BI201: protein purification

what is protein purification

procedures used to isolate proteins from cells or other complex mixtures

exploits differences in protein

• size

• charge

• sedimentation rate

• binding affinity

protein expression trials

small scale expression trials can be conducted prior to large scale expression to test effect of varying the growth conditions on protein expression level— can have a dramatic influence on the amount of protein produced

e.g. by optimising growth conditions in E.coli, protein of interest can represent up to 50% of the total protein in the cell

Pichia pastoris yeast can secrete the protein of interest into the medium— not possible with membrane proteins

Cell lysis

• if protein of interest is not secreted, next step is to break open cells to release the protein— cell lysis

• E.coli can be lysed by sonication. a machine called a sonicator is attached to a probe which emits high frequency sound waves into the sample that cause the cells to rupture

• anti-bacterial enzyme puridfied from egg white called lysozyme often used to break bacterial cell wall of E.coli

• Pichia pastoris yeast must be lysed using high pressure cell disruptors to generate enough shear force to break thicker cell wall

ultracentrifugation/sedimentation

• used to enrich cellular membranes and sub cellular organelles— wont achieve purity

• centrifuge tubes filled with broken cell extract and loaded into ultracentrifuge (600,00 times force of gravity)

• subcellular organelles containing protein of interest can be centrifuged into a pellet at the bottom of the tub e at appropriate speed

• alternatively, subcellular organelles in the pellet can be discarded, and overlying liquid, or supernatant can be retained e.g. to purify a cytoplasmic protein

solubilisation of protein using detergents

• sample fully solubilised before column purification

• detergents essential for solubilising membranes and membrane proteins

• detergents can also lyse mammalian cells (no cell wall)

• able to solubilise membranes as they are amphipathic— hydrophobic tail and polar head group

• hydrophobic tail: insert into plasma membrane and coat hydrophobic domains of membrane proteins

• polar head: faces aqueous solution and facilitates solubilisation

• detergents added after ultracentrifugation

• protein of interest in soluble sample can be now purified by column chromatography

column chromatography

• refers to any method where mixture of substances can be fractionated (separated) on a column

• columns have a porous glass disc inside that allow liquid smaples to flow through but retains insoluble material bigger than pore size

• sample applied to top— fractions collected from bottom

• typically filled with a resin e.g. agarose (small diameter beads) or cross linked matrix such as polyacrylamide

• resin or matrix filling the column— stationary phase

• liquid perlocating through— mobile phase

affinity chromatography

• method that exploits ability of proteins to bind tigthly to specific molecules

• theses molecules, or ligands, can be chemically linked to the resin or matrix to make affinity resin— column filled with affinity resin and impure protein solution is applied to top of column

• as sample perlocates through column, protein of interest binds to immobilised molecule while other substances are washed through column with buffer

• protein of interest can be recovered in highly purified form by adding a substance that releases the protein from affinity resin

immobilised metal affinity chromatography(IMAC) (Ni-NTA)

• Ni-NTA agarose most used affinity matrix

• NTA- nitrilotriacetic acid, 3 negative charged carboxyl groups— can immobilised nickel cations Ni²⁺

• Ni²⁺ has high affinity for histidine residues— PCR cna engineer protein to have row of histidine residues (multiple CAC or CAT sequences to clone gene)

• recombinant proteins routinely engineered to have 6 histidine residues in a row— His- tag (example of affinity tag)

• other peptide sequences exist that can be used to purify protein by affinity chromatography— different affinity tags bind to different affinity resins

• any protein with his tag can be purified to a high level of purity using a Ni-NTA agarose resin

• once protein of interest captured on Ni-NTA column can be washed to remove contaminating proteins— then washed from column with histidine residues which compete with his-tag for binding to Ni²⁺

gel filtration chromatography

• separates proteins according to size

• resin consists of gel beads containing different pore sizes

• smallest proteins enter all pores

• intermediate can enter some

• large are excluded

• larger proteins therefore traverse column rapidly in comparison

ion exchange chromatography

• separates proteins according to charge

• resins consit of matrices such as cellulose or agarose that are modified with charged molecules such as diethylaminoethyl (DEAE) or carboxymethyl (CM) groups

• positive charge resins like DEAE-cellulose bind anions— known as anion exchange resins

• negative charge resins like CM-cellulose bind cations— known as cation exchange resins

• binding affintiy of a protein depends on pH of solution

• acidic— solution more H+ ions and proteins more positive charged

• basic— solution more OH- ions and proteins more negative charged

estimating protein purity by SDS-PAGE

• sodium dodecyl sulfate polyacrylamide gel elctrophoresis

• proteins unfolded by mixing with detergent called SDS

• SDS— anionic surfactant (negative charge; compund that lowers surface tension)— disrupts non covalent bond sin protein molecules

• mixture applied to top of the gel and electric current applied to gel and proteins migrate through gel according to size only

• small proteins migrate faster than large proteins

• proteins in gel visulaised by immersing gel in blue dye to stain proteins

• known molecular weight proteins (markers) loaded in first lane

• whole cell extract in lane 1 contains many proteins of which protein of interest represents only a small fraction