Expression and Purification of Recombinant Protein …bbartholomew/protein-expression.pdfDNA Vectors...
Transcript of Expression and Purification of Recombinant Protein …bbartholomew/protein-expression.pdfDNA Vectors...
Presented By: Puspa pandey,Mohit sachdeva &Ming yu
Expression and Purification ofRecombinant Protein in bacteria and Yeast
DNA Vectors
Molecular carriers which carry fragments of DNA into host cell.
Usually derived from plasmids, which are small, circular, double-stranded DNA molecules
Some widely used vectors:
1. Plasmids2. Cosmids3. Yeast Artificial Chromosomes4. Phage lambda vectors5. Bacterial Artificial Chromosomes6. Human Artificial Chromosomes7. Transposons etc.
1. Plasmids
Extrachromosomal DNA mainly found in bacteria, capable of autonomous replication
Typically circular and double-stranded
Advantages as a Vector
They are derived from natural plasmids that occur in bacterial cells so easier to manipulate and propagate than other vectors.
More stable vector to maintain a particular clone
Great variation in size( from 1 to over 400 kb)
Disadvantages
Small fragments (10-15 kb) of DNA can be inserted.
Low transformation efficiency
2. Cosmids
Artificially constructed cloning vectors containing the cos gene of the phage lambda.
Can be packaged into lambda phage particles for infection into Bacteria.
Advantages
It can carry Larger fragments (40-50 kb) of DNA
High transformation efficiency
Usually used in making the Genomic libraries.
Disadvantages
Unable to accept more than 40-50 kbp of DNA
Needs sophisticated process for preparation
3. Yeast artificial chromosome (YAC)An artificially constructed chromosome, contains the telomeric, centromeric, and replication origin sequences needed for replication.
Advantages
Capable of carrying a large DNA fragment (up to 2 Mb)
One can get directly the eukaryotic protein products
Disadvantages
Transformation efficiency is very low
Not possible to recover large amount of pure insert DNA from individual clone.
Unstable sometimes, producing chimeric effects
Making and Purification of Recombinant Protein in bacteria and Yeast
Recombinant ProteinA protein obtained by introducing recombinant DNA into a host (microorganism or yeast cell) and causing it to produce the geneproduct.
A protein whose amino acid sequence is encoded by a cloned gene.
OR
Basic steps to get recombinant Protein:
1. Amplification of gene of interest. ( Using PCR).
2. Insert into cloning vector. (Ex: PCR*8).
3. Sub cloning into expression vector. (Ex: pKK223-3 or PSVK 3)
4. Transformation into protein expressing bacteria (E coli) or yeast.
5. Test for identification of recombinant protein.( Western blot orFluoroscence)
6. Large scale production. (Large scale fermentor)
7. Isolation and purification.
Various factors to be considered:
1. Which expression vector ?
2. Which host system?
3. Properties of protein • Membrane bound• Solubility• Single or multidomain• Size?
4. Where it expressed?
5. How to identify, isolate and purification method?
Necessary Features of expression vector:
•Origin of replication•Drug resistance marker•A promoter•Transcription terminator•Restriction sites
Expression Vectors with or without specific tags……
Vectors for non-fusion proteins
pKK223-3 pSVK 3PSVL SV40
Vectors for fusion proteins
pGEXpQEpETpEZZ 18
Features of small tags (Fusion proteins):
1.Improved expression ex: sumo fusions2.Improved solubility3.Cell compartments can be targeted
4.Improved detection ex: GFP tag fluoroscence,specific antibodies detection in western
5.Improved purification ex: using ionexchange or affinity chromatography
For small peptides
Poly hisFlagMyc
Large peptides
Maltose binding protein( MBP)Chitin binding domainCellulose binding domainGlutathione S- transferase( GST)
Features of Host --prokaryotic systems ex: E coli
Advantages Disadvantages
•Many references and muchexperience available e.g. E. coli.
•Wide choice of cloning vectors.
•Gene expression easily controlled.
•Easy to grow with high yields.
•Product can be designed forsecretion into the growth media.
•No post-translational modification.
•Biological activity and immunogenicity may differ from natural protein.
•High endotoxin content in gram negative.
Features of eukaryotic systems: yeast
Advantages Disadvantage
•Lacks detectable endotoxins.
•Fermentation relatively inexpensive.
•Facilitates glycosylation and formationof disulphide bonds.
•Only 0.5% native proteins are secreted soisolation of secreted product is simplified.
•Well established large scale productionand downstream processing.
•Gene expression less easily controlled.
•Glycosylation not identical to mammalian systems.
Isolation of protein: Initial steps prior to purification:
Disruption of cells: OsmoticChemicalEnzymaticMechanical
Clarification: CentrifugationFiltration
Concentration: Precipitation Ultra filtration
Differential centrifugation
Differential centrifugation:
It’s a procedure in which a homogenate is subjected to repeated centrifugations each time increasing the centrifugal force.
Separation is based predominantly on particle mass and size with larger and heavier particles pelleting at lower centrifugal fields
For specific organelle sub cellular fractions.
Purification of isolated proteins
Various purification methods:
1. Charge: IEC/IEF
2. Size: size exclusion chromatography
3. Hydrophobicity: Hydrophobic Interaction Chromatography
4. Ligand specifity: affinity chromatography, nucleotide and coenzymes resins,phosphoprotein resins.
5. Avidin biotin matrices
6. Carbohydrate binding
7. Dye resins.
8. Solubility: Precipitation
Precipitation
Proteins tend to precipitate at their isoelectric point if the ionic strength of the solution is very high;First step in protein purification;Ammonium sulfate and Trichloroacetate (TCA) are the most common salt.
Buffer Exchange
Importance:Different purification techniques require the protein present in buffers of specific pH and ionic strengths.
Size Exclusion Chromatography
Ion Exchange Chromatography
Affinity Chromatography
Hydrophobic Interaction Chromatography (HIC)
It is based on hydrophobic attraction between the stationary phase and the protein molecules.
The stationary phase consists of small non-polar groups ( butyl, octyl or phenyl) attached to a hydrophilic polymer backbone (cross-linked dextran or agarose, for example).
The sample is loaded in a buffer containing a high concentration of a non-denaturing salt (frequently ammonium sulfate). The proteins are then eluted as the concentration of the salt in the buffer is decreased.
Purification of Tagged Recombinant Proteins
Ni-NTA Agarose To purify recombinant protein containing polyhistidine (6xHis) sequence.Streptavidin Agarose To purify biotinylated recombinant
protein.
Thanks!