Purification of (recombinant) proteins. Pekka Lappalainen, Institute of Biotechnology, University of Helsinki

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1 Purification of (recombinant) proteins Pekka Lappalainen, Institute of Biotechnology, University of Helsinki

2 Physical properties of proteins that can be applied for purification -size -charge (isoelectric point) -hydrophobicity -heat stability -solubility -specific sequence features (proline-rich sequences, affinity to metal ions etc.)

3 Preparation of cell extracts from bacteria homogenization (sonication, french press, bead-beating) add protease inhibitors centrifugation soluble proteins membrane proteins and inclusion bodies -due to increased stability, it is typically important to carry out all these steps at low temperature

4 Important matters to consider before starting the purification Sample and target protein properties Temperature stability ph stability Detergent requirement Co-factors for stability or activity Sensitivity to metal ions Redox sensitivity Strategy Use low temp. Selection of buffers Consider choice of detergent Select additives, salt etc. Add EDTA or EGTA to buffers Add reducing agents

5 Methods for separating proteins

6 Size-exclusion chromatography (gel-filtration) -it is important to know the molecular weight of the target protein when selecting the gelfiltration media (e.g. Superdex 75 for proteins of 5-70 kda, and Superdex 200 for proteins of kda) -because some gel-filtration media are also weak ion-exchangers, it is typically useful to add some salt to buffers

7 Ion-exchange chromatography -is based on interactions of proteins with charged ion-exchange matrix -two forms: 1. Anion-exchange (negatively charged proteins interacting with positively charged matrix). 2. Cation-exchange (positively charged proteins interacting with negatively charged matrix). -before the experiment it is important to calculate the isoelectric pint of the protein of interest -the ph of the buffer in anion-exchange has to be >1 units higher than the pi of the protein of interes -in cation exchange the ph of the buffer has to be >1units lower than the pi of the protein of interest

8 Ion-exchange chromatography -elution in ion-exchange chromatography is typically carried out by using a salt gradient -e.g. in anion-exchange column the proteins with weaker negative charge (i.e. higher isoelectric point) elute first and the protein with srtonger negative charge elute later in higher salt concentration salt gradient (0-1 M NaCl) fraction no. salt A 280 (protein)

9 Hydrophobic interaction chromatography (HIC) -proteins are bound to HIC columns by moderately high concentrations of salt (which favors bind of hydrophobic proteins to matrix) -elution is achieved by a linear or stepwise decrease in the concentration of salt

10 Reverse phase chromatography -like in HIC, proteins bind to matrix through hydrophobic interactions -proteins are then eluted by by linear decrease in the polarity of the buffer (increase in the concentration of hydrophobic organic modifier) -similarly to HIC, proteins with less hydrophobic character elute first, and strongly hydrophobic proteins bind stronger to the matrix

11 Affinity chromatography -separates proteins on the basis of a reversible interaction between a protein and a specific ligand coupled to a matrix -to elute the targed protein the interaction must be reversed by specifically using acompetetive ligand or non-specifically by changing the ph ionic strength or polarity examples: -enzyme substrate analogue -antibody antigen -lectin polysaccharide -hormone/ receptor growth factor -protein target sequence domain (e.g. poly-pro)

12 Widely used affinity tags in protein purification His-tag -contains a poly-histidine stretch -binds strongly to metal-ions (Cu2+, Zn2+, Ni2+ etc.) that are immobilized to the matrix (e.g. chelating sepharose) -His-tagged proteins can be eluated form the matrix by imidazole Glutathione-S-transferase (GST) fusion proteins -30 kda protein that binds strongly to glutathione -can be eluated from glutathione-agarose by addition of blutathione to the elution buffer -alternatively, the target protein that is fused to GST can be first removed from GST by specific protease clevage and then eluted from the column that contains GST bound to glutathione-agarose beads

13 Concentrating protein samples -widely used methods for protein concentration are 1. Salt (ammonium sulfate) precipitation, and 2. Use of concentration devices with semipermeable membranes/ filter systems. -the benefit of using concentration devices is that the buffer conditions do not change. -AmSO 4 precipitation results in an increase in the salt concentration during the concentration procedure. -the most reliable method for detecting the concentration of the purified protein is to measure A 280 using a scanning spectrophotometer -the extinction coefficient of the protein can be calculated by the following equation: ε 280 (M -1 cm -1 ) = #Tyr x #Trp x #Cys x 120

14 Refolding insoluble proteins from inclusion bodies -many proteins are not soluble after expression in E.coli. Instead these proteins are misfolded and accumulate to large insoluble particles, inclusion bodies -inclusion bodies can be purified from the pellet-fraction of E.coli lysate by detergent treatment (membrane proteins are soluble in the presence of detergent, whereas inclusion bodies remain insoluble. -the purified inclusion bodies can be solubilized to high concentrations of Urea (8 M) or guanidium hydrochloride (6 M). -these proteins can then in many cases be refolded by a stepwise decrease in the Urea concentration -in refolding procedure it is important to add possible cofactors of the protein (e.g. nucleotide, metal-ion) and try different salt and ph conditions.

15 Examples of protein purification I: purification of DNaseI by conventional chromatography methods E.coli lysate Q-sepharose FF (anion-exchange) Hydroxyapathite column Q-sepharose HP (anion-exchange) Superdex-75 (gel-filtration)

16 Examples of protein purification II: purification of twinfilin-gst fusion protein E.coli lysate (from GST-twinfilin expressing strain) GST-twinfilin was bound to glutathione-agarose beads cell lysate material bound to glut. beads pooled fractions from gel-filtration sup. after thrombin digestion Separation of twinfilin from GST by thrombin digestion GST-twf Superdex-75 gel-filtration twf Pure twinfilin