Protein Purification

1. Protein Purification

2. Compartmentalization provides an opportunity for a purification step e Protein profile for compartments of gram-negative prokaryotes

3. Cell Disruption • Chemical: alkali, organic solvents, detergents • Enzymatic: lysozyme, glucanases, chitinase • Physical: osmotic shock, freeze/thaw • Mechanical: sonication, homogenization, wet milling, French press

4. Chemical Disruption Detergents such as Trition X-100 or NP40 can permeabilize cells by solubilizing membranes. Detergents can be expensive, denature proteins, and must be removed after disruption

5. French Press Cells are placed in a stainless steel container. A tight fitting piston is inserted and high pressures are applied to force cells through a small hole.

6. Homogenization Cells are placed in a closed vessel (usually glass). A tight fitting plunger is inserted and rotated with a downward force. Cells are disrupted as they pass between the plunger and vessel wall. Also, shaking with glass beads works, BUT: Friction = Heat

7. Sonication A sonicator can be immersed directly into a cell suspension. The sonicator is vibrated and high frequency sound waves disrupt cells.

8. Differential centrifugation

9. Inclusion bodies provide a rapid purification step Inclusion bodies provide storage space for protein, carbohydrate and lipid material in prokaryotes However, proteins exist as aggregates in inclusion bodies thus special precautions must be taken during purification

10. 10% Glycerol 40% Glycerol Even proteins can be separated by their sedimentation properties Function of both size and shape

11. Proteins have unique properties resulting from their amino acid composition • Localization • Charge • Hydrophobicity • Size • Affinity for ligands Arbitrary protein

12. The charge on a protein is dependent upon pH • The content of amino acids with ionizable side chains determines the overall charge of a protein • Thus, a protein containing a majority of basic • residues (ie. R and K) will be positively charged • and will bind to a cation-exchange support Ion exchange column Supports (examples)

13. Separation based on surface charge

14. Cation exchange chromatography • Protein samples are applied to this column at low ionic strength, and positively charged proteins bind to the column support • Proteins are eluted using a gradient of increasing ionic strength, where counterions displace bound protein, changing pH will also elute protein • Choice of functional groups on distinct column supports allow a range of affinities Na+Cl- - - - Protein - - - - - - - - - - - - - - - - -

15. Conversely, at a pH two orders of magnitude above their pKa, acidic amino acids will be negatively charged, thus proteins with a majority of acidic amino acids (D and E) will be negatively charged at physiological pH • Negatively charged proteins can be separated using • anion exchange chromatography

16. Anion exchange chromatography • Protein samples are applied to this column at low ionic strength, and negatively charged proteins bind to the column support • Proteins are eluted using a gradient of increasing ionic strength, where counterions displace bound protein, changing pH will also elute protein • Choice of functional groups on distinct column supports allow a range of affinities • Bead size affects resolution in both anion and cation exchange Na+Cl- Protein + + + + + + + + + + + + + + + + + + + +

17. Hydrophobic Interaction Chromatography • Although most hydrophobic amino acids are buried in • the interior of proteins, many proteins have hydrophobic • surfaces or patches which can be used for separation • A protein’s hydrophobic character is typically enhanced • by addition of high salt concentrations • Proteins are eluted from HIC columns via a gradient of • high salt to low salt concentrations

18. Isoelectric Focusing For any protein, there is a characteristic pH at which the protein has no net charge (isoelectric point). At the isoelectric pH, the protein will not migrate in an electric field.

19. Isoelectric focusing

20. Protein Precipitation • Precipitation is caused by changes that disrupt the solvating properties of water • Changes in pH, ionic strength, temperature, and the addition of solvents can cause precipitation (loss of solubility) • Most proteins have a unique set of conditions that result in precipitation

21. Precipitation with Salt • In practice, most procedures use the salt ammonium sulfate (NH4)2SO4 to precipitate proteins • The amount of salt required is directly related to the number and distribution of charged and nonionic polar amino acids exposed on the surface of the protein

22. Salt effects on protein solubility At low ionic strengths, the charges on the surface of a protein attract counter ions, decreasing electrostatic free energy and increasing solubility. Addition of low concentrations of salt, then, increase solubility of proteins ("salting in"). At high salt concentrations, however, protein solubility decreases ("salting out"). This is due to electrostatic repulsion between the surface ions and the hydrophobic interior of the protein and to the avid interaction of salts with water. This disrupts the ordered water in the hydration layer. Salts vary in their ability to salt out proteins and generally follow the Hofmeister series: Cations: NH4+ > K+ > Na+ > Mg++ > Ca++ > guanidium Anions: SO4-- > HPO4-- > acetate > citrate > tartrate > Cl- > NO3-

23. Salting out provides a purification step

24. Proteins can be separated on the basis of size • Gradient centrifugation • Gel filtration

25. Gel Filtration provides a molecular sieve Figures from Scopes, Protein Purification on Reserve

26. Gel Filtration Chromatography Proteins that enter porous beads will migrate slower than proteins that are excluded from the pores. Separation is a function of relative size and shape

