate, and then add sufficient salt to precipitate the desired protein.

Ammonium sulfate precipitation


Example: Your protein will remain soluble at 30% (w/v) ammonium sulfate, but precipitates at 40% ammonium sulfate. You slowly add the salt to your protein extract until it precipitation p to reaches a concentration of 30%, allow p occur, and then centrifuge the solution to remove the precipitate. You take the supernatant, and add ammonium sulfate until it reaches a concentration of 40%, allow precipitation to occur, and d th then centrifuge t if the th solution l ti to t remove the th precipitate. You dissolve the precipitate in a buffer and dialyze it to remove the salt.

Dialysis y The protein is put in a bag of cellulose membranes having small pores of controlled size. Proteins bigger than the pores are retained, while smaller molecules may diffuse out. As the volume of the buffer surrounding the bag is many times (100-1000x) the volume within the bag, the smaller molecules can be effectively removed after several changes of the outer buffer.

Dialysis

Gel filtration chromatography g p y


(a.k.a. "molecular sieve", "size exclusion") The protein is applied to the top of a column consisting of porous beads made of a hydrated material, such as agarose, dextran, , or p polyacrylamide. y y The pores of the beads are of a controlled size and this regulates which proteins can enter the beads. The larger proteins will be excluded from the beads and will flow through the column faster than the smaller molecules, which experience a much larger volume. In practice, i there h exists i a range of f pore sizes, i and d proteins i are separated by their sizes (and shapes): the largest are eluted , and the smallest elute last. first,

Gel filtration chromatography

Gel filtration chromatography


Vbed = Vbeads + Vvoid The void volume is the volume surrounding the beads. The bed volume is the total volume of the column. Vvoid id is typically about Vbed b d /3 A protein can be characterized by its elution volume (Velution), which hi h is i the th volume l of f solvent l t required i d to t elute l t it from f the th column. The relative elution volume (=Velution / Vvoid) is a quantity independent of the particular column used. By standardizing the column with proteins of known size, one can use this technique to estimate molecular weight (assuming that the shape is close to that of the standards more or less globular).

Commonly Used Gel Filtration Materials

I exchange Ion h chromatography h t h


This technique uses the charge on a protein to separate it from other proteins. In the process of ion exchange, ions in solution replace ions that are electrostatically bound to an inert support carrying groups with the opposite charge. Cation exchangers bear negatively charged groups. Anion exchangers bear positively charged groups. Polyelectrolytes, such as proteins, can bind to either cation or anion exchangers, depending on their net charge (i.e. depending on the pH).

Ion exchange chromatography


In most cases, a column is prepared with the ion exchanger, which is then equilibrated with the same buffer used to dissolve the protein. The exchanger and pH are chosen such that the protein will bind relatively tightly to the ion exchanger. The protein solution is loaded to the top and the column is washed with the buffer, , removing g all p proteins with the opposite charge or low charge. The protein can be eluted from the column either by changing the pH or by increasing the salt concentration, which shields the charges and thus decreases their attraction. Elution is most often f carried i d out by b applying l i a salt l gradient di to the h column. l

Ion exchange chromatography using stepwise elution.

Device for generating a linear concentration gradient.

Molecular formulas of cellulose based ion exchangers. exchangers

S Some common i ion exchangers.

Hydrophobic Interaction Chromatography (HIC)

Protein surface hydrophobicity Adsorption of biomolecules to a weakly hydriophobic surface at high salt concentration, concentration followed by elution with decreasing salt concentration gradient. gradient At high salt concentration Proteins hydrophobic surface f i exposed is d (solubility ( l bili is i reduced d d in i water) )

Hydrophobic Interaction Chromatography (HIC) matrix

5000PW

TSKgel Phenyl-5PW O

5000PW

TSKgel Ether-5PW

NPR

O TSKgel Butyl NPR

Affinity y chromatography g p y
This technique takes advantage of the fact that many proteins specifically bind other molecules as part of their function. One can use this information to construct a column containing the ligand g covalently y attached to a matrix. Upon passing the protein solution through such a column, only the proteins that can bind the ligand will be retained on the column. Then h the h conditions di i can be b adjusted dj d to effect ff release l from f the h ligand. Often this can be done simply by eluting with the soluble g version of the ligand.

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