Optimization of Heterologous Protein Production in Escherichia coli

This assignment delves into the realm of recombinant protein production, primarily focusing on the use of Escherichia coli (E. coli) as a microbial cell factory. The paper explores the advantages of using E. coli, including its rapid growth rate, ease of genetic manipulation, and availability of inexpensive media components. It examines the selection of the most suitable plasmid vectors, considering factors like copy number, promoter strength, and selection markers. The assignment also discusses the choice of host strains, with a comparison of different E. coli strains and their respective pros and cons. Furthermore, it addresses the crucial aspects of troubleshooting protein production, including strategies to overcome issues like low expression levels, protein toxicity, and protein misfolding. The paper concludes by emphasizing the versatility of the E. coli system and highlighting the ongoing efforts to improve protein expression efficiency and address challenges in recombinant protein production.

Protein 1
Heterologous Protein Production
Students Name
Name of Class
Professor
Name of School
The City and State
The Date

Protein 2
Introduction
It is clear that the generation of recombinant proteins inside the microbial structures has led to a
revolution in biochemistry. The capability of purifying and expressing the preferred recombinant
protein in more significant amounts enables it to be applied in industrial procedures, its
biochemical features and the production of commercial products (Lee & Hsu, 2018).
In the specialization of purifying and expressing the recombinant protein, some improvements
have been achieved. Therefore, in this paper, we suggest on the newly made progressions in the
Escherichia coli generation of recombinant protein. This paper also focuses on the modest
encounters while generating the heterologous proteins outlines various mechanisms and choices
successfully used in the expression of a substantial amount of proteins for many previous
decades (Pathak, et al., 2018).
Question one: the Organism to be used
The benefit of applying E.coli as the core organism are so well recognized. First, it contains
unparalleled kinetics that grows steadily, provided that there are optimal conditions in the
surrounding glucose and salts pathway the increasing duration is around 20 minutes (Rovere et
al., 2018). However, it should be put in mind that when expressing the recombinant protein there
could be some metabolic challenge effect to the microorganism, leading to a substantial decrease
in the period in production (Agah et al., 2018). Secondly, cultures with increased cell density are
attained with ease. It is approximate at around 2oo grams dry weight/I of the cell (Lim & Stacey,
2018). However, the rapid developments in the medium complexities cause densities far that
number. Thirdly, rich media complexion can be produced from the inexpensive and the already

Protein 3
available elements. Fourth, it is easy and fast to transform with the exogenous DNA (Amiri et al.,
2018).
Question 2: which is the most preferred Plasmid.
The most commonly used plasmid expression of recent come about through multiple integrations
of promoters, fusion protein or protein fusion, replicons, multiple cloning faces, and selection
markers removal approaches as shown in the table below (table 1). Following this, the present
vector expression catalogs are extensive and can be lost easily while selecting the most preferred
one. To come up with the best decision, there is the need for undertaking a careful evaluation in
regards to the requirements of individuals (Díaz-Dinamarca et al., 20118).
Table 1
Replicon
Genetic components which go through replication as independent units for example plasmids
consists of a replicon. The copy number is an essential guideline to consider during the selection
of a preferred vector (Gunnarsen et al., 2018). The regulation of the copy number is contained in
the replicon. It makes sense to have a thought that the increased dosages of plasmid are similar to
the recombinant proteins produced due to the high number of expression units found in the cell.

Protein 4
However, quantities of plasmid may cause a metabolic weight which lowers the development of
bacteria and could result in the plasmid being unstable. This will lead to a decrease in the number
of healthy organisms used in the synthesis of the proteins (Baron et al., 2018).
The most preferred vector, the pET series, contain pMB1 origin whereas the mutated type of
pMB1 origin is found inside pUC series. The wild-version of CoIE1 may be present inside
Qiagen. They are members of similar incompatibility class which means that they can't be
together propagated inside the same cell because they compete with each other during the
process of replication (Zhao et al., 2018).
Multiple recombinant protein expression can be attained with the use of 2 plasmids, pBAD
plasmids series, available p15A ori systems and 10 to 12 copies for every cell as stated by (Le et
al., 2018). However, triple expressions may rarely be attained using pSC101 plasmid. This
plasmid undergoes stringent replication control. Using plasmids containing such replicon could
be essential instances when there are high amounts of cloned genes or when the aftermath
generates an impact that is deleterious on the cell (Obeng et al., 2018).
Promoter
In Prokaryotic promoter study, the staple lac promoter is the primary element of lacoperon (da
Gama Ferreira et al., 2018). Where glucose and lactose are available, there is no complete
induction of the lactpromoter expression until the utilization of all the glucose. To attain
expression where glucose is present, a mutant which lowers sensitivity was applied to catabolite
control known as the lacUV5 (Troshagina et al., 2018). Although when a series of copy
plasmids are present, all the promoters are at a loss of at times experiencing increased rates of
expressions when there is no inducer.

