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Cell factories for Insulin production

Microbial Cell Factories 2014, 13:141 doi:10.1186/s12934-014-0141-0
Nabih A Baeshen ([email protected])
Mohammed N Baeshen ([email protected])
Abdullah Sheikh ([email protected])
Roop S Bora ([email protected])
Mohamed Morsi M Ahmed ([email protected])
Hassan I Ramadan ([email protected])
Kulvinder Singh Saini ([email protected])
Elrashdy M Redwan ([email protected])
Sample

ISSN
Article type

1475-2859
Review

Submission date

29 June 2014

Acceptance date

16 September 2014

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http://www.microbialcellfactories.com/content/13/1/141

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Cell factories for insulin production

Nabih A Baeshen1
Email: [email protected]
Mohammed N Baeshen1
Email: [email protected]
Abdullah Sheikh1
Email: [email protected]
Roop S Bora1
Email: [email protected]
Mohamed Morsi M Ahmed1,2,*
Email: [email protected]
Hassan I Ramadan1,3
Email: [email protected]
Kulvinder Singh Saini1
Email: [email protected]
Elrashdy M Redwan1,4
Email: [email protected]
1

Department of Biological Sciences, Faculty of Science, King Abdulaziz

University, P.O. Box 80203, Jeddah 21589, Saudi Arabia

Nucleic Acids Research Department, Genetic Engineering and Biotechnology

Research Institute (GEBRI), City for Scientific Research and Technology
Applications, Alexandria, Egypt
3

Cell Biology Department, Genetic Engineering and Biotechnology Division,

National Research Centre, Tahrir St.Dokki, Cairo 12311, Egypt

Protein Research Department, Genetic Engineering and Biotechnology Research

Institute, City for Scientific Research and Applied Technology, New Borg ALArab, Alexandria, Egypt

Corresponding author. Nucleic Acids Research Department, Genetic

Engineering and Biotechnology Research Institute (GEBRI), City for Scientific
Research and Technology Applications, Alexandria, Egypt

Abstract
The rapid increase in the number of diabetic patients globally and exploration of alternate
insulin delivery methods such as inhalation or oral route that rely on higher doses, is bound to

escalate the demand for recombinant insulin in near future. Current manufacturing
technologies would be unable to meet the growing demand of affordable insulin due to
limitation in production capacity and high production cost. Manufacturing of therapeutic
recombinant proteins require an appropriate host organism with efficient machinery for
posttranslational modifications and protein refolding. Recombinant human insulin has been
produced predominantly using E. coli and Saccharomyces cerevisiae for therapeutic use in
human. We would focus in this review, on various approaches that can be exploited to
increase the production of a biologically active insulin and its analogues in E. coli and yeast.
Transgenic plants are also very attractive expression system, which can be exploited to
produce insulin in large quantities for therapeutic use in human. Plant-based expression
system hold tremendous potential for high-capacity production of insulin in very costeffective manner. Very high level of expression of biologically active proinsulin in seeds or
leaves with long-term stability, offers a low-cost technology for both injectable as well as
oral delivery of proinsulin.

Keywords
Recombinant insulin, E. coli, Yeast, Expression system, Posttranslational modifications,
Transgenic plants

Introduction
The pioneering work of Stanley Cohen and Herbert Boyer, who invented the technique of
DNA cloning, signaled the birth of genetic engineering, which allowed genes to transfer
among different biological species with ease [1]. Their discovery led to the development of
several recombinant proteins with therapeutic applications such as insulin and growth
hormone. Genes encoding human insulin and growth hormone were cloned and expressed in
E. coli in 1978 and 1979 respectively. The first licensed drug produced using recombinant
DNA technology was human insulin, which was developed by Genentech and licensed as
well as marketed by Eli Lilly in 1982.
There are more than 300 biopharmaceutical products including therapeutic proteins and
antibodies in the market with sales exceeding USD100 billion [2,3]. Therapeutic monoclonal
antibodies have captured the major market share (>USD18 billion) followed by the hormones
(>USD11 billion) and growth factors (>USD10 billion) [4]. Biopharmaceuticals approved by
the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) from
2004 to 2013 are (56%) largely derived from mammalian cells, (24%) Escherichia coli,
(13%) Saccharomyces cerevisiae, (3%) transgenic animal and plant cells and (4%) insect
cells as shown in Figure 1 [5-13]. At present, insulin is being produced predominantly in E.
coli and Saccharomyces cerevisiae for treatment of diabetic patients.
Figure 1 Percentage of Biopharmaceuticals produced in different expression systems [513].
Since the early 1920s, diabetic patients were treated with insulin, which was purified from
bovine or porcine pancreas. The development in the field of genetic engineering allowed the
production of insulin in E. coli and yeast, which have been approved for therapeutic
applications in human by FDA [14,15].

