Methods of Protein Characterization

Need to characterize Protein:

Protein characterization is necessary for biological development and in research:

• To determine protein structure
• To determine the actual amino acid composition
• To sequence the protein product
• To examine the post-translation modifications on the proteins which can affect their functions
• To determine disulfide bonds

Steps for protein characterization:

• Cloning
• Expression
• Purification
• Characterization

Expression cloning:

• A technique in DNA cloning that uses expression vectors to generate a library of clones, with each clone expressing one protein.

An example would be using an expression library to isolate genes that could confer antibiotic resistance.

  Expression vector:

• A specialized type of cloning vector in which the transcriptional and translational signals needed for the regulation of the gene of interest are included in the cloning vector.
• The transcriptional and translational signals may be synthetically created to make the expression of the gene of interest easier to regulate.

Purpose of Expression cloning:

The aim of expression cloning is

• to produce large quantities of specific proteins
• to enhance translation of the gene of interest’s mRNA
• a transcription termination sequence,
• in eukaryotes specific sequences to promote the post-translational modifications of the protein product.

Protein purification:

• A series of processes to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms.
• Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest.
• The purification process may separate the protein and non-protein parts of the mixture, and finally, separate the desired protein from all other proteins.
• The pure result may be termed protein isolate.

Protein Separation & Detection

• Separate proteins with 1D or 2D Gel Electrophoresis
  • Isoelectric focusing
    • Immobilized pH gradients (IPG DryStrip)
    • Tube gel (carrier Ampholytes)
  • SDS PAGE
    • Reduced and alkylated Stain proteins
• Protein Characterization:

3D Structure Determination:

Can be done either by

X-ray crystallography or

NMR ( nuclear magnetic resonance)