Recombinant protein Recombinant protein production in Eukaryotic cellsproduction in Eukaryotic cells

Dr. W. McLaughlinBC35C

Recombinant protein Recombinant protein production in Eukaryotic cellsproduction in Eukaryotic cells

! rHuman protein must be identical to the natural protein

! Prokaryotes are generally unable to produce authentic eukaryotic proteins due to the absence of appropriate mechanisms for carrying out the necessary post-translational modification to the protein

PostPost--translational Modificationtranslational Modification

! Correct disulphide bond formation. Reaction mediated by the enzyme disulphide isomerase. An improperly folded protein is unstable and lacks activity

! Proteolytic cleavage of a precursor form. Selected segments of amino acid sequences are removed to yield a functional protein

PostPost--translational Modificationtranslational Modification

! Glycosylation. Gives a protein with stability and, in some instances, its distinctive properties. The most common protein glycosylations occur by the addition of specific sugar residues to serine or threonine(O-linked) or to asparagine (N-linked)

! Addition of amino acids within proteins. Modification of this type includes phosphorylation, acetylation, sulfation

Eukaryotic expression vectors Eukaryotic expression vectors

! A selectable eukaryotic marker gene! A eukaryotic promoter sequence ! The appropriate eukaryotic

transcriptional and translational stop signal

! A sequence that signals polyadenylationof the transcript messenger RNA (mRNA)

p=promoter, t=termination & polyadenylation sequence, cs=cloning site, EMS=eukaryotic marker system, ori=origin of replication

SaccharomycesSaccharomyces cerevisiaecerevisiae

! A single cell ! Well characterized genetically and

physiologically! Can be readily grown in both small

vessels and large scale bioreactors! Several strong promoters have been

isolated and characterized

Advantages of Yeast Advantages of Yeast expression systemexpression system

! Carry out many post-translational modifications (phosphorylation, glycosylation and targeting)

! Readily grown in small and large scale bioreactors! secretes few proteins, the product can easily

be purified! generally recognized as safe (GRAS)! extensive screening of products is not

required.

S. S. cerevisiaecerevisiae expression vectorsexpression vectors

! Episomal, or plasmid vectors! Integrating vectors! Yeast artificial chromosomes (YACs)

Yeast expression vectorYeast expression vector

! Most widely used are the E. coli/yeast shuttle plasmids

! Mitotically stabilized by autonomously replicating sequences – ARS/CEN region, 2! locus or by integration into the yeast genome

EpisomalEpisomal vectorsvectors

! Introduced by transformation into competent cells or electroporation

! Used extensively for the production of heterologous proteins

! Such plasmid-based expression systems are often unstable under conditions of large scale growth (>10 litres)

YACYAC

! The YAC is designed to clone large fragments of DNA (100 kb)

! The YAC is maintained as a separate chromosome in the host yeast cell

! The YAC is highly stable

USES of USES of YACsYACs

! the physical mapping of human genomic DNA

! the analysis of large transcription units! the formation of genomic libraries

containing DNA from individual human chromosomes

YAC cloning system

Recombinant ProteinsRecombinant Proteins--S. S. cerevisiaecerevisiae

Direct expression in yeastDirect expression in yeast

! Produces proteins that accumulate in the cytoplasm

! Production of human enzyme superoxidedismutase

HeterologousHeterologous gene expression gene expression in S. in S. cerevisiaecerevisiae

! Clone the human Cu/Zn-SOD cDNA into yeast episomal expression vector to obtain this authentic enzyme.

! The cDNA is used as yeast cells do not remove introns.

! If this was done in an E. coli vector there would be a problem with post transcription modification as the E. coli host cell only removes the initiator N-terminal methionine f-met from the Cu/Zn-SOD protein and the next amino acid alanine is not actylated

S. S. cerevisiaecerevisiae expression expression vectorvector

Secretion of Secretion of heterologousheterologousproteinsproteins

! In yeast only secreted proteins are glycosylated

! Facilitated by pre pro-"-factor or leader peptide

! Active proteins are released to the extracellular environment

! Leader peptide is removed by a yeast endoprotease

Purification of proteinsPurification of proteins

! Including an export signal in-frame with His-tag

! Purified by Ni-NTA affinity chromatography

Limitations of Yeast Limitations of Yeast expression systemsexpression systems

! Loss of plasmid even when inducible promoters are used

! The heterologous protein is often hyperglycosylated > 100 mannose residues

! Protein retained within the periplasmicspace, this makes it difficult to purify the protein

Cultured Insect Cell Cultured Insect Cell Expression systemExpression system! Baculovirus vectors used to

heterologously express proteins in insect cells

! Based on the ability of the virus to infect and multiply in cultured insect cells

BaculovirusBaculovirus vectorsvectors

! Most widely used virus Autographacalifornica nuclear polyhedrosis virus (AcNPV)

! A lytic virus that infects lepidopterans! E.g. the fall armyworm, Spodoptera

frugiperda cell lines Sf9 and Sf21

Advantages of insect cellsAdvantages of insect cells

! Recognizes most vertebrate protein-targeting sequences and thus express a wide variety proteins

! Many post-translational modification (phoshorylation, glycosylation, precursor processing, and targeting)

! Recombinant protein can either be produced within the cell or secreted into the culture medium

BaculovirusBaculovirus Expression Vector Expression Vector SystemSystem! Foreign gene cloned into a transfer vector

based on E. coli plasmid that carries a segment of the DNA from AcNPV

! Co-transfected along with ds-baculovirusDNA into insect cells

! Homologous recombination of the transfer vector with insert DNA with viral genome leads to the cloned gene being transferred into the AcNPV DNA.

! Heterologous proteins after 4-5 days

BaculovirusBaculovirus systemsystem

! Heterologous gene-expression levels can vary approx 1000-fold

! Depends on the intrinsic nature of the gene and encoded protein

Recombinant protein Recombinant protein produced by produced by baculovirusbaculovirus

Mammalian cellsMammalian cells

! Mammalian cells the best host for the expression or recombinant vertebrate proteins

! Produce the same post-translational modifications and recognize the same signals

! Expression levels are usually low

Mammalian cell expression Mammalian cell expression VectorsVectors! Contain an efficient promoter elements for

high level transcriptional initiation! Contain mRNA processing signals! Contain selectable markers! Plasmid sequences for propagation in

bacterial hosts

Mammalian cell expression Mammalian cell expression VectorsVectors

! contains a eukaryotic origin of replication from an animal virus, e.g. Simian virus 40 (SV40)

! Origin of replication from E. coli! promoter sequences that drive both the

cloned gene(s) and the selectable marker gene(s)

! transcription termination sequences -adenylation signals from animal virus e.g. SV40

Mammalian expression Mammalian expression vectorsvectors

! Versatile and effective! Used for the production of authentic

recombinant proteins for research and clinical trials

! Industrial production using engineered mammalian cells is costly

Reference Reference

! Molecular Biotechnology: Principles and applications of recombinant DNA. Glick and Pasternak 2nd edition. Chapter 7