Abstract

The present study focuses on the heterologous expression and purification of a recombinant protein
(~55 kDa) in Escherichia coli BL21 (DE3) using IPTG induction and Ni-NTA affinity
chromatography. The research aimed to optimize culture conditions to achieve high-yield, soluble
expression of the target protein, essential for downstream biochemical and functional analyses.

LB media was prepared for primary and secondary cultures, and IPTG was used to induce protein
expression. Following bacterial growth monitoring through OD600 measurements, cell pellets were
harvested and lysed using a Tris-based buffer containing Triton X-100 and PMSF. Sonication was
performed for homogenization. SDS-PAGE analysis confirmed that the expressed protein was
primarily found in the soluble fraction. Subsequent purification using Ni-NTA resin yielded a highly
purified protein, as confirmed by the appearance of a single ~55 kDa band in the elution fractions and
minimal loss in flow-through and wash.

The outcome confirms that E. coli remains a robust host system for high-yield recombinant protein
production when coupled with proper expression and purification strategies. The successful
purification of the protein with high purity and solubility offers a reliable protocol for further
structural, immunological, and pharmaceutical applications.

1
 Introduction
1.1 Background of the study
There are many hosts used for the production of recombinant protein, but the preferred choice is
Escherichia coli because it is easy to culture, has a very short life cycle and is easily manipulated
genetically due to its well-known genetics. E. coli was the first host used to produce a recombinant
DNA (rDNA) biopharmaceutical, enabling the approval of Eli Lilly’s rDNA human insulin in 1982.
We also use E. coli host often to produce therapeutic protein to minimize the cost of production. In E.
coli, we face two main problems; (1) is difficult or little expression of a foreign gene and (2) is
solubility of recombinant proteins for over expression. Structural, functional and biochemical studies
of protein requires an ample amount of good quality protein. There is no universal strategy to solve
this problem but there are a few methods that can improve the level of expression or non-expression or
decreased expression of a gene of interest in E. coli. Factors such as mRNA’s secondary structure, a
protein’s intrinsic ability to fold, its solubility, preferential codon use, its toxicity, and its need for post
translational modification have influence on expression of foreign gene in E. coli (Redwan, 2006). At
least four strategies can useful for increasing the expression and solubility of over-expressed protein;
Changing the vector, changing the host strain, and adding of some chemicals during the induction or
co-expression of other genes may help in the proper folding of the desired protein. If the above
strategies are unsuccessful, then one can alter the gene sequence without changing the functional
domain. Gene sequence alteration include the removal of a signal peptide coding sequence,
optimisation of codon according to E. coli, deletion of hydrophobic patch coding sequences, and
prevention of stable mRNA secondary structure formation by changing the sequence which forms
secondary structure etc. The main focus of this review is to describe various strategies for the
expression of foreign genes/proteins in E. coli with minimum difficulty.

1.2 Recombinant protein expression

Recombinant proteins are produced via recombinant DNA technology: They are encoded by
recombinant DNA clones into an expression vector. Subsequent transfer of the vector and the gene of
interest in a suitable host results in gene expression, transcription and translation of mRNA (messenger
RNA) to proteins.
The field of recombinant protein production is continuously evolving thanks to the culmination of
research dating back to the early discoveries in DNA and protein synthesis to genetic engineering
optimizations and breakthroughs in molecular biology. Today, recombinant technology is responsible
for most of the protein therapeutics on the market, which are developed for cancer, immune disorders
and other diseases.
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 Figure 1.1: Recombinant proteins
Benefits of recombinant proteins
Recombinant protein expression has benefits over the conventional, historical methods. Prior to the
rise of molecular biology and high-throughput recombinant protein technology, proteins had to be
purified from animal sources. Recombinant protein expression is defined at the sequence level,
therefore enabling high batch-to-batch reproducibility and is associated with fast and simple
workflows. Historical methods would have purified protein from different sources, which was not only
laborious, time-consuming and required large amounts of animal organs but also prone to
contamination from other products that hampered the purity of the final manufactured product.
One example is insulin. Prior to the approval of the first biosynthetic human insulin product in 1982,
and in concert with advancements in chromatographic processing, companies were able to produce
purer insulin. The caveat of the method was the tremendous quantities of animal pancreases needed,
which originated from cows or pigs. Using these methods, glands from 23,500 animals were needed to
produce sufficient insulin to treat 750 patients with diabetes for a period of one year. 3 Another
significant factor that affected insulin was the availability of animal glands. The unsustainability of
these methods was exemplified in the 1970s when the availability of animal glands declined, and the
cost of beef and pork fluctuated.
Major benefits of recombinant proteins are:
 High Purity: Recombinant proteins can be produced with a high degree of purity, reducing the
risk of contamination or impurities in experimental applications.

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  Customization: Researchers can design and produce recombinant proteins with desired
specificities, such as modifications, tags, or mutations to suit their experimental needs.

 Scalability: The possibility to scale up recombinant protein production makes it suitable for
large-scale industrial and therapeutic applications.

