This document discusses commonly used protein expression systems. It begins by explaining how recombinant DNA technology has enabled the cloning and expression of mammalian genes in different systems to produce therapeutic and vaccine proteins. It notes that the suitable expression system depends on factors like productivity, bioactivity, purpose and protein characteristics. The document then examines various prokaryotic (bacterial) and eukaryotic expression systems in detail, including E. coli, Bacillus subtilis, Lactococcus lactis, Pseudomonas, Corynebacterium, yeast, mammalian cells and baculovirus cells. It highlights advantages and disadvantages of each system for recombinant protein expression.

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Chapter:07
CommonlyUsedProteinExpressionSystems
Amjad Khan Afridi

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With the beginning of recombinant DNA technology,
cloning and expression of numerous mammalian
genes in different systems have been explored to
produce many therapeutics and vaccines for human
and animals in the form of recombinant proteins.
But selection of the suitable expression system
depends on productivity, bioactivity, purpose and
physicochemical characteristics of the protein of
interest.
INTRODUCTION

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 However, large scale production of recombinant proteins
is still an art in spite of increased qualitative and
quantitative demand for these proteins.
 Researchers are constantly challenged to improve and
optimize the existing expression systems, and also to
develop novel approaches to face the demands of
producing the complex proteins.
 Expression host systems are used for the expression of
recombinant proteins both for therapy and research work.

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 The quality of the product is very essential, especially in
medicine where production of human pharmaceuticals is
regulated under strict safety feature.
 Thus, suitable host system needs to be selected
depending on the purpose.
 rDNA technology has necessary tools to produce desired
viral / bacterial proteins in large quantities in a native
form.

5. PROKARYOTIC EXPRESSION SYSTEMS
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Bacterial expression system is widely used for the
expression of rDNA products.
They offer several advantages viz.,
-High level of recombinant protein expression
-Rapid cell multiplication and
-Simple media requirement
Bacterial Expression System

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 Escherichia coli is a Gram negative,
facultative anaerobic, rod-shaped
bacteria.
 E. coli are found in the environment,
foods, and intestines of human and
animals.
 E. coli are a large and diverse group
of bacteria.
 The harmless strains produce
vitamin-K and prevent colonization
of the intestine by pathogenic
bacteria.
1. EscherichiaColi Introduction

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 E. coli is a typical prokaryotic expression system and
one of the most attractive heterologous protein producer.
 To date improved E. coli is the extensively used cellular
host for foreign protein expression because of its rapid
growth rate which is as short as 20-30 minutes.
 Have the capacity for continuous fermentation and
relatively low cost.
1. EscherichiaColi ExpressionSystem

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 The expression of proteins in this system is the;
 Easiest,
 High-level expression,
 Quickest (Fast expression),
 Cheapest (Low cost),
 Simple culture conditions
1. EscherichiaColi ExpressionSystem

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Disadvantages
 Easy to form inclusion bodies
 Different codon systems
 Few post-translational modifications; Endotoxin
Applications
Production of purified protein (structure, enzyme, drug
discovery); Drug protein production.
1. EscherichiaColi ExpressionSystem

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 Bacillus subtilis is Gram-positive rod-
shaped, spores forming bacterium
found in the soil
 Also the normal flora of the body and
can found gastrointestinal tract of
ruminants and humans.
 Catalase positive and obligate aerobe
 B. subtilis, also known as hay
bacillus or grass bacillus
2.Bacillus subtilis

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 It is an alternative to the E. coli expression system.
 It can secrete degradative enzymes or antibiotics,
produce spores and can become competent for genetic
transformation
 Its biotechnology companies secreted enzyme producing
bacteria which produce a large scale of industrial
products.
 It is estimated that Bacillus species, including B.
subtilis, produce 60% of commercially available
enzymes.
2.Bacillus subtilis Expression System

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 The major advantage of B. subtilis is that it does not
produce Lipopolysaccharides (LPS), which may
otherwise cause degenerative disorders in humans and
animals.
 B. subtilis can also be transformed readily with many
bacteriophages and plasmids due to its genetic
characteristics.
 It is capable of secreting functional extracellular
proteases directly into the culture medium, facilitating
further purification steps.
 In some time these extracellular proteases degrade
heterologous proteins (therapeutic proteins)
2.Bacillus subtilis Expression System

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 Lactococcus lactis is Gram-positive, catalase-negative,
facultative anaerobic, non-motile, and non-spore-forming
bacterium.
 Used extensively in the production of buttermilk and
cheese
 Also famous as the first genetically modified organism to
be used alive for the treatment of human disease.
 L. lactis is widely used in biotechnology for large-scale
production of heterologous proteins.
3.Lactococcus lactis Expression System

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 It is considered as a potential alternative to E. coli.
 Pseudomonas fluorescens has been recognized to
function as a recombinant protein producer.
 Producing soluble proteins, while E. coli produces
insoluble protein
 P. fluorescens has low stability for the expression of
plasmids to produce heterologous proteins
 While, P. putida has high plasmid stability and proved to
be potential recombinant protein producer for industrial
use with growth properties similar to E. coli during fed
batch fermentations.
4. Pseudomonas Expression System

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 Corynebacterium are Gram-positive, slender, non-spore-
forming rods , facultative anaerobic and catalase positive.
 Non-pathogenic species of Corynebacterium are used for
the commercial production of various amino acids.
 The C. glutamicum is used for producing glutamate and
lysine, components of human food, animal feed and
pharmaceutical products.
 Expression of functionally active human epidermal
growth factor has been done in C. glutamicum
 Thus demonstrating a potential for industrial-scale
production of human proteins.
5.Corynebacterium Expression System

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 Eukaryotic genes are not really “at home” at prokaryotic
cells, even when they are express under the control of the
prokaryotic vectors.
 Prokaryotes do not carry out the same kinds of
posttranslational modification as eukaryotes do.
 Eukaryotic system for the expression of protein include:
 Yeast
 Mammalian cells
 Baculovirus cells (Insects)
 All these systems are great eukaryotic systems for the
expression of recombinant proteins.
Eukaryotic Expression System

18. 19/05/2021