Download Expression and Purification of Recombinant Protein in bacteria and

Expression and Purification of
Recombinant Protein in bacteria and Yeast
Presented By:
Puspa pandey,
Mohit sachdeva &
Ming yu
DNA Vectors
†Molecular carriers which carry fragments of DNA into host cell.
†Usually derived from plasmids, which are small, circular, doublestranded DNA molecules
Some widely used vectors:
1.
2.
3.
4.
5.
6.
7.
Plasmids
Cosmids
Yeast Artificial Chromosomes
Phage lambda vectors
Bacterial Artificial Chromosomes
Human Artificial Chromosomes
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
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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 Protein
A protein obtained by introducing recombinant DNA into a host
(microorganism or yeast cell) and causing it to produce the gene
product.
OR
A protein whose amino acid sequence is encoded by a cloned gene.
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 or
Fluoroscence)
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
Vectors for fusion proteins
pKK223-3
pSVK 3
PSVL SV40
pGEX
pQE
pET
pEZZ 18
Features of small tags (Fusion proteins):
1.Improved expression ex: sumo fusions
2.Improved solubility
3.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
Large peptides
Poly his
Flag
Myc
Maltose binding protein( MBP)
Chitin binding domain
Cellulose binding domain
Glutathione S- transferase( GST)
Features of Host --prokaryotic systems ex: E coli
Advantages
•Many references and much
experience available e.g. E. coli.
•Wide choice of cloning vectors.
•Gene expression easily controlled.
•Easy to grow with high yields.
•Product can be designed for
secretion into the growth media.
Disadvantages
•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
•Lacks detectable endotoxins.
•Fermentation relatively inexpensive.
•Facilitates glycosylation and formation
of disulphide bonds.
•Only 0.5% native proteins are secreted so
isolation of secreted product is simplified.
•Well established large scale production
and downstream processing.
Disadvantage
•Gene expression less
easily controlled.
•Glycosylation not identical
to mammalian systems.
Isolation of protein: Initial steps prior to purification:
Disruption of cells: Osmotic
Chemical
Enzymatic
Mechanical
Clarification: Centrifugation
Filtration
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!