Recombinant Hormones - Insulin

Question 1

Describe the insulin processing

Answer 1

preproinsulin: insulin precursor protein and contains a signal sequence for export into the ER which is proteolytically removed to give proinsulin
proinsulin: transported into secretory vesicles where prohormone convertases act on dipeptide cleavage sites to generate insulin
insulin: mature secreted hormone held together mainly by S-S bonds

Question 2

Events upon insulin binding to its receptor

Answer 2

conformation change in receptor
B chain tyrosine kinase is activated by self phosphorylation
mitogenic effects are stimulated by MAP kinase pathway
metabolic effects are stimulated through PI3-K pathway
GLUT4 glucose transport is upregulated

Question 3

How was insulin first made?

Answer 3

expressing A and B chains separately then refolding them into a mature insulin molecule

Question 4

Insulin prb production

Answer 4

insert the nucleotide sequence coding proinsulin into E. Coli
proinsulin is purified and proteolytic excision of C peptide is done in vitro
popular method due to single fermentation and purification scheme

Question 5

What is a mutein?

Answer 5

a protein with altered amino acid sequence compared to the natural product, usually enough to alter physiochemical and biological activity properties

Question 6

Site directed mutagenesis of plasmid DNA

Answer 6

to mutate bases in plasmid:
use 2 complementary primers that contain the mutated site
copy the plasmid so that the PCR product is the mutated plasmid
destroy the plasmid template using restriction enzyme Dpnl that will only cut methylated DNA isolated from the bacteria
the plasmid is ligated in the cells