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Cloning and purification of green fluorescent protein and its study

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  1. Introduction of GFP
    1. Glutathione S-transferase (GST)
    2. Poly histidinetag
    3. Calmodulin binding Protein (CBP)
    4. Green Fluorescent Proteins (GFP)
  2. Overview literature of GFP
    1. Osamu Shimomura
    2. Douglas Prasher
    3. Marty Chalfie
    4. Sergey A Lukyanov
  3. The 3d structure of GFP
    1. The Beta-Can structure
    2. Topology of folding
    3. Cysteins
    4. The environment of the fluorophor
    5. Tryptophan fluorescence
  4. The fluorophore of green fluorescent protein (GFP)
    1. Fluorescent spectrum of GFP
    2. The fluorophore center of GFP
  5. The excited state dynamics of GFP
  6. Fluorescence of GFP mutants
  7. Cloning of GFP
    1. Experiment 1
    2. Experiment 2
    3. Experiment 3
    4. Experiment 4
    5. Experiment 5
    6. Experiment 6
  8. Purification and physical properties of GFP
    1. Purifications and physical properties of green fluorescence protein
    2. Hydorphobic chromatography for purification of GFP proteins
    3. Protein electro phoresis of green fluorescent proteins
    4. Physical properties determination
  9. Results
  10. Conclusions

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. [...]

[...] The table below contains links to entries in the PDBsum database for 1gfl and other GFP variants, the structure of which have also been subsequently determined: PDBsum Link GFP Variants 1bfp blue variant of green fluorescent protein 1c4f green fluorescent protein s65t at ph 4.6 1ema green fluorescent protein from aequorea victoria 1emb gfp from aequorea victoria, gln 80 replaced with arg 1emc green fluorescent protein from aequorea victoria, mutant 1eme green fluorescent protein from aequorea victoria, mutant 1emf green fluorescent protein from aequorea victoria, mutant 1emg gfp (65-67 replaced by cro, s65t substitution, q80r) 1emk green fluorescent protein from aequorea victoria, mutant 1eml green fluorescent protein from aequorea victoria, mutan 1emm green fluorescent protein from aequorea victoria, mutant 1gfl structure of green fluorescent protein 1yfp structure of yellow-emission variant of gfp 2emd green fluorescent protein from aequorea victoria, mutant 2emn green fluorescent protein from aequorea victoria, mutant 2emo green fluorescent protein from aequorea victoria, mutant 2yfp structure of yellow-emission variant of gfp GFP has a cylindrical fold. [...]

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