A key step in proteomics the study of proteins function and structure is the purification of proteins. The ability to isolate and purify specific proteins is an essential feature of Modern biochemistry as it allows scientist to study proteins in isolation from other proteins, which greatly aids the under standing of a particular proteins functions. Unfortunately there is no single ideal proteins purification procedure and often the purification of a protein involves several techniques. The main idea behind proteins purification is to select the best techniques to isolate a protein of interest based on differences in its physical properties from other unwanted proteins. The proteins purification aim to cover many of the common techniques used in protein purification.
One of the important aspects of biotechnology is the use of bacteria as living factories to produce genetically engineered proteins know as Recombinant proteins as opposed to purify specific proteins from biological sample is the vast quantities can be produced and scientist can attach affinity tag that allows for rapid and specific purification of the recombinant proteins. The science behind the purification of recombinant proteins with a specialized affinity tag is the use of affinity chromatography. Affinity chromatography involves the attachment of a specific ligand to a solid support or resin. The ligand and will bind the specific tag and all the untagged, non specific proteins are washed from the solid support. The solid support is then treated with an elution buffer that breaks the interaction between the ligand and the tagged recombinant protein. Below are a few example of the more common affinity tags used in science today with information on the tag the ligand the elution buffer.
[...] Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. The gene for GFP has been isolated and has become a useful tool for making chimeric proteins of GFP linked to other proteins where it functions as a fluorescent protein tag. GFP tolerates and C-terminal fusion to a broad variety of proteins. It has been expressed in bacteria, yeast, slime mold, plants, drosophila, zebrafish, and in mammalian cells. As a noninvasive fluorescent marker in living cells, it allows for a wide range of applications where it may function as a cell lineage tracer, reporter of gene expression, or as a measure of protein-protein interactions. [...]
[...] In 1992 he published a paper in Gene; it reported the cloning of GFP and the sequence of the 238 amino acids in GFP, shown below. Sadly it was only a two year grant and the funding ran out before he could express the GFP clone he had produced in a manner that would result in a fluorescent GFP. GFP Amino Acid Sequence: MSKGEELFTGVVPVLVELDGDVNGQKFSVSGEGEGDATYGKLTLNFICTTGKLPVPWPTLVTTFSYGVQCFSRYPD HMKQHDFFKSAMPEGYVQERTIFYKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNVYI MGDKPKNGIKVNFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLEFVTAAR ITHGMDELYK Marty Chalfie Prof. Martin Chalfie (from the Biological Sciences, Columbia University web- site) In 1988, just as Doug Prasher, had started to work on the sequencing and cloning of GFP, Martin Chalfie heard about GFP for the first time. [...]
[...] Lab safe gel stain CLONING OF GFP Experiment No.-1 Bacterial Culture and Growth This experimental activity uses the most widely used medium for common bacterial culture. LB broth consist of tryptone yeast extract and sodium chloride. LB agar consist of tryptone yeast extract and agar. Both LB broth and LB agar are rich in nitrogen, carbon, and salt to meet the requirement of bacterial growth. Fresh medium is prepared by adding water followed by sterilizations. Material Required 1ml Bacteria with plasmid 1 or plasmid flask 100 ml molten agar 4 Petri Disches 1 vial Ampicillin 1 vial sterile water 1 inoculating loop 1 vial loop wash PROCEDURE 1. [...]
[...] Bacterial Trans Formation In the bacterial transformation in the + ve control [cloning] GFP fluorescence [GFP gene + vector DNA + Amp resistance] ve control Growth, No fluorescene [No GFP gene + Novector Amp Resistant] DH52 No Growth [Photo] FIG 4 CLONED GFP UNDER UV RAYS Result Purification and Physical Properties of Protein Result 1 Bacterial culture and growth FIG 5 GLASS BEADS OF GFP UNDER UV RAYS Result Experiment No the molecular weight of green fluorescent protein is 33K.D. [...]
[...] The green fluorescent proteins is extremely hydrophobic compared to the bacterial proteins which are relatively hydrophilic this allows the use of hydrophobic chramato graphy for the purification of recombinant protein green fluorescent protein as a tag purification process and the visualization of the protein through out the experiment. Many other tags are available in the market and the selection is dependent on the proteins to be purified and personnel preference. Know as the TAP (Tandem Affinity Purification) has recently been developed and employed with great success. [...]
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