Importance of Proteins:

In the animal kingdom, peptides and proteins regulate metabolism and provide structural support. The cells and the organs of our body are controlled by peptide hormones. Insufficient protein in the diet may prevent the body from producing adequate levels of peptide hormones and structural proteins to sustain normal bodily functions. Individual amino acids serve as neurotransmitters and modulators of various physiological processes, while proteins catalyze most chemical reactions in the body, regulate gene expression, regulate the immune system, form the major constituents of muscle, and are the main structural elements of cells. Deficiency of good quality protein in the diet may contribute to seemingly unrelated symptoms such as sexual dysfunction, blood pressure problems, fatigue, obesity, diabetes, frequent infections, digestive problems, and bone mass loss leading to osteoporosis. Severe restriction of dietary protein causes kwashiorkor which is a form of malnutrition characterized by loss of muscle mass, growth failure, and decreased immunity.
Allergies are generally caused by the effect of foreign proteins on our body. Proteins that are ingested are broken down into smaller peptides and amino acids by digestive enzymes called "proteases". Allergies to foods may be caused by the inability of the body to digest specific proteins. Cooking denatures (inactivates) dietary proteins and facilitates their digestion. Allergies or poisoning may also be caused by exposure to proteins that bypass the digestive system by inhalation, absorption through mucous tissues, or injection by bites or stings. Spider and snake venoms contain proteins that have a variety of neurotoxic, proteolytic, and hemolytic effects.
Many structures of the body are formed from protein. Hair and nails are made of keratins which are long protein chains containing a high percentage (15%-17%) of the amino acid cysteine. Keratins are also components of animal claws, horns, feathers, scales, and hooves. Collagen is the most common protein in the body and comprises approximately 20-30% of all body proteins. It is found in tendons, ligaments, and many tissues that serve structural or mechanical functions. Collagen consists of amino acid sequences that coil into a triple helical structure to form very strong fibers. Glycine and proline account for about 50% of the amino acids in collagen. Gelatin is produced by boiling collagen for a long time until it becomes water soluble and gummy. Tooth enamel and bones consist of a protein matrix (mostly collagen) with dispersed crystals of minerals such as apatite, which is a phosphate of calcium. By weight, bone tissue is 70% mineral, 8% water and 22% protein. Muscle tissue consists of approximately 65% actin and myosin, which are the contractile proteins that enable muscle movement. Casein is a nutritive phosphorus-containing protein present in milk. It makes up approximately 80% of the protein in milk and contains all the common amino acids.
Peptide hormones are produced by the endocrine glands (pituitary, thyroid, pineal, adrenal, pancreas) or by various organs such as the kidney, stomach, intestine, placenta, or liver. Peptide hormones can have complex, convoluted structures with hundreds of amino acids. The following graphics illustrate the chemical structure of human insulin and its three-dimensional shape. Insulin is made of two amino acid sequences. The A-Chain has 21-amino acids, and the B-Chain has 30-amino acids. The chains are linked together through the sulfur atoms of cysteine (Cys). Peptide hormones are generally different for every species, but they may have similarities. Human insulin is identical to pig insulin, except that the last amino acid of the B-Chain for the pig is alanine (Ala) instead of threonine (Thr).
What holds a protein into its tertiary structure?
The tertiary structure of a protein is held together by interactions between the the side chains - the "R" groups. There are several ways this can happen.
Ionic interactions
Some amino acids (such as aspartic acid and glutamic acid) contain an extra -COOH group. Some amino acids (such as lysine) contain an extra -NH2 group.
You can get a transfer of a hydrogen ion from the -COOH to the -NH2 group to form zwitterions just as in simple amino acids.
You could obviously get an ionic bond between the negative and the positive group if the chains folded in such a way that they were close to each other.
Figure 1
Hydrogen bonds
Notice that we are now talking about hydrogen bonds between side groups - not between groups actually in the backbone of the chain.
Lots of amino acids contain groups in the side chains which have a hydrogen atom attached to either an oxygen or a nitrogen atom. This is a classic situation where hydrogen bonding can occur.
For example, the amino acid serine contains an -OH group in the side chain. You could have a hydrogen bond set up between two serine residues in different parts of a folded chain.
You could easily imagine similar hydrogen bonding involving -OH groups, or -COOH groups, or -CONH2 groups, or -NH2 groups in various combinations - although you would have to be careful to remember that a -COOH group and an -NH2 group would form a zwitterion and produce stronger ionic bonding instead of hydrogen bonds.
van der Waals dispersion forces
Several amino acids have quite large hydrocarbon groups in their side chains. A few examples are shown below. Temporary fluctuating dipoles in one of these groups could induce opposite dipoles in another group on a nearby folded chain.
The dispersion forces set up would be enough to hold the folded structure together.
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Sulphur bridges
Sulphur bridges which form between two cysteine residues have already been discussed under primary structures. Wherever you choose to place them doesn't affect how they are form

