29. Insulin

Your boss gives you $10 million to make Kirkland insulin. Q1: How would you proceed?
(1) Party like it’s 1922 (i.e., extract bovine insulin from pancreases)
(2) Party like it’s 1995 (i.e., clone Babe [the pig from the movie] and isolate his insulin)
(3) Party like it’s 2025 (i.e., just stand in the corner looking at your phone)
(4) Party like it’s 1978 (i.e., clone the insulin ORF into E. coli and overexpress/purify)
(5) Make the hotdogs from 100% bovine pancreases (....probably already are anyway)

Party like it’s 1978 (i.e., clone the insulin ORF into E. coli and overexpress/purify)

Q2: Which of the below is NOT something you need to worry about with regards to using E. coli
to express insulin?
(1) improper protein folding in E. coli
(2) loss of the expression plasmid from the E. coli cell
(3) intracellular accumulation of expressed proteins as inclusion bodies in E .coli
(4) alternative codon decoding rules between humans and E. coli
(5) lack of appropriate post-translational modifications in E. coli

alternative codon decoding rules between humans and E. coli

Plasmid vector component: MCS

sequence containing multiple unique restriction enzyme sites for easy foreign DNA insertion

Plasmid vector component: T7 promoter

drives high-level expression of a gene of interest in systems using T7 RNA polymerase, commonly used in protein production

Plasmid vector component: HIS-Tag

histidine residues added to proteins for purification purposes using metal affinity chromatography.

Q5: How could we test this is the
seq of the correctly spliced gene?

use primer 5’-ctggttcaagggcttta-3’
in conjunction with reverse
transcriptase to make a cDNA copy of
the mRNA, clone it, and then
sequence it

Q7: What do you need to
check before committing
to using NcoI and XhoI?
(1) that these enzymes do
not inhibit reverse
transcriptase activity
(2) that NcoI only cuts
phosphorylated DNA
(3) that a XhoI site is
present within the
antibiotic resistance gene
of the plasmid chosen for
overexpression
(4) that these enzymes do
not cut internal of your
gene of interest
(5) that these enzymes
function on DNA and not
RNA

That these enzymes do
not cut internal of your
gene of interest

A potential issue with producing a human protein in E. coli
involves the codons used

Human genes often use codons that are less frequently used or recognized by E. coli, leading to inefficient translation and lower protein expression

we’ve mentioned silent (aka. synonymous)
mutations a few times in this course. Q9: Do silent mutations
have any phenotype?

Maybe, while they change a gene codon to one that still
encodes the same amino acid, not all codons are equal and
hence there may be a change in phenotype

What could you do to boost insulin production in E. coli?

synthesize a codon-optimized version of the insulin gene

You add isopropyl β-D-1-thiogalactopyranoside (IPTG) to 0.5 mM. Q12: Group E: What is this
and why do we add it?

molecular analog of allolactose used to induce gene expression in strains with the DE3 prophage, leading to the activation of T7 RNA polymerase and subsequent transcription of the insulin gene

Why do we run samples from the different steps (U, I, P, S, Ni-NTA, SEC) on the gel rather than just the SEC sample since that is the important one?

ensures each stage of the purification process is working correctly and helps identify any issues

When attempting to overexpress and purify a protein in E. coli it is common to use an IPTG-based expression system, where the gene of interest is only transcribed following addition of IPTG to the culture.  Why is the gene of interest not significantly expressed until IPTG addition?

(A)  IPTG inhibits the phage lysin protein present within pLysS; this prevents cell lysis and therefore enables the expression of the protein of interest

(B)  IPTG binds to and inhibits the activity of LacI which results in the transcription of the T7 polymerase gene, which in turn results in the transcription of the gene of interest which is located downstream of a T7 promoter

(C)  IPTG enables the E. coli to utilize sucrose as an energy source; the greater energy input results in enhanced protein production

(D)  IPTG binds to lacZ which promotes the ability of the encoded protein, LacZ, to bind to the LacO region of the gene of interest’s promoter, enhancing transcription

(E)  IPTG promotes the activity of the T4 DNA polymerase; resulting in enhanced transcription and translation of the gene of interest

IPTG binds to and inhibits the activity of LacI which results in the transcription of the T7 polymerase gene, which in turn results in the transcription of the gene of interest which is located downstream of a T7 promoter