Proteins Manipulation

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Proteins - Manipulation

• A. Isolation and Purification
• obtaining a pure protein
• methods
• basis of methods
• B. Characterization
• determining the properties of a protein
• C. Synthesis
• making proteins in the lab

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Protein characteristics exploited in separations

• size
• charge/polarity
• location
• soluble or membrane bound, etc
• specific characteristics of protein
• activity or function

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Activity and Specific Activity I

• Purification protocol
• a set of steps by which a protein is purified
• Monitoring a purification
• a property specific for the protein of interest
• activity (for instance, the ability to
  catalyze the transformation of a certain
  substrate)
• a specific unique characteristic (special
  absorbance)
• amount of total protein
• Specific Activity activity/total protein

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Activity and Specific Activity II

• Assays for total protein
• Biuret, Lowry, Folin
• low sensitivity
• Bradford
• moderate sensitivity
• Silver
• high sensitivity
• as a protein is purified the ratio of the
  activity to total protein is going to increase

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Some Purification Methods

• Salt precipitation
• Centrifugation
• Dialysis
• Column Chromatography
• Electrophoresis
• Crystallization

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Purification Protocol

• A set of steps that leads to the purification of
  a protein
• Specific activity increases
• Total activity decreases
• Loss of protein of interest at each step
• Try to minimize losses by picking steps and order
  of steps carefully

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Purification Protocol - Results
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Salt precipitation - method

• separation on basis of charge
• often times this is an early step
• (NH4)2SO4 most frequently used
• ammonium sulfate cuts
• cuts of saturation by salt
• retain cut with highest specific activity
• amount of specific protein/amount of protein

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Salt precipitation - theory

• "salting out"
• salts compete with proteins for water molecules
  for solvation at higher salt concentrations
  proteins begin to associate with each other
• the hydration shells of some proteins are smaller
  (less thick) and more easily removed than those
  of others
• protein - protein interactions become
  energetically more favorable than protein -
  solvent interactions
• nature of the ammonium ion
• strong H-bonder therefore very competitive for
  water
• nature of ammonium sulfate
• three ions in solution needs lots of water to
  solvate

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Centrifugation

• Separate on the basis of size
• Useful as first step in removing debris
• Often early step to get the fraction that you
  want
• from soluble to some suspended organelle or
  membrane component
• differential centrifugation with increasing
  speed smaller materials pellet out
• used to determine MW, size and shape
• being displaced by other methods

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Differential Centrifugation
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Dialysis

• Separate on the basis of size
• not the same as osmosis
• Old way with membrane bag and changing solution
• Eventually smaller proteins are removed

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Pressure Dialysis

• New way different membranes with pressure
  dialysis cells that have different exclusion
  limits
• Can push through proteins that can get through
  pores in membrane retain larger proteins
• Using two different membranes can get proteins
  between two size limits
• Greater than 20kd but less than 50kd

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Column Chromatography

• general principle using differential affinity
  over a distance traveled to effect a separation
• types
• gel filtration (gel exclusion, molecular sieve)
• ion exchange
• hydrophobic
• affinity
• HPLC (high performance/pressure liquid
  chromatography)

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Gel Filtration I

• Basis of separation
• size and shape
• for globular proteins rough separation on basis
  of molecular weight (MW)
• Exclusion limit
• size limit of proteins that will not enter gel
  matrix and therefore all come out together first
• Different exclusion limits for different types of
  gel
• G25, G75, G200 exclusion limits of 25k, 75k and
  200k respectively

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Gel Filtration II

• Load protein mixture
• Allow to penetrate gel
• Add buffer
• Collect fractions of separated and partially
  separated proteins

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Gel Filtration II

• Gel bead pore size
• gel pore size varies if a protein has a
  molecular cross section that is smaller than the
  pore size, it can enter the gel bead
• entering beads holds up (retards) proteins
• of the proteins that can enter the beads, the
  smallest will be held back the most and will
  therefore be the last off the column
• log of MW (y-axis) vs elution volume (x-axis) is
  often a straight line

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Gel Filtration - determination of approximate
molecular weight
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Ion Exchange

• Basis of separation
• overall net charge on polypeptide
• Effecting a purification
• Changing the net charge on the protein
• change in the pH on the column
• Increasing competition for binding sites on the
  gel
• adding competitive salt (one with the same charge
  as the protein you want to elute)