 Consistency: The production of recombinant proteins can be tightly controlled, ensuring

consistent quality and reproducibility in experiments, but also in drug manufacturing requiring
large quantities.

 Cost-effectiveness: Compared to traditional methods that obtain proteins from natural sources,
recombinant protein production can be more cost-effective, especially for rare or complex
proteins.

Due to their manifold advantages, recombinant proteins are widely used tools all over the scientific
landscape. For instance, researchers can design a recombinant protein as an antibody, which can bind
to one specific target antigen in the body. This makes them unique weapons in the fight against several
cancer types and other diseases.
1.3 Importance of Escherichia coli as a host system
Escherichia coli is a Gram-negative, facultative anaerobic bacterium originally discovered in the
human colon in 1885 by German bacteriologist Theodor Escherich (Feng et al., 2002). Owing to
extensive investigation and development, E. coli has become the best characterized organism on earth,
the workhorse in molecular biology laboratories, and one of the most important organisms used in
industry. Because of its rapid growth, easy culture conditions, metabolic plasticity, wealth of
biochemical and physiological knowledge, and the plethora of tools for genetic and genomic
engineering, E. coli has also become one of the best host organisms for metabolic engineering and
synthetic biology. Commonly used E. coli strains are generally considered harmless. Laboratory
strains, such as K-12, are classified as Risk Group 1 for lack of O-antigens, virulence factors,
colonization factors, and association with disease in healthy human adults. These non-pathogenic
strains have been widely used for productions of pharmaceuticals, food, chemicals, and fuels.
E. coli by no means is the most versatile organisms for industrial purposes. However, the large body of
knowledge in E. coli biochemistry, physiology, and genetics has enabled rapid progress in E.
coli metabolic engineering and synthetic biology. As such, many previously perceived limitations have
been overcome, and new phenotypes have been engineered in E. coli that surpass the traditional native
producers. For example, E. coli has been engineered for industrial production of amino acids which

4
has traditionally been produced from the natural producer Corynebacterium glutamicum (Gusyatiner et
al., 2018). E. coli production of n-butanol has also been demonstrated to the level similar to that
produced in Clostridia (Shen et al., 2011, Ohtake et al., 2017). In cases where no natural producers
exist, E. coli has been among the top choices as the host for metabolic engineering. In addition to
serving as a proof-of-concept model organism, E. coli has been used extensively as an industrial
producer. Lysine, 1,3-propanediol (PDO) and 1,4 butandiol (Burgard et al., 2016) productions are
prominent and successful examples. As such, E. coli is clearly the most preferred prokaryote for both
laboratory and industrial applications.
1.4 Challenges in Recombinant Protein Expression Using E. coli and Other
Expression Systems

Recombinant protein expression is a cornerstone of modern biotechnology, enabling the production of

proteins for research, therapeutic, and industrial applications. Among various host organisms,
Escherichia coli (E. coli) remains the most commonly used due to its well-characterized genetics,
rapid growth rate, cost-effectiveness, and ability to produce high yields of protein. However, despite its
widespread use, expressing recombinant proteins in E. coli presents multiple challenges that can hinder
yield, activity, and solubility of the target protein.

One of the primary issues with E. coli is the limited ability to properly fold complex eukaryotic
proteins, especially those requiring post-translational modifications like glycosylation or disulfide
bond formation. This often results in the formation of insoluble aggregates known as inclusion bodies,
requiring subsequent solubilization and refolding steps that are laborious and inefficient
(Hashemzadeh et al., 2021). Additionally, codon usage bias, mRNA secondary structures, protein
toxicity, and poor secretion pathways can negatively impact expression levels (Redwan, 2006; Hayat
et al., 2018).

Strategies such as co-expression with chaperones, fusion with solubility-enhancing tags (e.g., MBP,
GST), codon optimization, and adjustment of induction parameters (e.g., temperature, IPTG
concentration) have been developed to mitigate these problems (Jeong et al., 2014; Luo et al., 2016).
Nevertheless, these solutions are often protein-specific and require empirical optimization.

Other expression systems, such as yeast (Pichia pastoris), insect (baculovirus), and mammalian cells,
offer advantages like eukaryotic folding machinery and glycosylation capabilities, making them
suitable for producing more complex proteins. However, these systems come with trade-offs such as
higher cost, longer cultivation times, and increased complexity in genetic manipulation and scale-up

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processes (Nag et al., 2022; Tripathi, 2016).

In conclusion, while E. coli remains a powerful and preferred system for recombinant protein
expression, overcoming its limitations requires tailored strategies. Choosing an appropriate expression
system—prokaryotic or eukaryotic—depends on the nature of the target protein and the intended
downstream application.

