Question

You are studying the HIV Protease protein and you want to isolate it for study. Describe the steps that you would take to express the protein in bacteria and purify it for future study. Describe one other technique concerning protein analysis and what that technique would tell you about the protein.

Answer 1

Steps to express the protein in bacteria

1.Amplification of gene of interest (Using PCR): The first step is to isolate the gene of interest by amplification of the gene of interest using PCR from the viral suspension. Once the gene is amplified it has to be inserted into a cloning vector
2.Insert into cloning vector/ expression vector. (Ex: pKK223-3 or PSVK 3) A suitable plasmid vector with origin of replication and specific restriction sites for restriction enzymes is chosen. Restriction enzymes cleave the plasmid at specific sites where gene of interest can be introduced. DNA ligase can be used to join the fragment gene of interest to the plasmid. It should also have selectable markers for identification of recombinants once introduced into vectors.
4.Transformation into protein expressing bacteria (E coli): The bacterial cells are made competant and the plasmid is introduced into the host bacterial cell. Using selectable markers, recombinants are chosen and cultivated in bioreactors for production of protein
5.Large scale production. (Large scale fermentor): The recombinant bacteria is allowed to grow, multiply and produce large quantities of proteins.
6.Isolation and purification: The proteins are then isolated and purified by various techniques. Protein seperation methods include, SDS PAGE, isoelectric focussing, chromatic methods (HPLC, TLC), two dimensional electrophoresis

7.Test for identification of recombinant protein.( Western blot or Fluoroscence)

Further protein identification can be done using Edman degradation or mass spectrometry.

Mass spectrometry can be used to determine protein sequences and thus to determine the identities of proteins in a manner conceptually similar to that described above based on Edman degradation. A more powerful method (because it can be applied to large numbers of samples more rapidly) is however to use mass spectrometry to determine the precise molecular weights of specific cleavage fragments derived from a given protein.

We can then feed our experimental results into a protein (or more often a genomic) database to determine what proteins in our organism of interest would give a pattern of fragments exactly matching those found in our digested sample. There are of course a huge number of proteins in any organism, and the pattern of cleavage fragments obtained by treating a given protein with a single endoprotease often does not allow the protein to be identified uniquely.
We can usually use the resulting digestion patterns (again, analyzed by mass spectrometry to provide highly accurate determination of the fragment molecular weights) to produce a unique identification of our unknown protein:
Again, mass spectrometry is uniquely well-suited for such analyses because it can yield very accurate determinations of molecular weights from very even very small amounts of fragments resulting from the digestion of a particular protein. Techniques have now been developed by which proteins separated in twodimensional gels can be digested within the gels and then injected directly into a mass spectrometer for analysis of the resulting fragments.