1. Protein Purification and Characterization Techniques
Chapter 5

2. How can proteins be extracted from cells?
Many steps/techniques are needed to extract and separate protein of interest from many contaminants
Separation techniques – size, charge and polarity
Before purification begins, protein must be released from cell by homogenization
Many different proteins exists within one cell. To get our protein of interest, we only need that particular protein in our sample and not anything else.

3. How do we get the proteins out of the cells?
Different purification procedures involved in purification of enzyme Xanthine dehydrogenase. This table actually shows the recovery and purity of protein obtained with each procedure. The specific activity column compares the purity of protein at each step and percent recovery shows how much protein has been retained at each step. The retention of protein drops at each step but the purity increases.

4. What are different ways of homogenization of cells?
Grinding tissue in a blender with a suitable buffer
Releases soluble proteins and various subcellular organelles
Potter-Elvejhem homogenizer – A thick walled test tube with a tight fitting plunger
Breaks open cells – organelles intact
Sonication – Sound waves to break open cells
Continuous freezing and thawing – Ruptures cells
Homogenization – breaking a cell open or opening a cell by breaking the cell membrane. If a protein is attached to a membrane then detergent should be added to detach proteins

5. Differential Centrifugation
Sample is spun, after lysis, to separate unbroken cells, nuclei, other organelles and particles not soluble in buffer used
Different speeds of spin allow for particle separation
After homogenization the sample is subjected to different speeds – 600 x g separates out nuclei and unbroken cells. If the proteins are not found in the pellet then pellet is discarded. Then it is spun at 100,000 g for 60 minutes the pellet will have mitochondria, lysosomes and microbodies. If proteins are not found in the pellet then it is discarded. Proceed with the supernatant to the next speed level and have the ribosomes, ER, golgi and plasma membrane fragments in this pellet. Work with this supernatant that has all the organelles removed to purify your protein.

6. What is Salting out? Ammonium sulfate (NH4SO4) - used to “salt out”
Takes away water by interacting with proteins
- makes protein less soluble because hydrophobic interactions increases among proteins
Addition of salt – increases saturation
Different set of proteins precipitate
Centrifuge and save the set of proteins
Based on solubility, proteins are subjected to crude purification. Leads to more ion-dipole bonds, decreases solubility of proteins in water and increases hydrophobic interactions among proteins. This salting out is preparing the proteins for effective procedures of purification

7. What is Column Chromatography?
Basis of Chromatography
Different compounds distribute themselves to a varying extent between different phases
2 phases:
• Stationary: Samples interacts with this phase
• Mobile: Flows over the stationary phase and carries along with it the sample to be separated
Chromatography – chroma meaning ‘color’ and graphein meaning ‘ to write’. To separate plant pigments with visible color.
The material that makes the stationary phase is packed in a column. The sample is a small volume of concentrated solution that is applied to the top of column – it is the mobile phase called eluent that is passed through column. The eluent eventually comes out of the column – its volume is increased in the column

8. Column Chromatography

9. What is Size-exclusion/Gel-filtration chromatography?
Separates molecules based on size (molecular weight).
Stationary phase composed of cross-linked gel particles (Beads).
Two polymers – Carbohydrate polymer such as dextran (Sephadex) or agarose (Sepharose)
Polyacrylamide (Bio-Gel)
Gel particles are in bead form with one or two kinds of polymers

10. What is Size-exclusion/Gel-filtration chromatography?
Extent of cross-linking can be controlled to determine pore size
Smaller molecules enter the pores and are delayed in elution time.
Larger molecules do not enter and elute from column before smaller ones

11. Advantages of Size-exclusion/Gel-filtration chromatography
Separate molecules based on size
Estimate molecular weight by comparing sample with a set of standards

12. What is Affinity Chromatography?
Uses specific binding properties of molecules/proteins
Stationary phase has a polymer that is covalently linked to a compound called a ligand
Ligands bind to desired protein or vice versa
Proteins that do not bind to ligand elute out
By addition of more buffer, proteins that do not bind to ligand elute out.

13. What is Affinity Chromatography?
Bound protein can be eluted from column by adding high concentrations of ligand in soluble/mobile form.
Competition – proteins bound to ligand in column will bind to mobile ligands
Recovered from column – Produces pure proteins

14. What is Ion-exchange chromatography?
Interaction based on overall charge (less specific than affinity)
Cation exchanger – negatively charged resin – bound to Na+ or K+ ions
Anion exchanger – positively charged resin – bound to Cl- ions
Figure 5.7 A and B
Ion exchange resin has a ligand with positive charge or negative charge

15. What is Ion-exchange chromatography?
Column is equilibrated with buffer of suitable pH and ionic strength
Exchange resin is bound to counterions
Proteins – net charge opposite to that of exchanger stick to column
No net charge or same charge elute - first
Proteins that have net charge opposite to that of exchanger exchange places and stick to column. Those that have no net charge or same charge elute out first. After all nonbinding proteins are eluted the eluent is changed to buffer with higher ph that removes the charge on bound proteins or to one with higher salt concentration. The latter outcompetes bound proteins for limited binding space on column.