Protein 5
Basal expression regulator may be attained when the lacI gene’s mutated promoter is introduced.
It is known as laclQ which causes an increase in the amounts of expression of LacI (Yanez et al.,
2018). The derivative lacUV5 of the lac promoter is usually weak hence not much vital in the
production of recombinant protein (Fischer et al., 2018).
Selection Marker
To prevent the development of free cells in the plasmid, a marker with resistance is placed to the
backbone of the plasmid. Inside the E.coli function, the antibiotic genes for resistance are
commonly used for such reasons. The bla gene offers resistance on the ampicillin whose
generation is by an enzyme called periplasmic that causes the β-lactam antibiotic’s β-lactam ring
to be inactive. However, the continued secretion of β-lactamase the antibiotics degrades and
after a while, ampicillin may deplete (Quaas et al., 2018). Under such instances, cells that do not
contain plasmid may be left to increase their in quantity at cultivation.
Affinity Tags
When developing a project and there is a need for a recombinant protein that is purified and
actively soluble, it is immaterial to have ways too;
 Detect it in the purification and expression plan
 Purify it efficiently from the cellular milieu of E.coli
 Achieve solubility maximally
The huge polypeptide and amino acids stretch expressions in tandem having the preferred protein
to produce chimeric protein will enable the three aspirations to be attained straightforwardly
(Kannaki et al., 2018).

Protein 6
Tag Removal
Where biochemical or structural research on the recombinant protein is required, then there must
be the removal of the fusion partner from recombinant protein. Peptide tags may also be
eliminated as they may impede with the structure or function of the protein (Mett et al., 2018).
However, they can be placed somewhere for crystallographic research (Bilikova & Simuth,
2018). According to Tchesnokov et al. (2018), tags may be removed through chemical cleavage
or enzymatic cleavage.
Question 3 which is the preferred host?
A quick study on the appropriate E.coli strain to be used as a host will produce a lot of eligible
candidates. Each of them has disadvantages and advantages. However, the idea to be considered
is that a lot of them are unique strains applied on specific occasions. During an initial expression
screening, only two strains of E.coli are needed, these are, K-12 lineage derivatives and BL21
(DE3) (Cho et al., 2018).
Question 4 which combination will be successful?
It was evident that there are a lot of choices when developing an expression system that needs to
be contemplated. Choosing the combination that is perfect is not an easy priority. Therefore
some conditions must be evaluated to get a suitable protein (Binder et al., 2017). Where the
project requires a double protein expression constructs, cloned in various six expressing vectors,
every single one to be transformed in multiple triple expression strains, it then means that there
are 36 experiments on expression to be conducted. The time consuming and try and error factors
could be done steadily when micro-expression tests are performed before scale-up (Mesa-Pereira
et al., 2017).

Protein 7
Production of Troubleshooting Recombinant
This part focuses on the various approaches for recombinant protein optimized generation in
E.coli. It is never predictable whether the protein will be produced in active soluble form and
large quantities even after choosing the host and plasmid carefully. Some approaches to be tried
are outlined in the table below.
Table 2
Problem explanations solution
Less or absence of
expression
Proteins could be harmful
prior to to be induced
Reduce the copy number in
plasmid
Regulate basal inducers by:
 Using proper medium
containing glucose as
carbon origin
 Adding glucose while
expression vectors are
in use having
promoters based with
lac
 Using promoters with
tight control
Protein could be harmful after
inducement.
Protein to be directed to the
periplasm
Regulate induction rates or by:
 Using strains that
enables the induction
to be regulated
 Using promoters that
can be tuned
Using suitable strain for
expression of the protein that
is toxic
Inactive protein Not fully folded
cDNA mutations
Supervise the formation of
disulfide bond and let folding
happen further
Sequence plasmid after or
before induction. Where

Protein 8
mutations have been
discovered, the protein could
be harmful.
Conclusion
In regards to recombinant expression, the most commonly used microbial cell factory is E. coli.
It is the most preferred host for the stably folded expressions, globular proteins from eukaryotes
and prokaryotes. However, the membrane of the protein containing molecular load higher than
60k-Da is hard to be expressed. Various studies have been successfully generated proteins
amounting to 90-95 kDa from plants (Freudl, 2018). Protein expression on a large scale
experiment has indicated that ˂ 15% proteins that are non-bacterial and ˂ 50% proteins with
bacteria could be expressed in the soluble state in E.coli this shows how the system is versatile
(Schmideder & Weuster-Botz, 2017). Although when encountering a protein that is hard to
express it usually complex. There is hope that some solutions will come up to solve the
difficulties experienced while expressing any new protein in E.coli.

Protein 9
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Protein 10
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Protein 11
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Protein 12
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