Nowadays, recombinant human insulin is mainly produced either in E. coli or Saccharomyces

cerevisiae. Using E. coli expression system, the insulin precursors (IP) are produced as
inclusion bodies and fully functional polypeptides are obtained finally by solubilization and
refolding procedures [16]. Yeast based expression system yield soluble insulin precursor (IP)
which is secreted into the culture supernatant [17-19]. Saccharomyces cerevisiae is the most
preferred and predominant yeast for large scale commercial production of insulin, however
several other alternate yeast strains have been explored for insulin production [20-24].
Besides, E.coli and yeast, mammalian cells, transgenic animals and plant expression systems
are also employed as a host for large-scale production of recombinant insulin [25-28].
The incidence of diabetes is increasing at an alarming rate and it has been speculated that the
number of diabetic patients worldwide would increase to approximately 300 million by the
year 2025 [29]. Consequently, the requirement for insulin will increase manifold
(approximately more than 16000 kg/ year) and the productivity of current insulin expression
system would not be sufficient to meet the future market demands. Efficient expression
systems for insulin production are also needed and novel route for insulin administration such
as oral or inhalation are to be developed.
Several recombinant protein based drugs, produced by various expression systems are
approved by FDA. Among prokaryotes, Escherichia coli has always been preferred for
production of recombinant proteins as it offered several advantages including high growth
rate, simple media requirement, easy to handle, high yield and very cost effective. However,
there are some disadvantages using E. coli expression system, such as loss of plasmid and
antibiotic property, unsolicited inducers for gene expression, intracellular accumulation of
heterologous proteins as inclusion bodies, improper protein refolding, lack of posttranslational modifications (including unable to form disulphide bonds), protein-mediated
metabolic burden and stress, endotoxin contamination, poor secretion, proteolytic digestion
and complexity in downstream process [30-32].
Among yeast strains, Saccharomyces cerevisiae, Hansenulla polymorpha and Pichia pastoris
are very commonly used for production of recombinant proteins [21,24,33-35]. Like E. coli,
they grow rapidly and are very easy to handle and amenable to various genetic manipulations.
Recombinant proteins produced in yeast are properly folded and glycosylated to a certain
extent similar to the one expressed in mammalian cells. Various human therapeutic proteins,
including therapeutic monoclonal antibodies are being produced in mammalian cell lines
such as Chinese hamster ovary (CHO) and Baby hamster kidney (BHK) cells. Recombinant
proteins expressed in mammalian cells are properly folded, glycosylated and generally yield a
functionally active protein [36]. However, the production cost of biopharmaceuticals using
mammalian expression system is very high due to expensive culture media. However, when
we look at the number of approved biopharmaceuticals by United States and/or the European
Union for 2013 recorded above average as compared to past five years. The mean rate of
approval is 13 as shown in the Figure 2 [5-13]. Remarkably, the number is same for both the
years 2009 and 2013.
Figure 2 Approval numbers of Biopharmaceuticals in United States and/or European
Union over the past six years with a trend line showing the mean approval rate [5-13].