6
 Review of Literature

The purpose of this work is to create a set of expression vectors for rapid screening of optimal
conditions for the production of recombinant proteins prone to aggregation in E. coli in soluble form.
The work used modern genetic engineering methods, including optimization and de novo synthesis of
nucleotide sequences encoding helper polypeptides. For the production and chromatographic
purification of proteins, the standard strain BL21(DE3) and the affinity chromatography method using
a metal chelate sorbent were used. As a result, a set of nine vectors with helper polypeptides was
obtained to increase solubility and increase product yield during the production of recombinant
proteins in E. coli cells. The efficiency of obtaining protein drugs prone to molecular aggregation
using this set of vectors has been demonstrated by the example of three cytokines: interleukin-31 (IL-
31), interleukin-33 (IL-33), and transforming growth factor beta (TGF-beta1). The resulting set allows
for rapid selection of a suitable helper polypeptide as well as optimization of conditions for obtaining a
soluble form of recombinant proteins in E. coli during the pharmaceutical development of therapeutic
drugs. Zakharova et al., (2024)
With the development of recombinant DNA technology in the 1980s, the heterologous expression of
proteins has emerged as a valuable tool for researchers and pharmaceutical scientists, as it is generally
challenging to obtain satisfactory yields from natural sources. Several prokaryotic and eukaryotic
systems, including bacteria, yeast, insect, and mammalian platforms, have been developed to produce
native-like proteins of an organism on a laboratory-scale and industrial-scale settings. E. coli has been
the most widely used bacteria for the production of recombinant proteins due to its low cost, well-
established cellular biochemistry and genetics, rapid growth, and good productivity, and it has become
the most popular expression platform. However, optimizing the protocols for adequate expression and
solubility remains a major disadvantage of this system. In this chapter, we discuss the approaches and
methodologies to optimize the protein expression and solubility in E. coli and examine the
troubleshooting practices for obtaining large quantities of soluble and stable recombinant proteins. Nag
et al., (2022)
Due to its high expression capability, recombination of Escherichia coli and pET vector has become
the bioengineering preferred expression system. Because β-lactamases mediate bacterial antimicrobial
resistance, these enzymes have a substantial clinical impact. Using the E. coli expression system,
several kinds of β-lactamases have been produced. However, previous studies have been focused on
characterizing target β-lactamases, and the effects of cultivation and induction conditions on the
expression efficiency of target enzymes were not addressed. Using pET-28a as the cloning vector
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and E. coli BL21(DE3) as the expression host, this study originally elucidated the effects of IPTG
concentration, culture temperature, induction time, and restriction sites on recombinant β-lactamase
expression. Moreover, the effects of the target protein length and the 6 × His-tag fusion position on
enzyme purification were also explored, and consequently, this study yielded several important
findings. (i) Only the signal peptide–detached recombinant β-lactamase could exist in a soluble form.
(ii) Low-temperature induction was beneficial for soluble β-lactamase expression. (iii) The closer to
the rbs the selected restriction site was, the more difficult it was to express soluble β-lactamase. (iv)
The short-chain recombinant protein and the protein with His-tag fused at its C-terminus showed high
affinity to the Ni2+ column. Based on our findings, researchers can easily design an effective program
for the high production of soluble recombinant β-lactamases to facilitate other related studies. Li et al.,
(2022)
The expression of fusion (F) protein of Newcastle Disease Virus (NDV) from Indonesian isolates in E.
coli BL21(DE3) by IPTG induction have shown that the sample was a part of F gene of NDV from
Galur, Kulon Progo, Yogyakarta, Indonesia (0663/04/2013) with a molecular size of 600 bp that was
synthesized and inserted into pBT7-N-His expression vector. The recombinant F protein with
molecular weight of 25.6 kDa was successfully expressed in E. coli BL21(DE3), purified using Ni-
NTA magnetic silica beads, and confirmed by western blotting. Optimization of expression showed
that recombinant F protein was optimally expressed by induction of 1.0 mM IPTG when the cells
reached OD600 = 0.6. The induction duration was 8 h. B-cell epitopes prediction showed that F protein
possessed four epitopes that possibly recognized by B-cell. Since recombinant F protein was
considered to possess immunogenicity, its potency as a candidate of NDV vaccine agent should be
investigated in the future. Wulanjati et al., (2021)
That the Recombinant protein production for medical, academic, or industrial applications is essential
for our current life. Recombinant proteins are obtained mainly through microbial fermentation,
with Escherichia coli being the host most used. In spite of that, some problems are associated with the
production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic
burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription
of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological
processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of
different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number
pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21
wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources.
Results showed that metabolic burden associated with transcription and translation of foreign genes