16. What is Electrophoresis?
Electrophoresis- Charged particles migrate in electric field toward opposite charge

17. Differences between agarose and polyacrylamide gels
Agarose - matrix for nucleic acids
Charge, size, shape
Agarose matrix has more resistance towards larger molecules than smaller
Small DNA move faster than large DNA
Polyacrylamide - proteins
Charge, size, shape
Treated with detergent (SDS) sodium dodecyl sulfate – gains –ve charge
Random coil – shape
Polyacrylamide has more resistance towards larger molecules than smaller
Small proteins move faster than large proteins
All proteins gain same negative charge and shape of random coil – SDS breaks all tertiary and quaternary structures. Once denatured all proteins attain the form of random coil. Based on molecular weight both DNA and proteins can be identified
Molecular weight of DNA and protein can be determined

18. What is Isolectric focusing?
Gel is prepared with pH gradient that parallels electric-field
Charge on the protein changes as it migrates across pH
When it gets to pI, has no charge and stops
Separated and identified on differing isoelectric pts. (pI)
Different proteins have different titratable groups, they also have different isoelectric points. It is the pH at which a protein has no net charge. The number of positive is balancing the no of negative charges.
As proteins migrate through the gel under the influence of electric field, they encounter regions of different pH so with migration the net charge also changes. Eventually it comes to pI where it has no net charge and stops – no migration. Each protein will remain at its pH without further migration

19. What is two-dimensional gel electrophoresis?
Isoelectric focussing in one dimension and SDS-PAGE running at 90 degree angle to the first

20. How is 1˚ structure determined?
Determine which amino acids are present and in what proportions (amino acid analyzer)
Specific reagents - determine the N- and C- termini of the sequence
Cleave - determine the sequence of smaller peptide fragments (most proteins > 100 a.a)
Some type of cleavage into smaller units necessary
Aminoacid analyzer separates proteins either by ion-exchange chromatography or by high pressure liquid chromatography (allows high resolution separation of many amino acids in short time)
Second step helps to know the number of polypeptides – whether one or two
Step 3 cleave protiens at specific sites to determine their sequence
Step 4 cleave at sites not cleaved in step 3 and determine their sequence. Combine information of 3 and 4 to get complete sequence of proteins

21. Primary Structure Determination

22. What is Edman degradation?
Cleaving of each amino acid in sequence followed by their subsequent identification and removal
Becomes difficult with increase in number of amino acids
Amino acid sequencing - Cleave long chains into smaller fragments
Data can become confusing

23. Protein Cleavage Enzymes or Chemical reagents
Trypsin- C-terminal of (+) charged side chains/R-groups
Chymotrypsin- C-terminal of aromatics
Figure 5.17

24. Cleavage by Chemical reagent
Cyanogen bromide
C-terminal of INTERNAL methionines
Sulfur of methionine reacts with carbon of cyanogen bromide to produce a homoserine lactone at C-terminal end of fragment
Sulfur of methionine reacts with carbon of cyanogen bromide to produce a homoserine lactone at C-terminal end of fragment

25. Use different cleavage reagents to help in 1˚ determination

26. Determination of primary structure of protein
After cleavage, mixture of peptide fragments are produced.
Sequences can overlap – peptides can be arranged in proper order after all sequences have been determined
Can be separated by HPLC or other chromatographic techniques

27. Peptide sequencing by Edman Degradation
Can be accomplished by Edman Degradation
Sequencer - relatively short sequences (30-40 amino acids in picomoles) can be determined quickly
N-/C-terminal residues - not done by enzymatic/chemical cleavage
Edman degradation has become so efficient that today you no longer require to do or no longer enzymatic/chemical cleavage

28. Peptide sequencing by Edman Degradation
Edman’s reagent – Phenyl isothicocyanate
Reacts with peptide’s N-terminal residue – cleaved
Leaves rest of peptide intact – phenylthiohydantoin derivative of amino acid
Same treatment to 2, 3,4….40
Automated Sequencer – Process repeated

29. DNA sequencing Preferred to amino acid sequencing
Easy to obtain sequence of DNA to that of protein
DNA sequencing does not determine positions of disulfide bonds or detect amino acids like hydroxyproline
Using genetic code of DNA one can determine the sequence of amino acids

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