Structure and function of insulin

The human insulin is comprised of 51 amino acids and has a molecular weight of 5808 Da. It
is produced by beta cells of the pancreas and plays a key role in regulating carbohydrate and
fat metabolism in the body. Insulin is synthesized as a single polypeptide known as
preproinsulin in pancreatic beta cells. Preproinsulin harbours a 24-residue signal peptide,
which directs the nascent polypeptide to the endoplasmic reticulum. The signal peptide is
cleaved as the polypeptide is translocated into the human of the endoplasmic reticulum
resulting in the formation of proinsulin. In the Endoplasmic reticulum, the proinsulin is
folded in proper confirmation with the formation of 3 disulphide bonds. Folded proinsulin is
then transported to the trans-Golgi network, where it is converted into active insulin by
cellular endopeptidases called as prohormone convertases (PC1 and PC2) and exoprotease
carboxypeptidase E. The endopeptidases cleaves at two positions, resulting in the release of a
fragment termed as C-peptide. The mature insulin, thus formed consists of an A-chain with
21 aminoacids and a B-chain containing 30 aminoacids and both polypeptides linked together
by two disulphide bonds. Besides, the A-chain has an intrachain disulphide bond [37,38].

E. coli expression system for production of insulin

E. coli is a preferred microorganism for large-scale production of recombinant proteins.
However, several disadvantages limit its use for production of recombinant
biopharmaceuticals. Various post-translational modifications (PTMs) such as glycosylation,
phosphorylation, proteolytic processing and formations of disulfide bonds which are very
crucial for biological activity, do not occur in E. coli [39,40]. N-linked glycosylation is the
most common posttranslational modification of proteins in eukaryotes. It has been discovered
that the bacterium Campylobacter jejuni possess the capability to glycosylate the proteins and
it was also shown that a functionally active N-glycosylation pathway could be transferred to
E. coli [41]. Although the structure of bacterial N-glycan is different from that observed in
eukaryotes, engineering of Campylobacter N-linked glycosylation pathway into E. coli,
provides an opportunity to express heterologous proteins in glycosylated form in E. coli.
Expression of Pglb oligosaccharyltransferase or (OTase) from C. jejuni in E. coli showed a
significant increase in glycopepetide yield [42,43]. Recently efforts has been made to produce
glycosylated proteins with substrates other than native and non-native to E. coli and C.jejuni
[44-48].
The codon usage of the heterologous protein also plays a major role in determining the
expression level of recombinant protein. If the codon usage of heterologous protein differs
significantly from the average codon usage of the E. coli host, it could result in very low
expression. Usually, the frequency of the codon usage reflects the abundance of their
corresponding tRNA. Therefore, significant differences in codon usage could result in
premature termination of translation, misincorporation of aminoacids and inhibition of
protein synthesis [49]. Expression of heterologous proteins in E. coli can be improved by
replacing codons that are rarely found in highly expressed E. coli genes with more favorable
major codons. Similarly, co-expression of the genes encoding for a number of the tRNA for
rare codon, may enhance the expression of heterologous proteins in E. coli. There are some
commercial E. coli strains available that encodes for tRNA for rare codons such as BL21
(DE3) CodonPlus-RIL, BL21 (DE3) CodonPlus-RP (Stratagene, USA) and Rosetta (DE3).
BL21 (DE3) CodonPlus-RIL harbors tRNA genes for rare codons like AGG, AGA (arginine),
AUA (isoleucine) and CUA (leucine). Similarly, Rosetta (DE3) strain harbors tRNA genes
for rare codons like AGG, AGA (arginine), CGG (arginine), AUA (isoleucine), CUA

