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1. Being an Oral Paper Presentation
At the 37th Annual Scientific Conference of
The Nigerian Society for Biochemistry and
Molecular Biology,
UMYU, Katsina.
November, 2019
BY
YAHAYA RIKO YUNUSA
&
ZUBAIRU DARMA UMAR
Department of Microbiology, Umaru Musa
Yar’adua University, Katsina.
Production of Human Insulin Hormone
(Humulin) Through Genetic
Engineering Using Escherichia coli
1

2. Background 2
 Diabetes mellitus is a hyperglycemic metabolic
disorder resulting from insufficient production of, or
resistance to insulin, with consequent metabolic
dysfunctions (Zielinski et al., 2019).
 There are four types:
1. Type I/IDDM/Juvenile diabetes, subdivided into
types 1A and 1B.
2. Type II/NIDDM/Adult-Onset Diabetes
3. Type III/GDM
4. Miscellaneous DM due to genetic mutations,
drug/chemical inducement, endocrinopathies,
etc (Akinlade et al., 2014; Okafor, 2014).
 Currently, some 415 million people suffer from
diabetes worldwide, and by 2030, that figure is
projected to reach 552 million, roughly 1 in 16
people (IDF, 2019).
 In Nigeria, prevalence figures vary widely due
to data collection obstacles, but they range from
0.8-23.1%, with a pooled prevalence of 6%,
some 12 million individuals (Sabir et al., 2018;
Aluko et al., 2018).
Figure 1: The Islets of
Langerhans
Image Source: 123rf.com
WHO standard Cut-off
Values for Diagnosing
DM
FPG = ≥
7mmol/L
RPG = ≥ 11.1
mmol/L
PG 2-h PGL
(75g) = ≥ 11.1
mmol/L
&
Glycated
hemoglobin
(HbA1c)/TLC
≥ 6.5%
Figure 2: WHO Standard
Cut-off Values for
Diagnosing DM
Source: Uloko et al. (2018)

3. Recombinant DNA
Technology/Genetic Engineering:
History and Overview
A Watershed in
Molecular
Biology: In
1973, Herbert
Boyer of the
University of
California at San
Francisco, and
Stanley Cohen
of the Stanford
University use
E. coli
restriction
enzymes to
insert foreign
DNA into
plasmids (GNN,
2012).
Figure 4: The rudiments of rDNA
technology
3
Figure 3: Fathers of rDNA
technology, a. Boyer and b.
Cohen
Image Source:

4. Insulin vs. Human Insulin
Hormone/Humulin
 Insulin is a 51 Amino Acid polypeptide weighing 5808 Da, consisting of A and
B subunits, connected by two disulphide bridges, with subunit A having an
intra-subunit bridge (Tariq et al., 2018; Zielenski et al., 2019).
 Proinsulin contains a C subunit which can be proteolytically cleaved by
endopeptidases (pro-hormone convertase and carboxypeptidase) (Akinlade et
al., 2014).
 It was first isolated by Frederick G. Banting and Charles H. Best in 1921 at
Canada (Rhodes et al., 2005).
 Human Insulin Hormone (Humulin) is the first product of recombinant DNA
technology, first synthesized in 1978 by Genentech (Shinde et al., 2018).
4
Figure 5: A Model of Insulin Structure
Image source: Akinlade et al. (2014).
Figure 6: The founding fathers of modern Insulin research
Image source: diabetes-children.ca

5. Why humulin? & Why E.
coli?
 Up to the mid 80s, insulin
for treating diabetes – the
7th cause of death in the
US – was obtained from
bovine and porcine
pancreases (CBRA, 2019;
Tariq et al., 2018).
 Genetic mutations and
other incidences
triggering abnormalities
in amino acid sequences
may result in production
of inactive or unintended
proteins, say, insulin
(Tariq et al., 2018).
 This necessitates the
introduction of such
proteins (e.g. insulin) the
body is deficient in to
counterbalance that
lacuna (Baeshen et al.,
2014).
5
5
Figure 8: Humulin
Image Source: ScienceDirect (2019).
Escherichia coli is preferred
because of :
1. Its fast growth rate
2. Its amenability to genetic
manipulation
3. Its high level of rate of
synthesis of recombinant
proteins (Rodriguez et al.,
2014)
Figure 7: E. coli k12 is a prototype for
insulin production
Image Source: Sciencesource.com

