
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:
- 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
- Isoelectric focusing
- Protein Characterization:
3D Structure Determination:
Can be done either by
X-ray crystallography or
NMR ( nuclear magnetic resonance)