(leucine), CCC (proline) and GGA (glycine). These rare codons have been associated with
low expression of proteins in E. coli, hence application of these genetically engineered E. coli
host strains may improve the expression level of heterologous proteins and thus might result
in higher yield of desired protein [50-52]. The use of protease-deficient E. coli strains, which
carry mutations that eliminate the production of proteases may also improve the yield of
recombinant protein by reducing proteolytic degradation. E. coli strain BL-21, is deficient in
two proteases encoded by the lon (cytoplasmic) and ompT (periplasmic) genes. Rather than
the external parameters, targeted methods such as modifications in protease or secretion
pathways can provide the insight into biology of recombinant proteins [53]. In E. coli,
complex and large therapeutic proteins can be secreted in periplasm as it provides an
oxidizing environment and help in forming disulphide bonds, which facilitate the proper
folding of recombinant proteins and likely to yield reliable N- terminus of expressed protein
[54]. Periplasm has advantages over cytoplasm in less protein concentration and proteolytic
activity, improve the production titer [55], and enhance the solubility of recombinant protein.
Altogether, with these advanced modifications and developments ease the process of target
protein production thus accelerating the drug development [56].
Heterologous proteins generally accumulate in E. coli as inclusion bodies, which comprise of
insoluble misfolded aggregates of proteins. Use of molecular chaperones may increase the
protein solubility and assist in proper folding of recombinant protein. Some of the chaperones
prevent aggregation of protein and some assist in refolding and solubilization of misfolded
proteins. The most important chaperones in E. coli are GroEL, GroES, DnaK, DnaJ, GrpE
and Trigger factor. These chaperones may be used singly, or in combination to enhance the
protein solubility in E. coli [57,58].
Recombinant human insulin was first produced in E. coli by Genentech in 1978, using a
approach that required the expression of chemically synthesized cDNA encoding for the
insulin A and B chains separately in E. coli [59]. After expressing independently, the two
chains are purified and co-incubated under optimum reaction conditions that promoted the
generation of intact and bioactive insulin by disulphide bond formation. The first commercial
recombinant insulin was developed for therapeutic use in human by this two-chain
combination procedure [60]. Another approach involves the expression of a single chemically
synthesized cDNA encoding for human proinsulin in E. coli followed by purification and
subsequent excision of C-peptide by proteolytic digestion. This approach was more efficient
and convenient for large scale production of therapeutic insulin as compared to the two chain
combination approach and has been used commercially since 1986 [60]. Eli Lilly followed
this technology to produce Humulin, the first recombinant insulin approved in 1982, for the
treatment of diabetic patients. These first generation recombinant insulins have an amino acid
sequence identical to native human insulin and are preferred over animal derived insulin
products [14]. However, advancement in the field of genetic engineering and development of
technology to chemically synthesize genes with altered nucleotide sequence, facilitated the
development of insulin analogues with altered amino acid sequence. It had been observed that
native insulin in commercial preparations usually exist in oligomeric form, as zinc-containing
hexamer due to very high concentration, but in blood, biologically active insulin is in
monomeric form [61]. Hence, this oligomeric complex should dissociate so that insulin can
be absorbed from the site of injection into the blood. Due to this, subcutaneously injected
recombinant insulin usually have a slow onset with peak plasma concentration after 2 hours
of injection and longer duration of action that last for 68 hours [62]. Hence, in order to
develop a fast- acting insulin analogue, it was required to modify the aminoacids residues
whose side chains are involved in dimer or oligomer formation. It has been shown that amino

acids residues in insulin B-chain particularly B8, 9,12, 13, 16 and 23-28 play critical role in
oligomerization [63,64]. Lispro, developed by Eli Lilly, was the first fast acting insulin
analogue to obtain regulatory approval in 1996, for therapeutic use [60]. Insulin Lispro is
engineered in such a way that it has similar amino acid sequence as the native insulin but has
an inversion of proline-lysine sequence at position 28 and 29 of the B-chain, which resulted
in reduced hydrophobic interactions and thus prevented dimer formations. For commercial
production of insulin Lispro, a synthetic cDNA encoding for Lys B28- Pro B29 human
proinsulin was expressed in E. coli and insulin Lispro was excised proteolytically from the
proinsulin by treating with trypsin and carboxypeptidase. Another rapid-acting insulin
analogue, produced in E. coli is Glulisine (Apidra) which was developed by Aventis
Pharmaceuticals and approved by US regulatory authorities in 2004. Insulin Glulisine have
been generated by replacing B3 asparagine by a lysine and B29 lysine replaced by glutamic
acid [14].
To avoid multiple injection, long-acting insulin analogues with prolonged duration of actions
have also generated. Insulin Glargine is one of such long-acting insulin analogues, which was
developed by Aventis Pharmaceuticals and approved by regulatory authorities of USA and
EU in 2000. Insulin Glargine was generated by replacing the C-terminal asparagine of the Achain with a glycine residue and the C-terminal of the B- chain was modified by adding two
arginine residues. These modifications resulted in increase of the isoelectric point (pI) from
5.4 to neutral values. Glargine was produced as proinsulin and expressed in E. coli and was
finally formulated at pH 4 in soluble form. However, after subcutaneous administration, it
precipitated due to neutral pH in the subcutaneous tissue. Resolubilization of insulin occur
slowly, resulting in longer duration for its release in the blood [14].

