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.

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