Gel Electrophoresis

• Proteins are charged molecules

• Charged molecules will move in an electric field

• Polyacrylamide gels provide a convenient matrix to “sieve” the protein molecules as they move

  • Polyacrylamide Gel Electrophoresis (PAGE)

  • Movement through gel depends on charged and shape of molecule

• This is “native” or “non-denaturing” PAGE

• In order for movement to be proportional to molecular weight, there must be a constant charge to mass ratio

Charge to Mass Ratio

• DNA

  • A regular polymer

• Proteins

  • Irregular polymers

SDS-PAGE

• Protein charge is dependent on the amino acid sequence

• In order to create a constant charge-to-mass ratio and constant “shape”, proteins can be dissolved in sodium dodecyl sulfate and treated with a reducing agent to break all disulfide bonds (and prevent any disulfides from forming)

• Most proteins bind 1-2 SDS molecules per amino acid; proteins with bound SDS are unfolded, denatured, “random coil” molecules that are negatively charged

• Sodium dodecyl sulfate is an amphipathic molecule

• Protein sample is heated in the presence of SDS and mercaptoethanol

• Mercaptoethanol is added to reduce -S-S- bonds if present to -SH and to prevent them from forming if not originally present

• Multi-subunit proteins are converted to their component peptide chains by this treatment

• Migration is inversely proportional to the log of molecular weight

Isoelectric Focusing

• The pl is the pH at which the net charge of the protein is zero

• At its isoelectric point (pI) a protein has a net charge of zero and will not move in an electric field

• pl depends on the amino acid composition of the protein

• Electrophoresis in a pH gradient will cause each protein to migrate to a pH = p and stop

• Process

  • A stable pH gradient is established in the gel in an electric field—meaning high pH at the top and low pH at the bottom

  • Protein solution is added

    • Proteins move to the positive pole and depending on their pKas will become protonated

  • Proteins distribute along pH gradient according to pl

• Potential charged groups in proteins in order of pKa: C-terminus, Asp, Glu, His, N-terminus, Cys, Lys, Tyr, and Arg

• When the net charge is negative, the pH will lower, so the protein can be protonated

• When the net charge is positive, the pH will raise, so the protein can be deprotonated

2-Dimensional PAGE

• Isoelectric focusing (IEF) and SDS-PAGE can be combined to allow high resolution separation of complex protein mixtures

• First dimension: IEF (doesn’t depend on size, only pI)

• Second dimension: SDS-PAGE (depends only on size)

• Example:

  • 2-D-PAGE

    • Proteins associated with the cell membrane of the organism that causes Lyme Disease

Proteomics

• The study of large groups of proteins

• Proteome = all of an organism’s proteins

• All of the spots on the previous 2-D gel cam be identified

  • Pick spots, then digest with trypsin (a protease that cuts only at Lys and Arg residues

  • Mass spectrometry can determine the masses of all the peptides produced from each spot very precisely

  • The genome DNA sequence of the organism is known so the computer can compare the masses of the peptides from each spot with masses of the peptides expected from gene’s coding region

  • The gene encoding each spot can then be identified

• “-omics”

  • Genome = the complete DNA sequence of an organism

    • Humans have about 20,000 genes but much more DNA

  • Transcriptome = the complete set of RNA molecules transcribed from a genome

  • Proteome = the complete set of proteins

    • About 100,000 proteins in the human

  • Metabolome = the complete set of small molecules found in a cell

• Protein Purification

  • To study individual proteins in detail you need relatively large amounts (milligrams) of pure proteins

  • Goal of purification is to obtain a pure, undenatured protein from a mixture that may contain hundreds or thousands of different proteins

  • Various techniques are used to separated proteins based on their properties, usually chromatography based on ONE of the following:

    • Size

      • Gel-exclusion resin

    • Charge

      • Ion-exchange resin

    • Hydrophobicity

    • Specific bonding

      • Affinity resin

  • You must have some assay to follow your protein through the purification

  • Separation Based on Charge: Ion Exchange Chromatography

    • Positively charged protein sticks to the beads and the negatively charged protein does not

      • The more positively charged a protein is, the tighter it will bind

    • Positively charged protein can be eluted from the column by increasing the concentration of salt in the elution solution

      • More positive proteins will bind tighter, so they will be harder to unbind—meaning the salt concentration needs to raise higher

  • Separation Based on Size: Size Exclusion or Gel Filtration Chromatography

    • The beads contain aqueous pores

    • Smaller proteins can explore the spaces—slowing their progress through the matrix

    • Larger proteins are excluded from some or all of the spaces

      • Larger proteins elute more quickly

  • Separation Based on Affinity: Affinity Chromatography

    • Idea: If you could put a tag on a protein that makes it bind tightly to something that proteins do not normally bind to, you could separate the tagged protein from all the other proteins in a miAA2-ture of proteins

      • Use molecular cloning

    • Process:

      • DNA encoding to the protein you want to purify

      • Region of vector encoding 6 histidines -a “His-tag'}

      • Histidine binds tightly to nickel

      • Plasmid will produce a new protein

        • The protein you want to purify with 6 His residues at its C-terminal

• Importance of Overexpression in Protein Purification

  • Many proteins in the cell are present at very low concentration

    • Important proteins may comprise 0.1% or less of the total protein in the cell

    • By cloning the gene encoding the protein you wish to purify, you can usually greatly increase the level of the protein; plasmids may be present in many copies to amplify the gene encoding the protein

      • It’s much easier to purify a protein that is 10% of the total protein than one that is 0.1%

    • Using cloning, proteins can be expressed in and purified from organisms that are convenient for purification

      • A human protein might be expressed in E. Coli in order to make purification easier