Yeast expression system for the production of insulin

Yeast is a preferred host for expression of various heterologous proteins that require posttranslational modifications for its biological activity. Yeast cell has the ability to carry out
numerous post-translational modifications such as phosphorylation, O-linked glycosylation,
N-linked glycosylation, acetylation and acylation. Recombinant proteins are expressed in
soluble form in yeast and properly folded in functionally active form. Production of
biopharmaceuticals using yeast expression system is also very cost effective and is amenable
to scale up using large bioreactors. However, one major concern for producing therapeutic
glycoprotein for human application is that yeast N-glycosylation is of the high-mannose type,
which confers a short half-life in vivo and hyperimmunogenicity and thus render the
therapeutic glycoprotein less effective. Various attempts have been made to humanize yeast
N-glycosylation pathways in order to produce therapeutic glycoproteins with humanized Nglycosylation structure [65].
The therapeutic proteins produced in yeast are specifically from Saccharomyces cerevisiae
and include hormones (insulin, insulin analogues, non-glycosylated human growth hormone
somatotropin, glucagon), vaccines (hepatitis B virus surface antigen), uprate oxidase from
Aspergillus flavus, granulocyte-macrophage colony stimulating factor, albumin, hirudin of
Hirudo medicinalis and human platelets derived growth factor [34]. Like E. coli, yeast
derived recombinant biopharmaceuticals majorly intended as therapeutics for infectious
diseases or endocrine, metabolic disorders. Alternate yeast strains, besides S. cerevisiae, are
being explored for large-scale production of biopharmaceuticals. Specifically, Pichia pastoris
has the ability to attain high cell densities by its robust methanol-inducible alcohol oxidase 1
(AOX1) promoter and simple developmental approaches contribute to high quality and

quantity of recombinant proteins production. In comparison to Saccharomyces cerevisiae,

Pichia pastoris provides a major advantage in the glycosylation of secreted proteins because
it does not hyperglycosylate the heterologous proteins. Both yeast strains have a majority of
N-linked glycosylation of the high-mannose type, but the length of the oligosaccharides chain
added to proteins in Pichia (around 814 mannose residues per side chain) is much shorter
than those expressed in Saccharomyces cerevisiae (approximately 50150 mannose residues
per side chain), suggesting that glycoproteins produced in Pichia pastoris may be more
suitable for therapeutic use in humans [66,67]. Moreover, very high level of expression of
heterologous proteins can be attained in Pichia pastoris, that might constitute about 30% of
total cellular protein which is very high as compared to S. cerevisiae [68,69]. Therefore,
Pichia pastoris can be an attractive alternate for large-scale production of recombinant
insulin and insulin analogues. Comparing the different insulin production systems where the
bacterial expression systems show higher average specific productivity and maximum
biomass concentrations are higher in yeast, the overall production space-time yield remains
similar as shown in Table 1 [70].
Table 1 Comparison of human insulin production systems [70]
Source
Destination of product
Biomass cell dry weight (g/l)

Typical spec. growth rate (1/h)

Typical spec. production rate
(mg/gh)
Product concentration (g/L)
Productivity (mg/l h)
Reference

E. coli
Cytoplasm
80, in bioreactor
with fed-batch
culture
0.08 0.12
14.2

E. coli
Secreted
1.2, in shake flask
with batch culture

S. cerevisiae
Secreted
5, in shake flask
with batch culture

not specified
3.4

< 0.33
0.21

P. pastoris
Secreted
59, in bioreactor
with fed-batch
culture
<0.03
0.375

4.34
1,085
[71]

0.009
4.01
[72]

