Subsections
[Cr:4, Lc:0, Tt:0, Lb:12]
- Cloning of the DNA segment encoding the protein of interest into an
expression vector: Knowing the online servers/software for DNA and
protein analysis: Acquiring DNA sequence encoding the protein of
interest (for example GFP) from online database like PUBMED and PDB.
Analysis of DNA sequence for presence of internal restriction
digestion sites etc. using software like `Ape'. Primer designing:
Designing of 5' forward and 3' reverse complementary primers
containing appropriate restriction digestion sites, affinity tags
(penta-His etc.). PCR amplification of the DNA segment of interest
from a suitable source, purification of the PCR product, restriction
digestion, and subsequent ligation into the suitable bacterial
expression vector (also containing an antibiotic resistant marker) of
interest. Transformation into suitable competent cells (BL21 etc.).
(Students will learn how to prepare E. coli competent cells).
Selection of the antibiotic resistant single colony. e.Plasmid
isolation from the transformed cells and sequencing it to confirm the
sequence of cloned DNA segment of interest. Testing of protein
expression upon induction in presence of IPTG (SDS PAGE/Western
Blotting).
- Large scale expression and purification of the protein: Bulk scale
bacterial cell culture and IPTG induction for protein expression.
Detection of the protein by western blotting in soluble and insoluble
fraction after bacterial cell lysis. Affinity purification of the
protein from the soluble fraction of the bacterial cell lysate (for
His-tagged protein, Ni-agarose matrix will be used).
- Biochemical and biophysical characterizations of the purified protein:
Purified protein will be assayed for its biological activity.
(Fluorescence from GFP). UV-VIS absorption and emission spectra
resulting from intrinsic Tryptophan and GFP chromophores. Fluorescence
quenching and polarization studies. Unfolding and refolding studies
using CD and fluorescence methods.
- J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular cloning: a
laboratory manual, 2nd Edn., Cold Spring Harbor Laboratory Press
(1989).
