Methods of Protein Characterization

Need to characterize Protein:

Protein characterization is necessary in biological development and in research:

To determine protein structure
To determine the actual amino acid composition
To sequence of 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:

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)