0.075
1.04
[19]

3.075
17
[73]

Saccharomyces cerevisiae has been extensively used to produce recombinant human insulin
since early 1980s [17,18] and a large proportion of recombinant commercial insulins are
produced by this yeast expression system [19,74]. For efficient expression and secretion of
recombinant proinsulin in yeast, insulin construct was engineered to contain the native Achain and a B-chain lacking the C-terminal B30 threonine, either directly fused or linked via
a short synthetic C peptide (like AAK). The cDNA sequence encoding for this construct was
fused with -factor signal sequence of Saccharomyces cerevisiae for secreted expression of
proinsulin which gave yield upto 80 mg/ml of insulin. The single chain proinsulin was
purified and converted to active insulin by a trypsin-mediated transpeptidation reaction in
presence of threonine ester [19]. Besides native recombinant insulin, various insulin
analogues are also being produced in S. cerevisiae. Insulin Aspart is another fast-acting
insulin analogue, which was produced in S. cerevisiae, developed by Novo Nordisk and
approved by US FDA in 2001 for therapeutic use in human. Insulin Aspart was generated by
replacing proline residue at position 28 with aspartic acid in the B-chain. This genetic
modification resulted in an increase in inter-chain charge repulsion, decrease in selfassociation and thus causing rapid entry into the blood from the site of subcutaneous injection
[63,75].
Insulin Detemir is another recombinant long-acting insulin analogue that was commercially
produced in S. cerevisiae, developed by Novo Nordisk and approved for therapeutic use in
human in 2004 by European regulatory authorities. Recombinant Detemir have been
generated by removing the threonine residue at the 30 position of the B-chain, and a C14

fatty acid chain covalently attached to the lysine residue at the 29 position of the B-chain.
These genetic alterations resulted in the binding of insulin to albumin in plasma, which
ensured the slow and constant release of insulin and thus prolonging its duration of action up
to 24 hours [76-78].
Sacharomyces cerevisiae has been reported for the production of beyond 40 different
recombinant proteins [79]. A few of which related to diabetes are illustrated in Table 2, along
with different characteristics. Few proteins secreted extracellularly by Sacharomyces
cerevisiae with -factor leader sequence being repeatedly used for adequate production of
recombinant proteins. Furthermore, a synthetic leader sequence had been developed by
Kjeldsen and associates at Novo Nordisk for more efficient protein secretion in yeast [79,80].
Table 2 Some of the biopharmaceuticals produced by S. cerevisiae [2]
Type
Hormones

Protein
Insulin Precursor
Glucagon

Therapeutic application
Diabetes
Diabetes

Leader sequence
Synthetic
-Factor

Titer
80 mg/L
17.5 mg/L

Transgenic plants as host for insulin production

Transgenic plants have been utilized to produce recombinant proteins because of their
advantage of cost effectiveness, high quality protein processing, absence of human
pathogens, ease of production and presence of eukaryotic machinery for posttranslational
modifications. Initially, the human growth hormone was the recombinant protein product
extracted from transgenic tobacco plant [81]. After that, numerous different products have
developed from plants such as Hepatitis-B-Virus surface antigen, antibodies, industrial
proteins and milk proteins.
Recombinant human insulin has been successfully expressed and produced in oilseeds of
plant Arabidopsis thaliana [27]. This technology involved the targeted expression of insulin
in subcellular organelles known as oilbodies that allowed very high level of expression with
easy recovery of recombinant insulin. Oilbodies are storage organelles inside the oilseeds,
which comprises of hydrophobic triacylglycerol core encapsulated by phospholipid
membrane and an outer wall of proteins known as oleosins. Genetically engineered oil seeds
have been generated with recombinant protein specifically targeted to oilbodies as oleosin
fusion [27,82,83]. Then the oilbodies are easily separated from other seed components by
liquid-liquid phase separation, which reduced the number of chromatography steps required
to obtain purified insulin. It has been observed that insulin accumulated to high level in
transgenic seed (0.13% of total seed protein). Recombinant insulin was cleaved from the
oleosin fusion partner and matured with trypsin digestion following oil body purification to
yield a biologically active insulin. This study clearly demonstrated that expression of insulin
as oleosin fusion protein in plant allow accumulation of large amount of recombinant insulin
within the seed and also provide simple downstream purification by centrifugation i.e.
oilbody purification. Subsequent maturation to obtain biologically active insulin can be
accomplished using standard enzymatic methods currently used for commercial production of
insulin from E. coli and yeast. Oilseeds also act as a natural cellular warehouse, where
recombinant insulin can be stockpiled until required [27].
In another approach, transgenic plants have been generated, in which, tobacco and lettuce
chloroplasts were transformed with human proinsulin comprised of A, B and C-chains fused
with the cholera toxin B subunit [28]. It has been observed that, old tobacco leaves