Classification of proteins:

1. a) primary structure - unique linear sequence of amino acids in a polypeptide chain
* genetically determined
* determines all other structural levels
* directionality is from amino (N-terminus) to carboxyl (C-terminus)
b) secondary structure - develops from local interactions between amino acids
* amino acids in a peptide can interact with one another causing the peptide to fold and twist
* due to geometry of the bond angle between amino acids
* hydrogen bonding between amino and carboxyl groups in nearby regions
* repetitive structure based on common local functional group features
* structures are alpha helix and beta sheet, as well as random coils, that are hydrogen bond stabilized

Protein purification:

Protein purification: the basics
Arvind Varsani
http://Protein purification

Reasons for protein purification

•To identify the FUNCTION of a protein
•To identify the STRUCTURE of a protein
•To use the use the purified product –INTERMIDIATE- in downstream reactions / processing
•To produce a COMMERCIAL product

Selection of protein source

•Starting material can be from
–Animal tissue
–Plant material
–Biological fluids (e.g. blood, milk, sera)
RECOMBINANT expression
–Fermentation cultures (yeast, fungi, bacteria)
–Cell cultures (animal cells, plant cells, insect cells)

Important

•Protein in low concentration in natural sources
–Need to induce expression
Or express recombinantly in various expression systems

Yields for multi-step protein purifications

•Limit the number of steps
•Optimise each step
•Be careful of the yield if the proceduce requires several steps

Key steps in purification

•Release of target protein from starting material
•Removal of solids to leave the protein in the supernatant
•Concentration of the protein
•Removal of contaminants to achieve the desired purity
•Stabilization of the target protein

Three phase purification strategy

The final purification process should ideally consist of sample preparation, including extraction and clarification when required, followed by 3 major purifications step. The number of steps will depend on the purification strategy, purity requirements and intended use of the protein

Protein analysis

•Tracking protein of interest and determining the yield during purification
–Intended use of protein / source of starting material
•Physical studies e.g. x-ray, NMR, EM
•End product – pharmaceuticals

Analysis of protein purity

•Total protein
•Specific quantification
–Activity assays
–Binding assays
•Detection of impurities
–HPLC
–Gel electrophoresis
•Protein mass spectrometry

Methods for quantification of proteins in solution

Assay method Useful range Comments

NanoOrange assay 100ng/ml to 10ug/ml ·Samples can be read up to six
hours later without any loss in
the sensitivity

·Low protein to protein signal
variability

·Detection not influenced by
reducing agents or nucleic acid

BCA method
(Cu reduction) 0.5ug/ml to 1.5mg.ml ·Samples must be read within
10mins

·Not compatible with reducing
agents

BSA assay (Bicinchoninic acid)

•The first step is a Biuret reaction which reduces Cu+2 to Cu+1
•In the second step BCA forms a complex with Cu+1 which it purple colored and is detectable at 562 nm

Bradford assay (coomassie dye binding)