8
involves a decrease in recombinant protein expression. It is necessary to find a balance between
plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The
results obtained represent an important advance on the most suitable expression system to improve
both the quantity and quality of recombinant proteins in bioproduction engineering. Lozano Terol et
al., (2021)
Escherichia coli has been most widely used for production of the recombinant proteins. Over-
expression of the recombinant proteins is the mainspring of the inclusion bodies formation. The
refolding of these proteins into bioactive forms is cumbersome and partly time-consuming. In the
present study, we reviewed and discussed most issues regarding the recovery of “classical inclusion
bodies” by focusing on our previous experiences. Performing proper methods of expression,
solubilization, refolding and final purification of these proteins, would make it possible to recover
higher amounts of proteins into the native form with appropriate conformation. Generally, providing
mild conditions and proper refolding buffers, would lead to recover more than 40% of inclusion bodies
into bioactive and native conformation. Hashemzadeh et al., (2021)
The Variable Heavy-chain Homodimer (VHH) or Nanobody is a recombinant dromedary antibody
fragment which is classified as the smallest antibody fragments with the highest binding affinity and
specificity of the original whole antibody. In this study the Expression of Nanobodies in E. coli WK6
cell periplasm was performed. The protein expression and purity was and analyzed by Affinity
Chromatography, SDS PAGE and Western Blot. Upon elution with Imidazole, the concentrations
observed using the OD280 nm of the eluted fractions EI, E2 and E3 were observed to be 0.42 μg/ml,
0.13 μg/ml and −0.46 μg/ml respectively. This gives an Antilog of 7.88 kDa which showed the
calculated molecular size of our band. The SDS-PAGE gel reading was confirmed using Western blot
analysis and illustrated as the specific binding of the mouse Anti-His antibody to the Histidine tag of
the Nanobody. The Nanobody protein expression was then analyzed further with western blotting
showed a strong signal at the region corresponding to the 15 kDa marker indicating presence of the
Nanobody gene. This was taken as further confirmation of the protein expression from the bacterial
cells. Jember (2021)
The Enterotoxigenic Escherichia coli is a common cause of diarrhea in animals. The development of
vaccines against enterotoxins can effectively control the infection. A previously constructed a
recombinant antigen SLS fused by STa, LTB and STb enterotoxin have shown a high immunogenicity
in mice. Herein, we evaluated the expression of SLS in three different E. coli cells with corresponding
plasmids. SLS proteins expressed in E. coli BL21 (DE3) and Rosetta-gami B (DE3) were aggregated
as inclusion bodies, and the proteins solubility were not obviously promoted in low temperature

9
combined with adjustment of inducer concentration. In contrast, SLS protein with maltose-binding
protein (MBP) yielded from TB1 (DE3) cells were partially soluble. After increasing the IPTG
concentration in the medium up to 2 mM and incubating at 37 ℃ for 4 h, the soluble protein yield
reached the highest level (4.533 mg/0.2 L culture), which was significantly higher than the expression
of SLS protein in Rosetta-gami B (DE3) (P < 0.05). Therefore, the TB1-pMAL expression system can
be used for mass extraction and purification of SLS antigen prior to measuring its immunogenicity in
pregnant mammals. Zhao et al., (2021)
Currently number of people with diabetes is estimated to be over 370 million, in 2030 it will increase
to 552 million. In Poland, the number of people with diabetes is estimated to be 3.5 million (9.1%).
According to the estimates of the International Diabetes Federation, the percentage of patients in the
adult Polish population will increase to around 11% over the next 20 years. Despite the appearance of
insulin analogues on the pharmaceutical market, insulin delivery is still the most effective method of
pharmacotherapy in cases of extremely high hyperglycemia. A new bacterial host strain (Escherichia
coli 20) was obtained at the Institute of Biotechnology and Antibiotics and a new pIBAINS expression
vector was constructed that provides greater efficiency in the production of recombinant human
insulin. In the IBA Bioengineering Department, successful attempts were made to produce
recombinant human insulin on a laboratory and quarter-technical scale, and several batches were
performed on a semi-technical scale. The production process has been divided into several stages: 1.
biosynthesis of insulin in the fermenter, 2. isolation, purification and dissolution of inclusion bodies, 3.
protein renaturation, 4. enzymatic reaction with trypsin, 5. multi-stage purification of insulin using
low-pressure and HPLC techniques. At each stage of insulin production, qualitative and quantitative
analyses were performed to confirm identity and purity. In particular, the molecular weight of insulin,
the amount of insulin and the content of protein impurities were studied. The results of these
experiments are presented in this work. Zieliński et al., (2019)
In a study, using bacterial surface display of recombinant proteins and ability of sortase A in site-
specifically cleavage of the amino acid sequences, a novel method for one-step purification of
recombinant proteins was developed. Using computational program tools, a chimeric protein
containing a metallothionein (mt) and chitin binding domain (ChBD) was attached to the C-terminal
domain of the truncated outer membrane protein A (Lpp′-ompA) using sortase recognition site (amino
acid residues: LPQTG) as a separator. The structure of the chimeric protein was simulated using
molecular dynamics to determine if the LPQTG motif is accessible to the sortase active site. The
designed chimeric protein was expressed and purified. The purified chimeric protein was also
displayed on the surface of E. coli cells. Both purified chimeric protein and the E. coli cells displaying

