Prokaryotic systems E. coli is a popular and well understood system for heterologous protein expression.

• Expression options:

• Direct expression. E. coli cytoplasm is a reducing environment - difficult to ensure proper disulphide bonds formation.

• Fusion expression.

– Ensures good translation initiation,

– Can overcome insolubility and /or instability problems with small peptides.

– Has purification advantages based on affinity chromatography.

• Secretion

– a fusion alternative when proteins are fused to peptides or proteins targetted for secretion. Periplasm offers a more oxidising environment, where proteins tend to fold better. Major drawbacks: limited capacity for secretion (0.1- 0.2% total cell protein compared to 10% produced intracellularly) and inability for posttranslational modifications of proteins.

To minimise proteolysis

For efficient and selective purification

To optimise translation efficiency

Disadvantages Insolubility of heterologous proteins produced in E.coli - one of the main problems

Inclusion bodies. Dense particles, containing precipitated proteins. Their formation depends on

protein synthesis rate, growth conditions. Advantages: proteolysis resistant, big yield, relatively

pure, easy to separate. Disadvantages: inactive product requires in vitro refolding and renaturation

Refolding of recombinant proteins

Solubilisation. High t0 C, detergents, high concentration of inorganic salts or organic solvents all

used. The most commonly used organic solutes such as urea or guanidine-HCl often used in the

presence of reducing agents (mercaptoethanol or DTT).

Solubilised proteins purified by ion-exchange chromatography or other conventional methods, prior

to refolding.

Refolding. If no S-S bonds present - remove denaturing agent to allow protein to fold correctly. If S-

S bonds present - their formation can be accomplished: by air oxidation, catalysed by trace metal

ions; by a mixture of reduced and oxidised thiol compounds - oxidised DTT, reduced DTT;

GSSG/GSH; cystine and cysteine, cystamine and cysteamine.

Isolation and characterisation of correctly folded proteins.

Biological activity. Purity. SDS-PAGE. Chromatography - reversed phase or ion-exchange. N-

terminus determination by sequencing. Peptide mapping.

Bacillus subtilis is a better choice for secretion of a prokaryotic protein than E.coli. Secretes

proteins to the medium, including own proteases - therefore there might be a problem with

proteolysis. Overcome with mutants. Problems with plasmid stability - overcome by

integration into the chromosome.

Potential Problems in Prokaryotic Expression Systems

• Low expression

• Degradation by bacterial proteases

• Improper folding

• Oxygen limitations

• Biofilm formation

• Prokaryotic systems work for the expression of most

all proteins.

• However, some eukaryotic proteins produced in

these systems lack biological activity, are degraded,

or co-purify with other compounds which make the

protein unusable.

• Due to improper posttranslational protein

processing, including cleavage, folding and adding

chemical group to specific sites.

• To avoid these problems, Eukaryotic systems have

been developed for protein production.

Gene Expression in Prokaryotes

• Eukaryotic protein synthesis occurs in cytoplasm

or on the endoplasmic reticulum.

• These proteins are further post translational

processed that is required for protein activity and

stability.

• Disulfide isomerase also makes sure that the

proteins produced have the correct configuration.

• The proper glycosylation that are necessary for

protein conformation, localization by interacting

with specific receptor and increase stability.

Posttranslational Modification

• The choice of an expression system depends

primarily on the quality of the recombinant protein.

• However, the yield of the product, ease of se, and

cost of production and purification are also

important considerations.

• The vector must be designed to be maintained in

the eukaryotic host.

• The vector must have eukaryotic promoter,

transcriptional, translational stop signals, a

sequence that enables polyadenylation and a

selectable marker gene.

Eukaryotic Expression Systems

• The major features of a eukaryotic expression vector

are a promoter, a multiple cloning site, DNA segment

for termination and polyadenylation, selectable marker,

origin of replication in E. coli and eukaryotic cell and

Ampr for marker in E. coli.

• Saccharomyces cerevisiae

• Pichia pastoris

• Baculovirus-insect cell lines

• Mammalian systems

Eukaryotic Expression Systems

• It is the most common eukaryotic system and there is

a great deal of study about this organism.

• It is a ingle-celled and behaves like a bacterial culture

and can be grown in relatively simple media in both

small and large-scale production.

• Well characterized with many strong regulatable

promoters with naturally occurring plasmids.

• Carry out post-translational modifications.

• Secretes very few of its own proteins.

• Recognized as safe by USDA and FDA.

Saccharomyces cerevisiae

• There are three main classes of S. cerevisiae

expression vectors.

• Yeast episomal plasmids (YEps).

• Yeast integrating plasmids (YIps)

• Yeast artificial chromosomes (YACs)

• Yeast episomal plasmids have been used extensively

for the production of eitehr intra- or extracellular

heterologous proteins.

• Typically, vectors function in both E. coli and S.

cerevisiae.

Saccharomyces cerevisiae

• The YEps vectors are based on the high-copy-number

2µm plasmids.

• The vectors replicate independently via a single

origin of replication.

• There are more than 30 copies per cell.

• Selection scheme rely on mutant host strains that

require a particular amino acid (histidine,

tryptophan, or leucine) or nucleotide (uracil).