6. Escherichia coli and Humulin
production
 The central dogma of
molecular biology applies
here.
 Additionally, a variety of
plamids exist that can be
introduced into
variegated strains of the
organism.
 The insulin precursors
(proinsulin) are produced
as inclusion bodies that
after solubilisation and
refolding yield the final
insulin (Akinlade et al.,
2014; Okafor, 2014).
 Yields are very attractive:
up to 30% of total
expressed protein
(Baeshan et al., 2014).
6
Figure 9: Why E. coli is preferred over other organisms for humulin
production.

7. Diagrammatic Methodology
7
Figure 10:
Stepwise
Methodolog
y for the
production
of
recombinant
humulin
Image
source:
eduhk.hk
7

8. Detailed Methodology
1. Obtain E. coli strains
having a selectable
marker
2. Construct the human
insulin gene
3. Grow E. coli in flasks,
and then in a fermenter
4. Harvest cells and isolate
inclusion bodies
5. Dissolve of inclusion
bodies
8
6. Renaturate the sample
(stirring, airing, pH
adjustment).
7. Perform Citraconylation
8. Carry out Tripsinisation
9. Elute insulin via Low-
pressure
chromatography on
DEAE.
10. Perform
decitraconylation
11. Precipitate insulin using
Adapted from: Zielinski et al. (2019).
12. Perfrom low
pressure
chromatography
13. Perform HPLC
14. Carry out
Sephadex G-25
Chromatography
15. Measure protein
levels
spectrophotometric
ally
16. Carryout UHPLC

9. Advantages of Humulin Production using E.
coli, Drawbacks and their Solutions.
 Drawback: Translational errors of ribosomal stalling due
to ‘rare codons’.
 Solution: Incorporation of extra copies of nrelevant
tRNAs, such as argU, ileY, and leuW tRNA genes, for
AGA/AGG (arginine), AUA (iso-leucine), and CUA
(leucine) respectively.
 Drawback: Demand for ‘short-acting’ insulin.
 Solution: Insulin analogs synthesis, such as Humalog.
 Drawback: Absence of post-translational modification
pathways in E. coli
 Solution: Incorporation of pathways from Campylobacter
jejuni
 Drawback: Strain ‘eccentricity’
 Solution: Use of stable strains
9
Advantages
Drawbacks and their
Solutions
Safety:
FDA
Approved
Accessibilit
y:
Worldwide
Supply
Affordability
Elimination
of Risk of
Allergies
Extirpation of
zoonoses
cross-transfer
risk
Market for
product expected
to reach $ 54
billion.
Sources: Baeshan et al. (2014), Zielinski et al. (2019); Change.org (2019);
9

10. Guessing what the future
holds.
 Novel delivery systems for humulin, e.g. insulin
pens, jet injectors, insulin pumps, inhalable
insulin, check insulin, insulin pills etc.
 Use of molecular chaperons such as DnaK and
DnaJ to prevent protein aggregation and
enhance proper, homologous folding.
 Production of refined products from the
biotechnology market triggerred by genetic
engineering-derived breakthroughs in
optimisation of humulin expression in E. coli
strains.
 Gene therapy
10
10
Sources: Baeshan et al. (2014), Tariqet al. (2019);
madehow.com (2019), etc.
Figure 11: An Insulin
Pen
Source: drugs.com

11. Conclusio
n
Human Insulin production using E. coli,
when fully applied in the Nigerian
research sphere, will contribute
immensely towards mitigating the
prevalence of diabetes, ameliorating the
disease burden and enhancing public
health.
11

