Drugs from Bugs (salivary components)

Question 1

The main steps for developing drugs from bugs

Answer 1

1. protein purification
2. amino acid sequencing
3. translate amino acid sequence into nucleotide sequence (primer)
4. DNA library
5. Use nucleotide primers to isolate target gene
6. Expression of recombinant protein insert gene into cells of another organism

Question 2

What does protein purification involve?

Answer 2

chromatography

suitable assay

Question 3

What does amino acid sequencing involve?

Answer 3

mass-spectroscopy fingerprinting

N-terminal by Edman degradation

Question 4

What does translating the amino acid sequence into nucleotide sequence (primer) involve?

Answer 4

may need to use degenerative primers (check codon usage)

Question 5

What is the DNA library?

Answer 5

genomic (gDNA) library

complementary DNA library (cDNA) - based on the mRNA (expressed RNA)

Question 6

What does using nucleotide primers to isolate target gene involve?

Answer 6

PCR, isolate correct clone, sequence gene

anchored-PCR using the DNA library

Question 7

What does the mass production of the isolated salivary component involve?

Answer 7

bacteria - basic and cheap
yeast - can fold better
insect/mammalian - most expensive, but can add sugar (glycosylation) and good folding

Question 8

What are degenerative primers?

Answer 8

Primers that use information from related species or known sequences of the same species to look at common codon usage for a particular amino acid.
E.g.) ticks always use UUG for leucine.

Question 9

What is the shotgun approach?

Answer 9

extract all mRNAs
package mRNAs into E.coli - each bacteria contains one mRNA ("gene")
grow the bacteria
put on agar plate at dilute conc.
-> seq. tick cDNA in each colony 
\+bioinformatics

Question 10

Example of a drug from a “bug” - anticoagulant (not tick)

Answer 10

Leech - hirudin (aka Angiomax (commercial))