• When a Yep with a wild-type LEU2 gene is

transformed into a mutant leu2 host cell, only cells

that carry plasmid will grow.

Saccharomyces cerevisiae

• Generally, tightly regulatable, inducible promoters are

preferred for producing large amounts of recombinant

protein at a specific time during large-scale growth.

• Most heterologous genes are provided with a DNA

coding sequence for signal peptide that facilitates the

secretion of protein through cell membranes and

external environment.

• Other sequence that protect the recombinant protein

from proteolytic degradation, and provide a affinity tag

is also used.

• These extra amino acid sequences are equipped with

a protease cleavage site so that they can be removed

from the recombinant protein.

Saccharomyces cerevisiae

• Plasmid-based yeast expression systems are often

unstable under large-scale growth conditions even in

the presence of selection pressure.

• A Yip vector is used to integrate a heterologous gene

into the host genome to provide a more reliable

production system.

• The plasmid does not usually carry an origin of

replication.

• The disadvantage is the low yield of recombinant

protein from a single gene copy.

Saccharomyces cerevisiae

• Proteins may also be produced for secretion.

• In this system, any glycosylated protein is secreted (O

or N-linked).

• The coding sequences of recombinant proteins must be

cloned downstream of a leader sequence, the yeast

mating type factor α-factor.

• Under these conditions, correct disulfide bond

formation, proteolytic removal of the leader sequence,

and appropriate posttranslational modifications occur,

and an active recombinant protein is secreted.

• The leader peptide is removed by endoprotease that

recognizes the Lys-Arg.

Secretion of Heterologous Proteins

• For example, a properly processed and active form of

the protein hirudin; a powerful anticoagulant protein

cloned from a leech, was synthesized and secreted by

an S. cerevisiae.

• A YEp vector that had the prepro-α-factor sequence

added to the huridin coding sequencea to allow

expression that is cleaved away in processing.

• Leaves active hirudin which is secreted.

• Producing a recombinant protein for use in human

therapeutics in yeast rather than in bacteria is to ensure

the proper folding.

Secretion of Heterologous Proteins

Secretion of Heterologous Proteins

• Though S. cerevisae is successfully used to produce

recombinant proteins for human, it has major

drawbacks.

• The level of protein production is low.

• There is the tendency for hyperglycosylation resulting

in change of protein function.

• Proteins are often retained in periplasm, increasing

time and cost for purification.

• It produces ethanol at high cell densities, which is toxic

to cells.

Pichia pastoris Expression Systems

• P. pastoris is a methylotrophic yeast that is able to

utilize methanol as a source of carbon and energy.

• Glycosylation occurs to a lesser extent and the

linkages between sugar residues are of the α-1,2 type.

• P. pastoris strain was extensively engineered with the

aim of developing a “humanized” strain that glycosylate

proteins in a manner identical to that of human cells.

• It does not produce ethanol.

• It normally secretes very few proteins, thus simplifying

the purification of secreted recombinant proteins.

Pichia pastoris Expression Systems

Baculovirus-Insect Cell Expression

• Baculoviruses are a large, diverse group of viruses that

specifically infect arthropods, and are not infectious to

other animals.

• During the infection cycle, two forms of baculovirus are

produced.

• A single nucleocaspid (virus particle) which can

infect more midgut cells.

• Clusters of nucleocaspids that are produced

outside of the cells (virions) in a protein matrix

(polyhedrin).

Baculovirus-Insect Cell Expression

Baculovirus-Insect Cell Expression

• The polyhedrin gene is replaced with a coding

sequence for a heterologous protein, followed by

infection of cultured insect cells, resulting in the

production of the heterologous protein.

Baculovirus-Insect Cell Expression

• Constructs have been made using the polyhedrin

promoter to produce large quantities of

extracellular protein.

• Most proteins are modified and secreted properly.

• Grows very well in many insect cell lines allowing

easy production.

• Minor problem that doesn’t process certain

mammalian glycosylation types correctly

(galactose and sialic acid; N-linked.)

Baculovirus Expression Vectors • The specific baculovirus that has been used

extensively is Autographa californica multiple nuclear

polyhedrosis virus (AcMNPV.)

• A gene of interest is inserted into the MCS and the

transfer vector is propagated in E. coli.

• Next, insect cells in culture are cotransfected with

AcMNPV DNA and the transfer vector carrying the

cloned gene.

Baculovirus Expression Vectors

Increasing the

Yield of

Recombinant

Baculovirus

Mammalian Cell Expression Systems

• Important for producing proteins with all post-

translational modifications.

• Many established cell lines are useful.

• Transient expression: African green monkey, baby

hamster, & human embryonic (all kidney tissue cell

lines.)

• Long-term expression: Chinese hamster ovary and

mouse myeloma cells.

Mammalian Cell Expression Systems

• Expression vectors in these systems are usually

derived from an animal virus such as SV40 (simian

virus 40).

• Can be used for expression of single polypeptides,

homooligomers, and heterooligomers.

• The latter is made possible by transforming with

two or more separate cloned genes.

• Industrial production is however costly.

