Recombinant protein production in

microbes

A presentation By:-

Anamitra Sen(BTB/16/332)
Neha Panwar(BTB/3C/16/109)
Ashlesha Gadkary(BTB/16/328)
Tanya Bisht(BTB/16/313)
What is a Recombinant Protein Production?
1) Recombinant Protein is a protein encoded by a
gene — recombinant DNA — that has been
cloned in a system that supports expression of the
gene and translation of messenger RNA

3) The formation of 4) Modification of the gene by

recombinant protein is recombinant DNA technology can
carried out in specialized lead to expression of a mutant
vehicles known as vectors protein.

2) The process of generation of the recombinant

protein to produce large quantities of proteins, by
the modification of gene sequences and
manufacture useful commercial products is termed
as Recombinant protein production
 Product is typically achieved by the
manipulation of gene expression in an
organism such that it expresses large
amounts of a recombinant gene. This
includes the transcription of the
recombinant DNA to messenger RNA
(mRNA), the translation of mRNA into
polypeptide chains, which are ultimately
folded into functional proteins and may be
targeted to specific subcellular or
extracellular locations.
Basic mechanism of Recombinant protein
production
●
To aid the study of proteins that are produced at a low level, the gene
encoding them generally has to be over-expressed.The most straightforward
way to achieve this is to fuse the target gene to a strong promoter.
●
The strong promoter, usually derived from a highly expressed gene, will drive
the expression of any gene placed under its control through the recruitment of
RNA polymerase to that gene.
●
Vectors will often contain a multiple cloning site located between a strong
transcriptional promoter and terminator sequence. Additionally, the expression
vector, like other plasmids, will contain an origin of replication and a
selectable marker such that the vector may be autonomously replicated and
maintained within cells.
 The architecture of an Expression Vector
● An expression vector should contain a
strong inducible promoter, a multiple
cloning site for the insertion of target
genes, and a transcriptional terminator.

● Additionally, a ribosome binding site

(RBS) is included to promote efficient
translation
Typical protein
expression
workflows.
 Different types of Expression Vectors
●
Escherichia coli
●
Saccharomyces cerevisiae
●
Pichia pastoris
●
Baculovirus-infected insect cells
●
Mammalian cells
 Escherichia coli

●
E. coli remains the host cell of choice for the majority of protein
expression experiments. Its rapid doubling time in simple defined media,
combined with an extensive knowledge of its promoter and terminator
sequences, means that many proteins of both prokaryotic and eukaryotic
origin can be produced within the organism
●
Additionally, E. coli cells are easily broken for the harvesting of the
proteins produced within the Cell.
●
E. coli cells are unable to process introns and do not possess the extensive
post-translational machinery found in eukaryotic cells that can glycolylate,
methylate, phosphorylate or alter the initially produced protein in other
ways, such as through extensive disulphide bond formation
 Many different promoter sequences have been used to
illicit inducible protein production in E. coli

Lac Promoter Tac

Promoter

The λPL
Promoter
 Some application with E.coli expression
Vector
● Production of
Factor VIII for
● Production of haemophilia ● Human Insulin
Human growth treatment
hormone
● Hepititis B
vaccine
 Sachharomyces cerevesae

Baker’s yeast, S. cerevisiae, is a single-celled eukaryote that

grows rapidly (a doubling time of approximately 90 min) in
simple, defined media similar to those used for E. coli cell
growth. Proteins produced in S. cerevisiae contain many, but
not all, of the post-translation modifications found in
highereukaryotic cells. For example, human α-1-antitrypsin, a
52 kDa serum protein involved in the control of coagulation and
fibrinolysis, is normally glycosylated
 The Gal system of
Saccharomyces cerevisiae
Factors which affect protein over-
expression
 Some factors to improve protein expression
● Expression vector levels.
● Transcriptional termination
● Codon usage
● Protein sequence
● Protein degradation
 Expression vector levels

One would imaging that increasing the

copy number of the expression vector would lead to an increase in the
accumulation of the protein it encodes. There are, however,
documented cases when a very high expression vector copy number (in
comparison to the levels obtained for pBR322) did not result in
increased protein production and others where increased vector levels
actually reduce the levels of protein production.
 Codon usage
● The degeneracy of the genetic code means that more than one codon
will result in the insertion of an individual amino acid into growing
polypeptide chain.
● The genes of both prokaryotes and eukaryotes show a non-random
usage of alternative codons. Genes containing favourable codons will
be translated more efficiently than those containing infrequently used
codons.
● This effect is particularly prevalent in genes that are highly expressed
in E. coli, where there is a high degree of codon bias.
 Challenges to Recombinant Protein production
●
Plasmid loss is the main cause of reduced recombinant
protein productivity in plasmid-based systems. An
unequal plasmid distribution upon cell division will
eventually lead to plasmid-free cells. This is called
plasmid segregational instability.
●
Protein folding is a complex
process in which two kinds
of molecules play an
important role: foldases,
which accelerate protein
folding; and chaperones,
which prevent the formation
of non-native insoluble
folding intermediates
●
On occasions, folding does not
proceed adequately. This
results in misfolded proteins
that accumulate in intracellular
aggregates known as inclusion
bodies. One of the main causes
of incorrect protein folding is
cell stress, which may be
caused by heat shock, nutrient
depletion, or other stimuli.
 In conclusion
●
Without a doubt, E. coli is the most widely used host for
heterologous gene expression. It has been used for this
purpose for more than 40 years, so there is much
accumulated knowledge about its advantages and
disadvantages as an expression platform
●
Microbes other than E. coli can be used for heterologous
protein production. The impact of yeasts on the biotech
industry is paramount, as 20% of biopharmaceutic proteins
are synthesized in yeasts. But still, they are not the first-
choice microorganism for recombinant protein production.
 In conclusion
●
Saccharomyces cerevisiae and Pichia pastoris should be
considered alongside E. coli in any project in need of a
recombinant protein
●
Filamentous fungi are excellent protein secretors, a
property that makes them ideal as a host at an industrial
scale.
●
Usefulness of microalgae as expression systems,
focusing on the production of recombinant vaccines has
also been researched.
Thank you!