Protein Purification and Expression MCB 130 L Lecture

Protein Purification and Expression MCB 130 L, Lecture 2

Why purify a protein? • • To study its function To analyze its physical properties To determine its sequence For industrial or therapeutic applications

Protein expression in E. coli p. GEX plasmid: -Plac promoter-induce with IPTG - Gene encoding affinity tag-glutathione S tranferase (GST) - Spacer between genes - encodes protease cleavage site (thrombin) - Polylinker for inserting gene of interest Figure 1: Diagram of the p. GEX expression vector. .

Requires ligation that inserts gene of interest in frame with gene encoding GST In frame in p. GEX-2 T Bam. HI CTG GTT CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACG L V P R G S P G I H R D * Insert into Bam. HI site Bam. HI insert Bam. HI CTG GTT CCG CGT GGA TCC CTG GGT GAG CGT GAA GCG GGA TCC CCG GGA ATT CAT CGT GAC TGA. . . L V P R G S L G E R E A G S P G I H R D * Out of frame in p. GEX-3 X -Bam. HI ATC GAA GGT CGT GGG ATC CCC GGG AAT TCA TCG TGA CTG ACT GAC I E G R G I P G N S S * Insert into Bam. HI site Bam. HI insert Bam. HI ATC GAA GGT CGT GGG ATC CCT GGG TGA GCG TGA AGC GGG ATC CCC GGG AAT TCA TCG TGA. . . I E G R G I P G * A * S G I P G N S S * * indicates stop codon

Cell lysis: rupture cell wall / plasma membrane, --> release contents (organelles, proteins…) 1. Homogenization (blender) 2. Sonication 3. Osmotic shock

Protein purification: Centrifugation: apply centrifugal force - Centrifuges: produce 100 -500, 000 xg (force of gravity) - Components sediment based on mass density, shape - Separates organelles, macromolecules Three types: 1. Differential 2. Velocity 3. Equilibrium

Protein purification: Centrifugation Differential: Separation primarily by mass 1, 000 xg 10 min Low g force, short time: large organelles pellet 20, 000 xg 20 min Medium g force, longer time: smaller organelles pellet 80, 000 xg 60 min High g-force, long time: large macromolecules pellet 150, 000 xg 180 min

Protein purification: Velocity sedimentation --> Separation by mass, density, shape

Protein purification: Equilibrium sedimentation --> Separation by density

Protein purification – column chromatography -Protein mixture applied to column -Solvent (buffer) applied to top, flowed through column - Different proteins interact with matrix to different extents, flow at different rates -Proteins collected separately in different fractions

Affinity chromatography separation by biological binding interactions thrombin site protein of interest apply sample GST Ex: GST - Glutathione porous bead GST-tagged proteins bind to gluthatione on beads glutathione Non specifically (weakly) bound proteins washed off GST-tagged proteins eluted with glutathione (competitor) or thrombin (protease) wash elute

Gel filtration chromatography - separation by size Beads have different size pores As column flows: - large proteins excluded from pores and therefore flow rapidly - small proteins enter pores, see more volume and therefore flow slowly

Ion exchange chromatography – separation by charge Beads have charged group: + charge binds acidic amino acids - charge binds basic amino acid Different proteins bind with different affinity Eluted with increasing amount of salt (Na. Cl or KCl) Different proteins elute at different salt concentrations

Protein purification by chromatography

Separating and visualizing proteins – SDS-PAGE 1. Heat sample with SDS and b -mercaptoethanol SDS = Detergent (ionic) - Denatures proteins - Coats proteins - Each protein has similar mass/charge ratio b-mercaptoethanol - reduces disulfide bonds 2. Separate on polyacrylamide gel - polymer of acrylamine/polyacrylamide - TEMED, amonium persulfate catalyst for polymerization - Protein migrates through gel matrix in electric field.

SDS-PAGE D= 1/log. M D= Distance migrated M = Molecular mass Staining: Coomassie Blue (G 250) Silver staining

Two-dimensional gel electrophoresis Advantage: higher resolution Disadvantage: costly 1 st dimension - isoelectric focusing (IEF) - Separates by charge - Isoelectric point = p. H at which protein charge is neutral (+ = -) - Ampholytes in gel - create p. H gradient in electric field - Protein migrates to p. H= isoelectric point 2 d dimension - SDS PAGE (+) (-)