•Absorbance shift in Coomassie Brilliant Blue G-250 (CBBG) when bound to arginine and aromatic residues
•The anionic (bound form) has absorbance maximum at 595 nm whereas the cationic form (unbound form) has and absorbance maximum at 470 nm

Lowry assay (Cu reduction)

The first step is a Biuret reaction which reduces Cu+2 to Cu+1
The second reaction uses Cu+1 to reduce the Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate). This is detectable in the range of 500 to 750 nm

Absorbance at 280nm

·Monitors the absorbance of aromatic amino acids, tyrosine and tryptophan or if the wavelength is lowered, the absorbance of the peptide bond. Higher order structure in the proteins will influence the absorption

Enzyme activity assays

•Continuous (kinetic assays)
–No separation step

Cell disruption / breakage for protein release

•Extraction techniques are selected based on the source of protein (e.g. bacteria, plant, mammalian, intracellular or extra cellular)
•Use procedures that are as gentle as possible. Cell disruption leads to the release of proteolytic enzymes and general acidification
•Selection of an extraction technique often depends on the equipment availability and the scale of operation
•Extractions should be performed quickly, at sub-ambient temperatures in a suitable buffer to maintain pH and ionic strength
•Samples should be clear and free of particles before beginning chromatography

Cell disruption: source variations

•Tissues – variable
•Mammalian cells – easy
•Plant cells – some problems
•Microbial cells – vary, common
•Yeast and fungal cells – more difficult

Cell disruption: methods

1 Chemical / enzymatic
•Cell lysis (osmotic shock and freeze thaw)
•Enzymatic digestion
Blood cells
Mammalian cells
•Fractional precipitation
•Extra cellular proteins

2 Mechanical
•Hand and blade homogenizers
tissue
•Sonicator / disruptors
•Grinding with abrasive
plant/yeast
•Bad beaters / mill
•French press
•micro fluidizer

Lytic enzymes and detergents

•Lysozyme: disrupts bacterial cell walls (hydrolysis of peptidoglycans) leading to cell rupture
–Effective with gram positive bacteria, gram negative generally require pre-treatment with a chelating agent such as EDTA
•Detergents: anionic and non-ionic detergents have been used to permeabilize gram negative cells. Detergents are required for the release of integral membrane proteins.

Simple shear methods

Glass homogenizer (dounce, ten-broeck)

sonicator

•sonicator

Sample clarification

•Centrifugal sedimentation
•Coagulation and flocculation
•Filtration

Sedimentation

•Operates on the basis of density difference between components in a mixture (e.g. solids and liquid)
•Rate of sedimentation is dependent on:
–Magnitude of different in component densities
–Particle size, shape and concentration
–Magnitude of centrifugal force
–Flocculating of coagulating cells or organelles

Sedimentation

•Operates on the basis of density difference between components in a mixture (e.g. solids and liquid)
•Rate of sedimentation is dependent on:
–Magnitude of different in component densities
–Particle size, shape and concentration
–Magnitude of centrifugal force
–Flocculating of coagulating cells or organelles

Coagulation and flocculation

•Coagulation
–Increase in particle size from the joining of like particles
–Promote by reducing charge repulsion
•Addition of multivalent ions (e.g. Al3+)
•Adjust pH to isoelectric point
•Flocculation
–increase in particle size by addition of agents acting as bridges between particles
–Generally polyelectrolytes that neutralise surface charges on particles and then link particles to form aggregates

Concentration of extracts

•Freeze drying
•Dialysis
•PEG precipitation
•Concentration / fractionation by salting out
–Ammonium sulphate precipitation
•Ultraflitration
–Desalting
–Size fractionation

Protein purification

•Affinity chromatography
–Binding to immobilised ligands e.g antibodies, co-factors
•Ion exchange chromatography
–Anion (-) and cation (+) exchanger
•Hydrophobic interaction chromatography
–Colum coated with hydrophobic fatty acid chains
•Size exclusion chromatography
–Gel filtration
•Electrophoresis
–SDS