27. Size exclusion can be used to determine oligomeric state Vo = Void volume (the excluded volume surrounding the beads) Ve = Intermediate volume (partially excluded) Construct a standard curve using known proteins of known sizes

28. Gel Filtration Chromatography Log Mol Wt Ve - Vo

29. A protein’s substrate preference can be used in a very specific purification step Intrinsic If a protein binds ATP, put over a column support that has ATP crosslinked on it, thus selecting for ATP-binding proteins (can be done or a wide range of substrates such as sugars, Proteins, etc.) Added Specific protein domains can be fused to proteins of interest at the gene level to facilitate purification (ie. Fuse a maltose binding protein domain to any random protein, then it will bind specifically to a maltose containing column)

30. Metal chelation is a popular affinity purification method Various “expression vectors” create fusions to poly-Histidine tags, which allow the protein to bind to columns containing chelated metal supports (ie. Ni+2) Figures from Qiagen Product literature

31. Examining your purified protein

32. Use of SDS-PAGE vs. Native gel electrophoresis

33. Two dimensional gel electrophoresis

34. Assessing your purification procedure

35. Total vs. specific activity

36. We can “control” protein expression With the notable exception of proteins such as those that compose the ribosome, many proteins are found only in low abundance (particularly Proteins involved in regulatory processes) Thus, we need to find ways to grow cells that allow ample expression of proteins that would be interesting for biochemical characterization.

37. Find conditions for cell growth that enhance a protein’s expression For example, cytochrome c2 is utilized by R.sphaeroides for both respiratory and photosynthetic growth; a slight increase in levels of this protein is observed under photosynthetic growth conditions. However, Light-Harvesting complexes are only synthesized under photosynthetic growth conditions; obviously if you want to purify this protein you need to grow cells under photosynthetic conditions

38. Molecular Biology allows us to manipulate genes • Understanding the basic mechanisms of gene expression • has allowed investigators to exploit various systems for • protein expression • Prokaryotic expression systems • Eukaryotic expression systems • Yeast • Mammalian • Viral expression systems Baculovirus and Insects

39. Terminator Promoter lamB Transcriptional unit What do we need to produce a protein? lamB A gene Ribosome binding site lamB Translational unit

40. Terminator Promoter lamB Molecular Biology presents an opportunity for useful genetic constructs Antibiotic resistance gene Origin of Replication ori bla Plasmid Can fuse gene to other sequences conferring affinity

41. Choice of promoter allows control over transcription levels • Intrinsic promoters can be sufficient for overexpression • in multi-copy plasmids • Constitutive promoters with high activity (ie. promoters for • ribosomal genes) can be useful for producing non-toxic • proteins • Inducible promoters allow control of expression, one can “titrate” the promoter activity using exogenous agents

42. ori bla lamB An expression system utilizing lactose and T7 RNA polymerase is a popular choice in prokaryotes Genome Plasmid T7 polymerase dependent promoter T7 pol Lactose-inducible promoter

43. Inclusion bodies provide a rapid purification step Proteins exist as aggregates in inclusion bodies thus special precautions must be taken during purification. Typically, inclusion bodies can be readily isolated via cell fractionation. following isolation the proteins must be denatured and renatured to retrieve active protein.

44. Additional concerns regarding protein expression Modifications Inclusion bodies Codon usage

45. Cells exhibit nonrandom usage of codons This provides a mechanism for regulation; however, genes cloned for purposes of heterologous protein expression may contain “rare” codons that are not normally utilized by cells such as E. coli. Thus, this could limit protein production. Codon usage has been used for determination of highly expressed proteins.

46. Molecular Biology allows us to manipulate genes • Understanding the basic mechanisms of gene expression • has allowed investigators to exploit various systems for • protein expression • Prokaryotic expression systems • Eukaryotic expression systems • Yeast • Mammalian • Viral expression systems Baculovirus and Insects

47. Non-prokaryotic expression systems have emerged due to increasing simplicity and the need for proper modifications. Although you can express a eukaryotic cDNA in a prokaryote is the protein you purify, what the eukaryotic cell uses? Invitrogen : www.invitrogen.com Gateway vectors Novagen: www.novagen.com

48. Several hyperthermophilic archaeal species have also been shown to be dependent on tungsten (W), also Cd important in diatoms

49. Fe is most abundant, followed by Zn

50. Metals in Biology • Enzyme co-factors Redox active centers in many enzymes Fe: Electron transport, SOD, Cytochrome P450 Zn: SOD Mg, Mn: Photosynthesis Cu: Electron transport Ca: Cell signaling Ca, Na, etc: Substrates in ion pumps • Structural components of enzymes Fe: Hemoglobin, Cell structure Zn: Zn fingers in transcription factors Ca: Bone structure, Cell structure