10
Lpp′-ompA-mt-ChBD carrier protein were then treated with sortase to evaluate the efficiency of
sortase-mediated cleavage of purified chimeric protein as well as surface displayed-chimeric protein. It
is shown that mt-ChBD protein was successfully cleaved and dissociated from Lpp′-ompA carrier and
released into the medium after treatment with sortase in both recombinant protein and surface
displayed-chimeric protein. The experimental results confirmed the molecular dynamics analysis
results. The presented method could be regarded as a novel strategy for one step expression and
purification of recombinant proteins. Fasehee et al., (2018)
Host, vector, and culture conditions (including cultivation media) are considered among the three main
elements contributing to a successful production of recombinant proteins. Accordingly, one of the
most common hosts to produce recombinant therapeutic proteins is Escherichia coli. A comprehensive
literature review was performed to identify important factors affecting production of recombinant
proteins in Escherichia coli. Results: Escherichia coli is taken into account as the easiest, quickest, and
cheapest host with a fully known genome. Thus, numerous modifications have been carried out on
Escherichia coli to optimize it as a good candidate for protein expression and; as a result, several
engineered strains of Escherichia coli have been designed. In general; host strain, vector, and
cultivation parameters are recognized as crucial ones determining success of recombinant protein
expression in Escherichia coli. In this review, the role of host, vector, and culture conditions along
with current pros and cons of different types of these factors leading to success of recombinant protein
expression in Escherichia coli were discussed. Successful protein expression in Escherichia coli
necessitates a broad knowledge about physicochemical properties of recombinant proteins, selection
among common strains of Escherichia coli and vectors, as well as factors related to media including
time, temperature, and inducer. Hayat et al., (2018)
Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for
recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion
protein could be expressed in soluble form and purified to near homogeneity in a single step from
Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’)
pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the
level of a recombinant protein expression in soluble form and its yield of recovery during each
purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-
Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined
method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved
recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a
new addition to the arsenal of existing affinity tags for recombinant protein expression and

11
purification, and is particularly useful where soluble expression and high degree of purification are at
stake. Luo et al., (2016)
Production of recombinant proteins in Escherichia coli has been improved considerably through the
use of fusion proteins, because they increase protein solubility and facilitate purification via affinity
chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and
purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on
the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-
terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified.
GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the
intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein
production in vivo showed that CusF produces large amounts of soluble protein with low levels of
inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF
contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins
can be purified with readily available IMAC resins charged with this metal ion, producing pure
proteins after purification and tag removal. We therefore recommend the use of CusF as a viable
alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant
proteins in E. coli. Cantu-Bustos et al., (2016)
Development of successful production strategies for recombinant proteins is a challenging task due to
problems associated with the scale-up of bioprocesses. Recent developments in fermentation
technology have provided a suitable platform to produce bioproducts for the development of
diagnostics, therapeutics and vaccines for bacterial, viral and other diseases. A bacterial system is the
commonly used expression system for production of recombinant products such as proteins, enzymes,
and antibodies. Escherichia coli is one of the most commonly used bacterial hosts for the production
of these products. A variety of recombinant proteins has been produced and cultivation studies have
been carried out at small scale. However, the majority of the recombinant proteins are not amenable
directly to large scale production processes due to various factors. To meet the growing demand of
potential bioproducts, there is a need to produce these products in large quantity employing
fermentation processes. Development of a suitable purification strategy is yet another important step to
recover biologically active products. The present paper reviews the current developments in
production and purification of recombinant products from E. coli. The bioproducts obtained after
fermentation and purification processes may fulfill the requirements for the development of various
diagnostics, therapeutics, or prophylactic agents against different kinds of diseases. Tripathi (2016)
Escherichia coli, is one of the most widely preferred organism for the production of recombinant

12
protein. Most of the FDA approved therapeutic proteins are produced in E. coli. The well-established
cell factory of E. coli makes it a perfect heterologous system of choice for the production of
recombinant proteins. In recent years, several advances have been taken place for modifying these cell
factories for easy production of therapeutic proteins with utmost precision. Several molecular tools and
protocols are available in hand for the high level protein production in heterologous expression system.
Adapting the best strategy for producing recombinant proteins in E. coli can be obtained using several
approaches. Combination of strategies will work well to enhance the expression and stability of
produced protein. In this review we try to collate different strategies and approaches that can enhance
the production as well as the stability of proteins expressed in E. coli. Joseph et al., (2015)
Protein purification of recombinant proteins was based on a variety of standard chromatographic
methods and approaches, many of which were described and mentioned throughout Current Protocols
in Protein Science. In the interim, there has been a shift toward an almost universal usage of the
affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can
complicate producing proteins under regulatory conditions. Regardless of the protein expression
system, questions are asked as to which and how many affinity tags to use, where to attach them in the
protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly
address some of these issues. Also, although this overview focuses on E.coli, protein expression and
purification, other commonly used expression systems are mentioned and, apart from cell-breakage
methods, protein purification methods and strategies are essentially the same. Wingfield (2015)
Bacterial cells can be engineered to express non-native genes, resulting in the production of,
recombinant proteins, which have various biotechnological and pharmaceutical applications. In
eukaryotes, such as yeast or mammalian cells, which have large genomes, a higher recombinant
protein expression can be troublesome. Comparatively, in the Escherichia coli (E. coli) expression
system, although the expression is induced with isopropyl β-d-1-thiogalactopyranoside (IPTG), studies
have shown low expression levels of proteins. Irrespective of the purpose of protein production, the
production process requires the accomplishment of three individual factors: expression, solubilization
and purification. Although several efforts, including changing the host, vector, culture parameters of
the recombinant host strain, co-expression of other genes and changing of the gene sequences, have
been directed towards enhancing recombinant protein expression, the protein expression is still
considered as a significant limiting step. Our protocol explains a simple method to enhance the
recombinant protein expression that we have optimized using several unrelated proteins. It works with
both T5 and T7 promoters. Chhetri et al., (2015)
The Human erythropoietin (hEpo) is an essential regulator of erythrocyte production that induces the

