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Lecture

BIOD60H3 Lecture Notes - Cyanogen Bromide, Carboxypeptidase B, Glutamine


Department
Biological Sciences
Course Code
BIOD60H3
Professor
Rongmin Zhao

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Primary structure - AA sequence
Secondary: local spatial arrangement of a polypeptide backbone atoms through H-bond
interactions
Tertiary 3D structure of entire chain
Quaternary: spatial arrangement of diff polypeptide chains in a protein
1 polypeptide can be cleaved to form more than 1 protein
1 gene can be transcribed into different mRNA's
1 mRNA can be translated into multiple proteins
PROTEIN PURIFICATION
Solubility:
- salting out: increase ionic strength increase solubility (salting in), then decrease (salting out)
Ionic charge: ion exchange chromatography
electrophoresis
isoelectric focusing
Polarity: hydrophobic interaction chromatography
Size: -gel filtration chromatography
-SDS-PAGE
Binding specificity: affinity chromatography
Only functional protein
All purification methods must be based on the properties of the target proteins
If the properties of the protein is unknown, different strategies have to be tried.
HOW ARE PROTEINS ISOLATED AND PURIFIED FROM CELLS?
1000's of proteins in cells can b separated and purified on basis of size and electrical
charge
Proteins tend to be least soluble at their pI
increasing ionic strength at 1st increases solubility of proteins (salting-in, then
decreases it (salting-out)
SALTING IN - decrease [salt] cause protein to precipitate (solubility decreases)?
These 2 are opposite processes
SALTING OUT - increasing [salt] cause protein to precipitate
High concentration of salt added to protein solution to reduce solubility of protein
Target protein will precipitate at a specific salt concentration, at which the other proteins
will remain soluble
Common salt: (NH4)2SO4 -> high solubility and not significantly affected by temperature
High concentration of (NH4)2SO4 will take away H2O molecule originally around the
protein molecule

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-then electrostatic interactions bn protein molecules make proteins precipitate
Initially all are soluble in buffer, add salt to solution, unwanted protein precipitates (mb
remove by centrifugation)
-supernatant on top
*proteins are least soluble at pI, it's a pH thing
COLUMN CHROMATOGRAPHY (general term)
Types:
1. Ion exchange
2. Hydrophobic
3. Gel-filtration
4. Affinity chromatography
Use a column, fill with matrix, apply protein mixture, let protein bind, wash away bound
or unbound protein, elute
Protein has diff affinity, elute using this
Properties define the types above
-alternative way to column chromatography is the Batch operation:
-the binding matrix is placed in a centrifugation tube, the binding, washing and elution of protein
is conducted by sequentially addition and removal of solution manually
-extremely useful for small scale operation
ION EXCHANGE CHROMATOGRAPHY
Binding: low salt concentration
Washing: low salt -> wash away unbound proteins
Elution: high salt
If matrix negatively charged, then +'ively charged group will remain
Decrease pH, proton will bind likely -> +'ive charge
At the end, can add high salt to compete with protein to make precipitate
Cation exchange: CM (carboxymethyl): Matrix-CH2-COO-
Mono S ®: matrix-O-CH2-CHOH-CH2-O-CHOH-CH2-SO3-
Anion exchanger: DEAE (diethylaminoethyl): matrix-CH2-CH2-N-H(CH2CH2)2
Mono S ®: matrix-O-CH2-CHOH-CH2-O-CHOH-CH2-N-(CH3)3
Matrix: usually inactive polymers
*for all protein purification, need a buffer system
GEL FILTRATION CHROMATOGRAPHY - separate based on size
A gel filtration chromatography column: beads filter purely on size, smaller molecules
enter beads
Larger molecules are excluded from gel beads and emerge from column sooner than
smaller mole's, whose migration is retarded b/c they can enter the beads

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SIZE-EXCLUSION CHROMATOGRAPHY/MOLECULAR SIEVE/GEL FILTRATION
Holes inside beads. Proteins get inside and come out continuously, smaller get stuck longer
(delayed). It’s a matrix inside bead like puzzle
-large bypass the beads
Usage of gel-filtration:
Purification of protein from contaminant proteins
Determination of protein size (using molecular weight standard and assuming the
globular size of protein)
Desalting
Usually last step of protein purification
AFFINITY CHROMATOGRAPHY
1. Binding, washing and elution involved
2. Commonly used affinity tags: 6xHis: binding to Ni2+ and eluted with imidazole
FLAG ® tag: peptide: DYKDDDDK. Binding to antibody and eluted with FLAG peptide
protein A: ~20kDa protein, binding to IgG and eluted with low pH
Does not depend on exactly structure of protein, can even denature protein -> still work
Easily elute protein using imidazole solution, side chain of histidine is imidazole,
compete with protein
Process to make more pure after each step
1. Crude extract
2. Salt precipate
3. Ion exchange chromatography
4. Molecular sieve chromatography
5. Immunoaffinity chromatography -> after this step, 1000x more pure
PROTEIN ANALYSIS
1. Analysis of chemical and physical properties of target proteins: MW, AA
composition, amino acid sequence, pI, molecule size and shape etc.
2. Analysis usually associated with purification
-itself can be a purification process
1. A focus will b given on protein primary sequence determination
ANALYSIS OF PROTEIN BASED ON SIZES (SEPARATION OF PROTEINS)
SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)
Reducing agent:
DTT (Dithiothreitol)
Beta-mercaptoethanol
SDS (sodium dodecyl sulfate) -> a strong ionic detergent
After binding to SDS, all proteins have the same charge:mass ratio so that separation is
only based on MW by using networked polyacrylamide gel
1 SDS molecule binds to 2 amino acid residues
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