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Ion Exchange Materials

• Anion exchangers
• gel material is charged - charged species will
  bind
• species can be knocked off of gel by making
  species positive or by adding competing negative
  salt ion
• DEAE-cellulose
• Cation exchangers
• gel material is - charged charged species will
  bind
• species can be knocked off of gel by making
  species negative or by adding competing positive
  salt ion
• CM-cellulose

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Ion Exchange Strategies

• Binding target protein
• protein of interest binds to gel at starting pH
• proteins with same charge as gel are repelled
  off of gel
• target protein is eluted off of gel by change
  in pH or salt
• change in pH or salt can be abrupt (step) or
  gradual (gradient)
• some proteins may still bind to gel after change
• result target protein is separated from
  non-binding and hard-binding proteins
• Not binding target protein
• protein of interest is same charge as gel and is
  repelled off of gel
• proteins of opposite charge stick to gel
• result target protein is separated from bound
  proteins

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Hydrophobic Chromatography

• groups such as octyl alcohol are attached to
  cellulose particles of gel material
• proteins are retarded on the basis of their
  attraction to hydrophobic group
• proteins can be eluted off of column by
  increasing octyl alcohol
• application to membrane bound proteins

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Affinity Chromatography

• presence on the column of covalently attached
  unit for which the protein has an affinity
• substrate, cofactor, inhibitor
• protein is retarded in its passage down gel or
  even held tight to gel
• protein is knocked off of gel by changing
  affinity
• pH for instance changes the nature of binding
  site

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HPLC

• high performance (pressure) liquid chromatography
  
• use same materials as with previous examples of
  chromatography but under conditions of high
  pressure
• enhanced flow, better resolution
• avoid diffusion

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Electrophoresis - Principles

• charged species move toward electrode of opposite
  charge
• separation is on basis of size/charge ratio
• if ratio is constant then separation is
  effectively on the basis of size although
  "pulling" on the species in electric field is
  responsible for the separation
• speed of movement of species is dependent on the
  nature of the media through which species moves

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Electrophoresis - Types

• Analytical
• SDS-PAGE
• PAGE
• Preparative
• Isoelectric focusing

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SDS-PAGE - the parts

• sodium dodecyl sulfate (SDS)
• SDS C12 aliphatic chain with doubly negatively
  charged sulfate at end
• effect disrupt all attractions within and
  between units on polypeptide chain based on
  hydrophobic or charged interactions
• Mercaptoethanol disrupt disulfide linkages
• polyacrylamide gel (PAG)
• cross linked matrix
• degree of cross linking can be varied
• electrophoresis (E) - principles apply!

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SDS-PAGE - how it works

• one SDS binds per 2 amino acid residues in
  protein chain
• each SDS has a charge of -2
• charge/ of amino acids -1 constant
• effectively swamps out other charges
• separation therefore based on size
• sled dog analogy smallest proteins can navigate
  PA forest fastest

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SDS-PAGE - determination of approximate
molecular weight

• log of MW (y-axis) vs migration distance
  (x-axis) is often a straight line
• size value from graph gives subunit MW
• if different subunits obtain MW of each subunit
• MW of native protein is multiple of subunit(s) MW

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Amino Acid Analysis
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Determining Native MW

• Native molecular weight is the weight of the
  intact active protein
• gel filtration can give a very rough native
  molecular weight (so can centrifugation and
  dialysis)
• Many proteins consist of subunits
• SDS-PAGE can give an approximate value for
  subunit weight
• Empirical weight is the smallest weight is
  based on the lowest ratio of amino acids
• since the mass of each amino acid residue is
  known, the sum of these masses gives the
  empirical weight
• amino acid analysis can give the exact empirical
  weight
• mass spec can now also be used to get this

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Determining Native MW - Example

• gel filtration gives a native MW of about 80 k
  SDS-PAGE gives a subunit weight of 37 k amino
  acid analysis gives a weight of 18.7 k based on
  one trp in the empirical unit
• of subunits native MW/subunit weight
• 80/37 2.16 2 subunits (nearest whole
  number)
• of empirical units in subunit
  subunit/empirical
• 37/18.7 1.98 2 units (nearest whole
  number)
• Native MW 74.8 k
• 18.7 x 2 empirical units/subunit x 2
  subunits/native protein

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Determining the sequence of amino acids in a
polypeptide

• Analyses on Polypeptide
• determination of N and C termini
• enzymatic/chemical cleavage into fragments
• (determination of N and C termini of fragments)
• determination of sequence of fragments by Edman
  degradation
• Solving the puzzle
• determining sequence by overlapping sections of
  sequenced fragments

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Edman degradation
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Merrifield Solid State Synthesis