13
division and differentiation of erythroid progenitor cells in the bone marrow into mature erythrocytes.
It is widely used for the treatment of anemia resulting from chronic kidney disease, chemotherapy, and
cancer-related therapies. Active hEpo, and hEpo analogs, have been purified primarily from
mammalian cells, which has several disadvantages, including low yields and high production costs.
Although an Escherichia coli (E. coli) expression system may provide economic production of
therapeutic proteins, it has not been used for the production of recombinant hEpo (rhEpo) because it
aggregates in inclusion bodies in the E. coli cytoplasm and is not modified post-translationally. We
investigated the soluble overexpression of active rhEpo with various protein tags in E. coli, and found
out that several tags increased the solubility of rhEpo. Among them maltose binding protein (MBP)-
tagged rhEpo was purified using affinity and gel filtration columns. Non-denaturing electrophoresis
and MALDI-TOF MS analysis demonstrated that the purified rhEpo had two intra-disulfide bonds
identical to those of the native hEpo. An in vitro proliferation assay showed that rhEpo purified from
E. coli had similar biological activity as rhEpo derived from CHO cells. Therefore, we report for the
first time that active rhEpo was overexpressed as a soluble form in the cytoplasm of E. coli and
purified it in simple purification steps. We hope that our results offer opportunities for progress in
rhEpo therapeutics. Jeong et al., (2014)

14
Objectives of the study:
1. To optimize expression conditions of antigen for maximum protein yield.
2. To validate the presence of recombinant antigen.
3. To isolate and purity the recombinant antigen.

15
 Chapter-3
Materials and Methods

1. Media Preparation
1. Primary Culture: For each experiment, a primary culture was initiated in 30 ml
of Luria-Bertani (LB) media. In Experiment 1, 0.465g of LB media was used ,
while in Experiments 2 and 3, 0.75g of LB media was used.
2. Secondary Culture: Secondary cultures were grown in 250 ml of LB media. For
Experiment 1, 3.875g of LB media was used , and for Experiments 2 and 3, 6.25g
of LB media was used.
3. Sterilization: All prepared media were sterilized by autoclaving at 121°C for 1
hour.
1.2 Bacterial Culture and Induction
 Primary Culture: Each 30 ml primary culture flask was inoculated with 40µl of
a glycerol stock and supplemented with 30µL of filtered kanamycin. The flasks
were then incubated for 16 hours in a shaking incubator at 37°C and 250 rpm.
 Secondary Culture: Each 250 ml secondary culture flask was inoculated with 6
ml of the primary culture and supplemented with 250µl of filtered Kanamycin.
The flasks were incubated at 37°C with shaking at 250 rpm.
 And in Experiments 1 and 3 50 ml feed of 5x concentrated media was added to
the secondary culture during the growth phase.
 Induction: Protein expression was induced by adding 125µl of IPTG to the
secondary cultures when the optical density (OD) reached the desired level.
Following induction, the incubation temperature was reduced to 25°C, while
shaking was maintained at 250 rpm for 16 hours.
1.3 Optical Density Measurement
The growth of the bacterial cultures was monitored by measuring the optical density
at a wavelength of 600 nm (OD600). For measurements where high cell density was
expected, samples were diluted 1:10 (300µl sample in 2700µl distilled water).

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 Chapter-3
2. Cell Harvesting
The bacterial cultures were divided into 45 ml aliquots in 50 ml falcon tubes. The
cells were harvested by centrifuging the tubes at 900 rpm for 30 minutes at 4°C.
After centrifugation, the supernatant was discarded, and the weight of the cell pellet in
each tube was determined by subtracting the weight of an empty falcon tube.
3.Cell Lysis and Homogenization
Table 1. A fresh 20 ml lysis buffer was prepared containing
Component Concentration/ Volume
NaCl 590mM
Tris 50mM (4.23ml)
5% Glycerol 1.25ml
1% Triton 200µl
1mM PMSF(from 100mM stock) 200µl
Autoclave water 19.02ml (to make upto 20ml)

 Cell Resuspension: The cell pellets were resuspended by adding a small volume of
lysis buffer and vortexing. The resulting suspension was collected in a clean falcon
tube on ice. This process was repeated until the pellet was completely dissolved.
4.Homogenization:
The resuspended cell solution was homogenized using a probe sonicator. The
sonication parameters were set to an ON time of 20 seconds, an OFF time of 15
seconds, a run time of 40 minutes, and a power rate of 30%. The sample was kept on
ice during sonication. The process was continued for approximately 15 minutes until
the sample turned from turbid to partially translucent. The homogenized sample was
then centrifuged at 11,000 rpm for 20 minutes at 4°C. The resulting supernatant and
pellet were stored at -20°C.

