Native and recombinant sources can be used to produce enzymes. Preparation of crude extracts involves homogenizing or lysing the source cells or tissues and preventing proteolysis. Various techniques can then be used to separate and purify proteins based on exploiting differences in their solubility, charge, size, or ability to bind specifically. Key steps include precipitation with ammonium sulfate or organic solvents, ion exchange chromatography, size exclusion chromatography, and affinity chromatography. Fractional precipitation provides a simple initial concentration and enrichment of proteins.

1. Production of Enzymes

2. Native sources
Bacteria
Tissues
Animal
Plant
Recombinant protein production
Prokaryotic expression systems
E.coli
Eukaryotic expression systems
Yeast
Insect cells
Mammalian cells
Cell free systems
Sources of Enzymes

3. Preparation of Crude Extracts
Homogenisation: Rupture tissue and cell structures
Subcellular Fractionation:
1.Animal tissue
Soft tissue (liver, kidney) and hard tissue (skeletal and cardiac muscle)
Liquid sheer Blenders and mincers
Soluble and insoluble proteins
Membrane-bound and extracellular matrix proteins
(Detergent for protecting (Breakage of covalent crosslinking by
hydrophobic interactions) chemical or proteolytic methods)
2. Plant tissue
Breaking the cell wall required to release contents
Often done by grinding in mortar and pestle with acid-washed sand
Inactivating agents such as phenolics and proteases
Most organs in plants have a complex mixture of proteins unlike the many
specialised animal organs, which are enriched in proteins of specific function.

4. Preparation of Crude Extracts
Contnd.
3. Bacteria expressing recombinant protein
Cell wall lysis with enzymatic methods (lysozyme), or mechanical means
Cell membrane lysis with detergents, osmotic shock, mechanical means, etc.
Overexpression can lead to inclusion bodies due to insufficient time and
inappropriate environment for folding
Strong hydrophobicity and charge propensity can promote inclusion body formation
Solubilisation of inclusion bodies often require detergents.
Not advisable for expressing active proteins
Lowering growth temp., lowering inducer concentration, coexpression of chaperone
proteins, inducing endogenous chaperones by heat-shock, etc. can reduce formation
of inclusion bodies.
Inclusion bodies give high yields, allows expression of proteins that are toxic to bacteria
Fusion construct with a highly soluble protein (eg. GST) can increase solubility

5. Preparation of Crude Extracts
Contnd.
Preventing Proteolysis
Proteolytic enzymes that are well-regulated in intact cells will go uncontrolled when the
cell is ruptured leading to the degradation of the protein of interest.
In heterologous expression systems, recombinantly expressed protein may undergo
degradation in vivo.
It is important to determine that loss of protein of interest is due to proteolysis and not
due to other causes such as thermal denaturation, oxidative damage, adsorption
onto surfaces, persistent binding to column matrix, binding of inhibitors, removal of
activators and cofactors, etc.
Denatured and flexible proteins are susceptible to proteolysis.

6. Preparation of Crude Extracts
Contnd.
Strategies for prevention of proteolysis:
Use a cell or tissue with less of endogenous proteases (eg. muscle rather than liver or
kidney, mutant becteria defective in endogenous protease)
Proteases co-exist with natural inhibitors, which when separated in a purification step
activates the protease.
In the crude state, several proteins compete for the protease. Upon purification
protease action on protein of interest may increase. Addition of a carrier protein
such as BSA-Bovine serum albumin (BSA) is a globular protein can be considered
to engage the protease.
Denaturation in SDS sample buffer can make protein of interest susceptible to SDS-
resistant protease. TCA precipitation to inactivate the protease followed by
dissolving in SDS-sample buffer is advised.
Addition of protease inhibitors
Mechanism-based inhibitors for irreversible inhibition and non-covalent inhibitors
(eg. PMSF, leupeptin, pepstatin, etc.) for reversible inhibition of proteases.
Supplementation of the reversible inhibitor at each purification step.