accumulated proinsulin upto 47% of total leaf protein and similarly, old lettuce leaves
amassed proinsulin up to 53% of total leaf protein. Proinsulin stored in leaves of lettuce was
found to be very stable as up to 40% of proinsulin was detected even in senescent and dried
leaves as shown in Table 3. Proinsulin from tobacco leaves was extracted with 98% purity
and cleaved by Furin protease to release insulin peptides. Oral delivery of unprocessed
proinsulin encapsulated in plant cell or by injection into mice revealed lowering of blood
glucose levels similar to commercially available insulins. Based on the yield (3 mg of
proinsulin/gm of leaves), it was estimated that one acre of tobacco plantation could yield upto
20 million daily doses of insulin per year [28]. C-peptide of proinsulin, which is not present
in current commercially available insulin and insulin analogues derived from E. coli and S.
cereviciae, would be a great advantage in long-term treatment of diabetic complications such
as stimulation of nerve and renal functions. Very high level of expression of biologically
active proinsulin in tobacco and lettuce leaves and long-term stability in dried leaves offers a
reliable low-cost technology for both injectable as well as oral delivery of proinsulin.
Table 3 CTB cholera toxin B subunit proinsulin expression in tobacco and lettuce
chloroplasts [28]
Type of transgenic plant
Destination of the product
Percentage of Total leaf protein
Protein yield per gram of leaf tissue
Reference

Tobacco
Chloroplast
47%
2.92/gm
[28]

Lettuce
Chloroplast
53%
3.28/gm

Conclusion
Over the next 20 years, WHO has estimated that insulin sale would grow from $12 billion to
$54 billion globally. Dietary and lifestyle changes are causing dramatic increase in diabetes
incidence all over the world. Both Type I and Type II diabetic patients use insulin, however
late stage Type II diabetes patients require large doses of insulin as they develop insulin
resistance. The dramatic increase in the number of diabetic patients globally and exploration
of alternate insulin delivery methods such as inhalation or oral route is bound to escalate the
demand for recombinant insulin in near future. Current manufacturing technologies will not
be able to meet the growing demand of insulin due to limitation in production capacity and
high production cost. Recombinant human insulin is produced predominantly using E. coli
and Saccharomyces cerevisiae for therapeutic use in human. However, there is an upmost
need to increase the production by several fold of a biologically active insulin and its
analogues from E. coli and yeast using latest novel and efficient technologies. Another
strategy, using a different expression host other than E. coli and Saccharomyces cerevisiae
could be employed. Plant-based expression system hold tremendous potential for highcapacity production of insulin in very cost-effective manner. Very high level of expression of
biologically active proinsulin in seeds or leaves with long-term stability, offers a low-cost
technology for both injectable as well as oral delivery of proinsulin. Moreover, transgenic
seeds can also act as warehouse where recombinant insulin can be stockpiled until required.

Competing interest
The authors declare that they have no competing interests.

Authors contributions
All authors contributed in writing, design and figures of this review. All authors read and
approved the final manuscript.

Acknowledgments
This work was supported by the NSTIP strategic technologies program in the Kingdom of
Saudi Arabia (Project No. 10-BIO1257-03). The authors also, acknowledge assistance from
the Science & Technology Unit, Deanship of Scientific Research and Deanship of Graduate
Studies, King Abdulaziz University, Jeddah, KSA.

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