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5.SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Chapter-3

 Buffer Preparation

Table 2. Standard buffers for SDS-PAGE was prepared including

Buffer/ Reagent Preparation
1X100 SDS Running Buffer 100ml 10X SDS buffer was dissolved in
900ml distilled water

30% Acrylamide and Bis-Acrylamide 14.5g Acrylamide and 0.5g Bis-

Solution Acrylamide dissolved in 50ml distilled
water

Tris-HCL(1.5M; ph 8.8) 9.085g of Tris base was weighed and

dissolved in 30ml distilled water, and ph
was set to 8.8 using 5N HCL. And the
volume was made upto 50ml

Tris-HCL(0.5M; ph 6.8) 3.028g of Tris base was weighed and

dissolved in 30ml distilled water, and ph
was set to 6.8 using 5N HCL. And the
volume was made upto 50ml

10% SDS – 5g of SDS 5g of SDS was dissolved in 50ml

distilled water

10% APS 10mg APS was dissolved in 1ml

distilled water

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 Gel Preparation: Chapter-3
Table 3. Resolving gel composition
Solution Component Component volume(ml)/gel mold volume
of
Distilled Water 4.0
30% Acrylamid 3.3
1.5M Tris (pH 8.8) 2.5
10% SDS 0.1
10% APS 0.1
TEMED 0.004

Table 4. Stacking gel composition

Solution Component Component volume(ml)/gel mold volume
of
Distilled Water 1.4
30% Acrylamid 0.33
1.5M Tris (pH 8.8) 0.25
10% SDS 0.02
10% APS 0.02
TEMED 0.002

 Resolving and stacking gels were prepared and cast between glass plates. Propanol
was used to overlay the resolving gel to ensure a flat surface and prevent drying.

 Sample Preparation: Uninduced and induced cell samples were collected. The
pellets were resuspended in 1X PBS. The sonicated pellet was dissolved in lysis
buffer.

 Gel Loading and Running: Remove the comb carefully and the gel was set up in the
running apparatus along with a dummy plate. Running buffer was filled between the
plates and sample were loaded

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 Table 5. Sample was Loaded Chapter-3
Sample Name Volume of Sample + Dye
Ladder 3µl
Uninduced A 20µl+5µl,dye
Induced A 20µl+5µl,dye
Supernatant A 20µl+5µl,dye
Pellet A 20µl+5µl,dye
1:10 Induced A 20µl+5µl,dye
1:10 Supernatant A 20µl+5µl,dye
1:100 Induced A 20µl+5µl,dye
1:100 Supernatant A 20µl+5µl,dye

 Staining and Destaining: After electrophoresis, the gels were washed with water and
stained with Coomassie Brilliant Blue solution for 30 minutes on a rocker. The gels
were then destained overnight in a destaining solution with gentle agitation.
6. Affinity Chromatography
 Buffers: Equilibration Buffer: 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM Imidazole
Wash Buffer: Same as equilibration buffer with 20–30 mM Imidazole
Elution Buffer: Same base buffer with 250 mM Imidazole
 Column Preparation: Ni-NTA resin equilibrated with 5 column volumes of equilibration
buffer.
 Loading Sample: Supernatant (soluble lysate) loaded onto the column using a syringe.
Flow-through collected.
 Washing: Column washed with 10–15 mL wash buffer.
Weakly bound proteins removed.
 Elution: Elution buffer with 250 mM imidazole used.
Fractions collected in separate tubes (~500 μL each).
 Sample Preparation for SDS-PAGE: Eluted fractions mixed with Laemmli buffer.
Heated at 95 °C for 5 minutes before loading on gel.

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 Chapter-4

Results and Discussion

1. Bacterial Growth and Pellet Weight

The optical density of the cultures was measured at various stages, and the final pellet weights
were recorded.
Table 6. Comparison of Primary and Secondary OD and Final Pellet Weight.
Experiment Primary OD Secondary OD Pellet Weight
Experiment 2.617 4.178 3.82
1(HiMedia)
Experiment 5.878 4.379 2.79
2(Invitrogen)
Experiment 4.513 5.085 3.38
3(Invitrogen)

Graphical Representation
1. Graphical Representation of Primary and Secondary OD on Different Media

7
5.878
6
5.085
5

4 4.379 4.513
4.178
3
2.617
2

0
Experiment 1 (HiMedia) Experiment 2 (Invitrogen) Experiment 3 (Invitrogen)

Primary OD Secondary OD Pellet weight

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 Chapter-4

Fig. 1 Experiment 1 HiMedia Primary OD Fig. 1 Experiment 2 Himedia Secondary OD

Fig. 2 Experiment 2 Invitrogen Primary OD Fig. 2 Experiment 2 Invitrogen2 Secondary OD