Selectable Markers for mammalian

Expression Vectors

Strategy to Increase Yields of

Recombinant Mammalian Cells

Restriction Enzyme Analysis

In situ Hybridization • Type of hybridization that uses a labeled complementary DNA, RNA or

modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough (e.g., plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH), in cells, and in circulating tumor cells (CTCs).

• This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.

• Basic Steps – permeabilization of cells with proteinase K to open cell membranes (around 25 minutes,

not needed for tissue sections or some early-stage embryos)

– binding of mRNAs to marked RNA probe (usually overnight)

– antibody-phosphatase binding to RNA-probe (some hours)

– staining of antibody (e.g., with alkaline phosphatase)

Marker and Reporter genes

• A reporter or marker gene is a gene, which produces a specific phenotype, in turn enables the differentiation of the cells possessing this particular gene from those without this gene. Hence, the transformed cells can be selected easily among the thousands of non transformed cells.

• Reporter genes form specific protein products, which are easily detectable and quantifiable, sometimes even without destroying the tissue.

• Reporter genes are an invaluable tool to track and study another associated gene in bacterial and mammalian cell culture, animals and plants. One can easily find out the expression patterns of a gene within the cell by fusing its promoter with one of the several reporter genes and transfecting inside the living cells.

• So, it is very useful in for the monitoring and detection of plant transformation, for studying the activities of regulatory elements such as promoter and enhancer.

Features of an ideal reporter gene

• Easily quantifiable

• Relatively rapid degradation of the enzyme

• High signal to noise ratio (Low endogenous background)

• Should not be toxic to cells

• Products of the reporter gene should be resistant to the chemicals used in the processing

• Assay should be sensitive and reliable.

Types of reporter genes

• Selectable marker – The cells that contain this type of marker gene show the ability to

survive under selective conditions. These selective conditions would otherwise result in the death of the cells lacking that specific gene. Most commonly used selective agents are antibiotics. Out of the millions and billions of cells, only few of them get transformed by the foreign DNA. It is practically impossible to check every individual cell, so a selective agent is required to eliminate the non transformed cells, leaving only the desired ones.

• Scorable marker – Expression of this kind of marker gene results in a quantifiable

phenotype i.e., it will make the cells containing it to look different. The main principle behind the use of these reporter genes for the study of molecular processes in living cells means that in natural genes, synthetic modification have introduced in order to either simplify the detection of the product or to distinguish it from similar genes in the genome. These reporter genes were assayed at the level of protein.

Selectable markers Scorable markers (reporter genes)

• Typically used to recover transgenic plant cells from a sea of non-transgenic cells

• Antibiotic resistance markers and herbicide resistance markers are most common

• Can help visualize transient expression

• Can help visualize if tissue is stably transgenic

• Useful for cellular and ecological studies

Selectable marker

Non-selectable maker gene systems—Reporter genes/ Scorable marker

Luciferase

• As a reporter, offers several advantages including the capability of monitoring gene expression patterns non-destructively in real time with great sensitivity.

• The firefly (Photinus pyralis) luciferase catalyzes the ATP-dependent oxidative decarboxylation of luciferin. After the reaction occurs the luciferase is inactive until the oxyluciferin is released from the enzyme complex.

• This is a slow process and the LUC half life is very short; thus, it is believed that LUC activity more accurately reflects transcriptional activity than some other reporter genes that are more stable and accumulate over time

Green fluorescent protein

• The green fluorescent protein (GFP) from jellyfish (Aequorea victoria) has become a powerful reporter gene to complement selectable marker genes and can be used to select for transformed material alone.

• The great advantage of GFP as a non-conditional reporter is the direct visualization of GFP in living tissue in real time without invasive procedures such as the application or penetration of cells with substrate and products that may diffuse within or among cells.

• GFP has not been extensively used as a reporter for studies in the regulation of gene expression or the study of regulatory elements; however, it has been a very useful tag for monitoring intracellular location and transport when fused to other proteins of interest.

Phytoene synthase

• The bacterial gene coding for phytoene synthase from Erwinia herbicola can act as a non-conditional reporter gene by altering the carotenoid biosynthetic pathway in chloroplasts so that coloured carotenoids accumulate.

• The coloured tissues expressing the reporter gene can then be manually removed and cultured to generate transgenic plants.

• Phytoene synthase catalyses the synthesis of phytoene from geranylgeranyl pyrophosphate and phytoene is a precursor of lycopene, the carotenoid that imparts the red colour to tomato.

• E. herbicola phytoene synthase targeted to the chloroplast, generated transgenic orange callus as a visual marker for transgenic tissue at about 50% efficiency and may be used to monitor transgenic plants

Fusion Proteins

• Protein fusion done at level of DNA (genes)

– coding region of cellular protein fused in frame to coding region of target protein gene

– when transcribed and translated generates a fusion protein

Fusion Proteins

gene of interest

Note: translation stop of cellular gene must be removed

Note: reading frame of fusion protein must be contiguous

Fusion proteins • Used to avoid problems with digestion of foreign proteins by proteases

• Used to aid purification of foreign proteins, often by affinity chromatography

• Often a rare protease cut site is added to the fusion partner