Recombinant Protein Production

By:

Majid Mojarrad

• synthesis and purification of proteins

• Proteins produced in two types:

– Native proteins

– Fusion proteins

• We need – Appropriate promoter

– Appropriate host• Prokaryotic host

– E.Coli

– B. Subtilis

• Eukaryotic– Yeast

– Fungi

– Insect

– Mamalian cell line

– Transgenic animal

Cloning and Expression OptimizationDominic Esposito, Group Leader

Cloning•PCR Gateway Sequencing•Restriction enzymes and ligase•Multiple expression vectors (>50)

Expression optimization (E. coli)•Strains•Temperature•Fusion tags•Induction time, level

Expression optimization:E. coli strain (rare codons)

Expression optimization:Temperature after induction

Expression optimization:Fusion tags

Expression optimization:Fusion tags and solubility

Microbial FermentationDavid Miller, Group Leader

•E. coli, yeast•Shake flasks to 60 liters•Batch and fed batch•Cell lysis and processing (membranes)•Isotopic labeling

Microbial Fermentation:• Computer controlled 80 liter fermenter, workingvolume 20 – 60 liters

• 4 x 20 liter fermenters, computer controlled, 5 –15 liter working volumes

Microbial Fermentation:Batch (tank) vs. fed batch

Yeast systems

• Saccharomyces cerevisiae

• Schizosaccharomyces pombe

• Pichia pastoris

• Hansela polymorpha

• Kluyveromyces lactis

• Yarrowia lipolytica

S. Serviciae or

GRAS (Generally Regarded As

Safe)• Intracellular expression

– higher protein yields

– But• more difficult extraction

• and purification.

• co- and post-translational processing of proteins at N- and C-termini.

• proteolytic degradation

• addition of tags might result in aggregation and insolubility

• Secretion

– N-terminal signal sequences for co-translational translocation

– Then removed by a signal peptidase

– Examples of popular signal sequences• Pho5, Suc2 and the a -factor.

• Vectors based on a small multi-copy plasmid

(called the 2μ plasmid)• YEps (for yeast episomal plasmids)

• Use auxotrophic host

Disadvantage of yeasts

• Hyperglycosylation which leads to • Reduce activity

• Immunogenicity

– To solve this problem

• Glycosylation mutants

• Remove of glycosylation sites

• Using of some other type of yeast such as P. pastoris

P.pastoris

• highly developed fermentation technology

• can be grown to higher cell densities in culture and in fermentors

• it can secrete proteins to much higher levels than S. cerevisiae

• Yields of heterologous proteins of around 12g/L in P.pastoris

• Use methanol as carbon source

• alcohol oxidase promoter (AOX1) for heterologous protein expression by adding of methanol to medium

• This promoter strongly repressed in absence of methanol

• Integrative and autonomously replicating (PARS1 and PARS2) vectors available

• Protein can produce by intracellular expression or secretion

• A better secretor than S.c

• Glycoproteins are often less mannosylated than those, of Saccharomyces

• About 35% of N-linked oligosaccharides have less than 14 mannose units

• Pichia's secretion signal from its own acid phosphatase

• Proteolysis in Pichia– Can be minimised by buffering the pH of the medium

• significant progress has been made in “humanizing” the

glycosylation pathways of P. pastoris

Baculovirus system

• Uses insect cells from Spodopterafrugiperda

• infected with baculoviruses Autographa

californica

• Use polyhedrin promoter to expression

Baculovirus Vector

• Vector has sequences for expression using po

• lyhedron gene expression system

• Sequences also present for integration into baculovirus

(AcMNPV) genome via recombination

• Prokaryotic sequences not shown

Baculovirus Transfer Vector

Done in cell

culture

Screening for

recombinants

tedious by PCR

Examples of Proteins Successfully

Produced by Baculovirus Systems

E. coli Baculovirus Shuttle

Vector - Bacmids

• Shuttle vectors allow ease of transfer between systems

• Genetic manipulations in one system, expression in another

Bacmid Construction (2)

• attR and attL are lambda sequences to give high

efficiency and specific transposition

Bacmid Construction (3)

• Bacmid now operates efficiently in both E. coli

and insect cells

Modifying the Insect Cell Host

• Some enzymes simply not present

• Genetic engineering of the host for proper

expression

• Add missing glycosylation enzymes

• Add proteolytic processing enzymes

AdvantagesThe polyhedrin gene is not required for the continuous production of infectious virus in

insect cell culture. Its sequence is replaced with that of the heterologous gene.

The polyhedrin gene promoter is very strong. This determines a very high level of

production of recombinant protein.

Very late expression allows for the production of very toxic proteins.

This system is capable of post-translational modifications.

Disadvantages

•Expensive.

•Glycosylation in insect cells is different (insect cells unable to produce complex N-linked

side chains with penultimate galactose and terminal sialic acid) from that in vertebrate

cells, therefore, a problem for therapeutic proteins.

•A large fraction of the RP can be poorly processed and accumulates as aggregates.

•Discontinuous expression: baculovirus infection of insect cells kills the host and hence

the need to reinfect fresh cultures for each round of protein synthesis.

•Inefficient for production on a commercial scale.

Mammalian Systems

• Sometimes insect cells simply don’t carry

out proper/necessary glycosylations

• Other processing may also not occur

• Mammalian cell systems are more

expensive by may be required for active

product

Mammalian Expression Vector

• “I” is an intron that enhances expression

• Other signals similar to insect and prokaryotic

vectors

Translation Control Elements

• K - Kozak Sequence (equiv. To rbs)

• S - for secretion signal peptide

• T – tag peptide for purification

• P – proteolytic cleavage sequence

• SC – stop codon for translation

• 3’UTR – proper sequences for efficient translation and

mRNA stability (e.g. polyadenylation sequence)

Two Vector Expression System

•Useful for proteins of two different polypeptides

Two Gene Expression Vector

Bicistronic Expression Vector

IRES from mammalian virus

•Gives more uniform level of expression of two genes

Selectable Markers for Mammalian

Systems

• Most commonly used to select for

transformed cells (killing nonresistant ones)

• Can be used for increasing expression of

heterologous proteins

Selective marker gene systems for mammalian cells

Use of Selectable Markers for

Increasing Heterologous Protein

Production in Mammalian Systems

• Methotrexate (MTX ) inhibits dihydrofolate

reductase (DHFR)

• DHFR- host cell with DHFR gene on cloning

vector (i.e. linked to target gene)

• Gradually increase MTX concentration in culture

• Gene copy number of DHFR and linked target

gene increase to compensate for inhibition of

DHFR (more protein that is less active gives cell

enough metabolic through put to survive)