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 Chapter-4

Fig. 3 Experiment 3 Invitrogen Primary OD Fig. 3 Experiment 3 Invitrogen Secondary OD

2. Impact of Feeding Strategy on Bacterial Growth

Experiments were conducted with and without the addition of a concentrated feed to the secondary
culture.
Table 2: Comparison of Secondary OD with and without Feed.
Before Induction After Induction
With Feed 1.557 5.085

2.066 4.178
Average 1.8115 4.6315
Without Feed 2.087 4.379

Graphical Representation
2. Graphical Representation of Secondary OD on the basis of with Feed and Without Feed

5 4.6315
4.5 4.379
4
3.5
3
2.5 2.087
2 1.8115
1.5
1
0.5
0
With Feed without feed

Before Induction After Induction

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 Chapter-4

Fig. 4 Secondary OD with Feed

3.Bacterial Growth Over Time

The progression of optical density in the secondary cultures was monitored over time.

Table 3: Secondary OD at Different Time Points.

Experiment At t=0 At t=4hr At t=5hr At t=16hr

Experiment 1 0.267 - 1.557 4.178
Experiment 2 0.1410 1.915 2.087 4.388
Experiment 3 0.193 1.767 2.066 5.085

Graphical Representation
3. Graphical Representation of Secondary OD at Different Time

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 Chapter-4

6
5.085
5
4.388
4.178
4

1.9152.087 2.066
1.767
2 1.557

1
0.267 0.141 0.193
0
Experiment 1 AtExperiment
t=0 At t=4hr
2 At t=5hr At t=16hr
Experiment 3

4. SDS-PAGE Analysis of Protein Expression

The expression of the target protein was analyzed by SDS-PAGE. The expected protein size is
approximately 55kDa.
The samples were noted to be highly concentrated, with faint bands around 55kDa. A clear band
at approximately 55kDa was visible in the 1:10 diluted supernatant sample and a faint band in the
1:10 diluted induced sample, indicating the presence of the target protein. Very light bands were
seen in the 1:100 supernatant sample, and the band was fainter still in the 1:100 induced sample.

Fig. 5 SDS-PAGE

5. Affinity Chromatography
 SDS-PAGE Analysis of eluted fractions:
 A strong band at ~55 kDa observed in elution fractions, indicating successful purification.
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 Chapter-4
 No significant bands in flow-through, suggesting good binding to resin.
 Wash fractions showed faint bands, indicating minimal loss of target protein.
 Purity: The eluted protein appeared as a single prominent band, indicating high purity post-
affinity chromatography.

Fig. 6 After Purification

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 Conclusion
The present study successfully demonstrated the expression and purification of a recombinant protein
of approximately 55 kDa using Escherichia coli as the host system and Ni–NTA affinity
chromatography as the purification strategy.

The optimized expression conditions—using LB medium, IPTG induction at 25 °C, and incubation at
250 rpm—led to robust bacterial growth, with post-induction OD600 values reaching up to 4.388.
SDS–PAGE analysis confirmed that the target protein was expressed efficiently and primarily in the
soluble fraction, which is crucial for downstream purification and application.

Harvesting of the biomass and subsequent lysis using Triton X-100-based buffer followed by
sonication allowed for effective cell disruption and protein release. The purification of the His-tagged
protein via Ni–NTA chromatography yielded a highly enriched product, as indicated by a single,
strong ~55 kDa band on SDS–PAGE gels. The absence of significant protein in the flow-through and
only faint bands in the wash fractions confirmed the binding specificity of the resin and the minimal
loss of target protein during the purification process.

Overall, this work validates the use of a standard but powerful system for the recombinant expression
of proteins in E. coli, yielding high purity and solubility without requiring complex refolding steps or
additional purification tags. The methods established here lay a solid foundation for future applications
of the purified protein in structural, biochemical, or immunological

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 Future Prospects
While this study has demonstrated effective expression and purification of a recombinant ~55 kDa
protein using E. coli and Ni-NTA chromatography, several avenues exist for future improvement and
application:

1. Protein Yield Quantification and Stability

Quantitative protein assays such as Bradford or BCA should be employed to assess total yield.
Stability tests at various storage conditions and pH ranges can be performed to evaluate long-
term usability.

2. Functional Validation

Future experiments should include functional or enzymatic assays to confirm the biological
activity of the purified protein.

3. Tag Removal and Structural Analysis

The His-tag can be cleaved post-purification for studies requiring native conformation, and
further purification can be carried out using size-exclusion chromatography. Structural studies
via X-ray crystallography or NMR could provide deeper insights into function.

4. Expression Optimization

Further tuning of IPTG concentration, induction timing, and use of fusion tags or chaperone co-
expression may enhance solubility and expression levels.

5. Scale-Up for Industrial Applications

The current protocol can be scaled up for larger fermenters to meet preclinical or industrial
demands, particularly in the development of diagnostic kits or biotherapeutics.

6. Alternative Hosts for Complex Proteins

If post-translational modifications are required, future studies can explore alternative

expression systems such as yeast or mammalian cells.
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