7. Protein Separation Techniques

8. Why to separate proteins?
1.To detect different proteins
Analytical
2. To purify protein(s) of interest
Preparative

9. 1. How much is needed?
2. Should biological activity be retained?
3. What degree of purity is needed?
4. What source should be used?
5. Is previous literature available?
Designing Method for Purification
Considerations

10. 1. How much is needed?
Physico-chemical analysis such as CD-ORD spectroscopy,
microcalorimetry, crystallography, etc. - Hundreds of mg.s of
purified protein
Kinetic analysis of enzyme activity, Raising polyclonal
antibody, etc - 1 mg or below
Sequencing – Less than microgram
In general, the fewer the steps, higher the final yield
Designing Method for Purification
Contn.d

11. 2. Should biological activity be retained?
Neutral aqueous buffers at low temp.s retain activity.
Gel electrophoresis for eg. is not suitable to purify active
protein.
Designing Method for Purification
Contn.d

12. 3. What degree of purity is needed?
Absolute homogeneity is difficult to achieve.
The level of purity depends on the final application.
For eg. Immunisation requires high purity whereas an
enzyme activity analysis may be conducted with lower level
of purity
Designing Method for Purification
Contn.d

13. 4. What source should be used?
Abundance of the protein in the source
Plants are difficult sources. Microorganisms are better
while animal tissues are most preferred.
Designing Method for Purification
Contn.d

14. 5. Is previous literature available?
If yes, start by using the same protocol. Make minor
modifications as necessay.
Spending lot of time and efforts to improve published
methods is not generally fruitful.
Designing Method for Purification
Contn.d

15. STRATEGY
Exploiting differences
Differences in properties between proteins is the basis of
separation techniques
1. Solubility
Surface amino acids determine the solubility.
Addition of salts or organic solvents can selectively
precipitate a set of proteins with similar solubility.
Low degree of purity, High yield, Concentration of proteins,
Large scale operation

16. Exploiting differences-Contn.d
2. Charge
Charge of a protein depends on the pH. Different proteins
with different charges at the same pH can be separated by
ion exchange chromatography
Proteins with different isoelectric points (pH at which net
charge is zero) can be sperated by chromatofocusing or
isoelectric focusing

17. 3. Size
Size-exclusion chromatography
Ultrafiltration
SDS-PAGE
Exploiting differences-Contn.d

18. 4. Specific binding
Affinity chromatography
5. Special properties
Heat stability
pH stability
Engineered binding properties
Exploiting differences-Contn.d

19. Documenting Purification
Protein Purification Table
Fraction Volume
(ml)
Protein
conc.
(mg/ml)
Total
Protein
(mg)
Activity
(U/ml)
Total
Activity
(U)
Specific
Activity
(U/mg)
Purification
factor
Overall
Yield
(%)-
TA’/TA0
Homogenate 8500 40 340,000 1.8 15,300 0.045 1 100
45-70%
Ammonium
sulfate pellet
530 194 103,000 23.3 12,350 0.12 2.7 81
CM-Cellulose 420 19.5 8190 25 10,500 1.28 28.4 69
Ligand affinity
chromatograp
hy
48 2.2 105.6 198 9,500 88.4 1,964 62
DEAE-
Sepharose
12 2.3 27.6 633 7,600 275 6,110 50

20. Fractional Precipitation
Manipulating the solubilities of proteins
Solubility depends on solvent composition and pH. Different proteins differ in their
solubilities.
Addition of salt increases ionic strength, removes water of solvation from protein
thereby exposing hydrophobic patches that interact each other leading to
aggregation and precipitation of proteins.
Different proteins precipitate at different salt concentration ranges.
This cannot achieve major purification of a protein, but provides a simple
procedure to concentrate and enrich a protein in the fraction.
Salts such as NaCl, Na2SO4, KCl, CaCl2 and MgSO4 can be used but the most
commonly used one is (NH4)2SO4 because of its high solubility, low heat of
solution, low density and general harmless nature towards proteins.
Organic solvents such as acetone, ethanol, etc. when added to protein solutions
cause precipitation by changing dielectric constant of the solvent and by removal
of water of solvation.

21. Ammonium Sulfate Saturation Chart