Lecture 19: Protein Overexpression, DNA Oligonucleotides, PCR

Question 1

Ways to overexpress protein in E. coli

Answer 1

• inducible promoter
• inducible RNA polymerase

Question 2

How to overexpress protein in E. Coli (inducible promoter)

Answer 2

1. insert cDNA of protein of interest after lac promoter
2. add IPTG (induce the lac promoter)
3. transciption and translation
4. lyse cells
5. purify proteins

Question 3

How to overexpress protein in E. Coli (inducible RNA polymerase)

Answer 3

1. T7 polymerase gene gets inserted into host genome after the lac promoter
2. transcription and translation of T7 RNA polymerase
3. T7 RNA polymerase binds to T7 late promoter (on plasmid expression vector) and transcribes target gene
4. overexpression of protein.

Question 4

Parts of bacterial plasmid

Answer 4

• multiple cloning (restriction) sites
• T7 RNA Polymerase Promoter (strong)
• T7 Transcription Terminator
• Lac Operator (inducible with galactose/IPTG) • Synthetic Epitope (HIS6 or HA) at NH or COOH
• used in cells that express phage T7 RNA polymerase and lac repressor

Question 5

Purification and Detection Protein Tags

Answer 5

• Protein Tags - are small peptides that bind to protein
  
  • used for affinity chromatography
• examples
  
  • His6: Histidine tag on protein binds to Ni-NTA (nickel) (bound to column).
    
    • release protein with imidazole.
  • Glutathione-S-Transferase: GST tag on protein binds to glutathione (bound to column.
    
    • release protein with protease or glutathione
  • Epitope: elute protein with antibody
    
    • examples
      
      • Myc
      • HA

Question 6

Epitope Tagging and GFP Fusion Proteins

Answer 6

• green fluorescent protein plasmids can be transfected into eukaryotic cells if they have a eukaryotic promoter
• GFP - fluoresces - in nucleus or in cytosol

Question 7

Insulin

Answer 7

• lack of insulin causes type I diabetes
• produced in pancreas
• affect sugar metabolism
• insulin pre-protein: 2 polypeptides - A chain (21AA) and B chain (30AA)
  
  • joined by disulphide bonds

Question 8

Synthetic DNA oligonucleotide uses

Answer 8

• Hybridization
• Gene Arrays: like northern blot but looks at all genes in a genome at one time
• Polymerase Chain Reaction
• Mutagenesis: introduces restriction sites to make changes to genes
• DNA sequencing

Question 9

Chemical Synthesis of DNA

Answer 9

1. On a solid phase nucleotide support, 3’ linkage is made with the 5’ side protected
2. deprotect the 5’OH end
3. add next nucleotide with protected 5’ end
4. repeat steps 2 and 3 until done
5. cleave from support
6. purify

• forms single stranded DNA up to 100 nt in length
• sequential addition of reactive nucleotide derivatives 3’ to 5’

Question 10

Oligonucleotide hybridization probes for Southern blotting and library screening

Answer 10

• oligonucleotide probe - around 20 nt sufficient
• mix all possible sequences to make a degenerate probe

Question 11

Polymerase Chain Reaction (PCR) - properties and uses

Answer 11

• amplifies DNA by repeated rounds of DNA syntehesis
• highly sensitive
  
  • can be used for mutagenesis
  • detect single molecules?
• rapid - every round doubles amount
• many uses
  
  • obtain genes
  • introduce restriction sites by PCR
  • site-directed mutagenesis of genes
  • detection, diagnosis, forensics
  • quantitation of nucleic acids
    
    • eg SYBER Green intercalation into dsDNA - fluorescence - measure nucleic acid amount

Question 12

Polymerase Chain Reaction (PCR) - process

Answer 12

1. Denature dsDNA by heat (95C)
2. Add Primers, lower temp (42C) to allow annealing (Hybridization)
3. Add thermostable DNA polymerase, synthesize DNA at 72C
   
   • Taq polymerase from Archea Thermus aquaticus ​
4. Denature double-stranded DNA - Cycle 2
5. Anneal and polymerize - Cycle 2

Question 13

PCR uses: Introduce restriction sites and cloning PCR product

Answer 13

1. add restriction sites at before primers
2. PCR -
   
   • adds EcoRI and BamHI restriction sites
3. Digest PCR product and the plasmid vector (which already has restriction sites) with EcoRI and BamHI
4. mix and ligate

Question 14

PCR use: Gene Mutagenesis

Answer 14

• add sequences (that want to be added to the genome) to end of primer
  
  • the sequences should be complementary to each other and include desired new sequence
  • sequence will bind to itself. then will become site of synthesis
  • then put in reaction with primers without the new sequence