12. References - I
Akinlade, A.T., Ogebra, A.O., Fasanmade, O.A. and Olameyegun, M.A. (2014). Serum C-peptide assay of
patients with hyperglycemic emergencies at the Lagos State University Teaching Hospital (LASUTH),
Ikeja. International Archives of Medicine, 7(50): 1-26.
Baeshen, N.A, Baeshen, M.N., Sheikh, A., Bora, R.S., Ahmed, M.M.M., Ramadan, H.A.I, Saini, K.S.,
Baeshan, N.A. and Redwan, E.M. (2014). Cell factories for insulin production. Microbial Cell Factories.
13(1): 1.
Baeshan, A.M., Al-Haejin, A.M., Bora, R.S., Ahmed, M.M.M.,Ramadan, H.A.I., Saini, K.S., Baeshen, N.A.
and Redwan, E.M. (2014). Production of Biopharmaceuticals in E. coli: Current Scenario and Future
Perspectives. Journal of Microbiology and Biotechnology, 25(7): 953–962.
California Biomedical Research Association (CBRA) (2019). Genetically Modified Organisms and
Biomedical Research. Available Online. Accessed 05:11:2019.
Change.org. (2019). We are against cow and pig insulin in india. Available online via:
https://www.change.org/p/ministry-of-health-we-are-against-cow-and-pig-insulin-in-india, accessed
06:11:2019.
Genome News Network (GNN) (2018). Genetics and Genomics Timeline. Available Online via:
www.genomenewsnetwork.org. Retrieved 5:11:2019.
Madehow.com (2019). How Products are Made – Insulin. Available online via:
www.madehow.com/Volume-7/insulin.html, accessed 6:11:2019.

13. References - II
Shinde, S.A., Chavhan, S.A., Sapkal, S.B. and Shrikhande, V.D. (2018). Recombinant DNA Technology and its
Applications: A Review. International Journal of MediPharm Research, 4(2): 79-88.
Sabir, A.A., Balarabe, S., Sani, A.A., Isezuo, A.S., Bello, K.S., Jimoh, A.O. and Iwuala, S.O. (2018). Prevalence of
diabetes mellitus and its risk factors among the suburban population of Northwest Nigeria Sahel Medical Journal,
20(4): 168-172.
Uloko, A.E., Musa, B.M., Ramalan, M.A., Gezawa, I.D., Puepet, F.H., Uloko, A.T., Borodo, M.M. and Sada, K.B.
(2018). Prevalence and Risk Factors for Diabetes Mellitus in Nigeria: A Systematic Review and Meta-Analysis.
Diabetes Therapy, 9:1307–1316.
Zielinski, M., Romanik-Chruścielewska, A., Mikiewicz, D., Łukasiewicz, N., Sokołowska, I., Antosik, J., Sobolewska-
Ruta, A., Bierczyńska-Krzysik, A., Zaleski, P. and Płucienniczak, A. (2019). Expression and purification of
recombinant human insulin from E. coli 20 strain. Protein Expression and Purification, 157: 63–69.
ScienceDirect (2019). Insulin Derivative. Available Online via: https://www.sciencedirect.com/topics/medicine-and-
dentistry/insulin-derivative; Short Acting Insulin. Available Online via
https://www.sciencedirect.com/topics/medicine-and-dentistry/short-acting-insulin, and Humulin. Available Online
via https://www.sciencedirect.com/topics/neuroscience/humulin, accessed 06:11:2019.
Rodriguez, V., Asenjo, J.A. and Andrews, B.A. (2014). Design and implementation of a high yield production system
for recombinant expression of peptides. Microbial Cell Factories, 13(65).
Tariq, F., Khan, M.A.U., Shahzad, S., Chaudry., W.B., Arif, A. and Gharib, G. (2019). Production of Remedial Proteins
through Genetically Modified Bacteria. Advancements in Life Sciences – International Quarterly Journal of
Biological Sciences.,5(2): 37-45.