HETEROLOGOUS PROTEIN PRODUCTION IN YEASTS

PAPER- 6Genetic Engineering Techniques And Its Applications.

UNIT- 2 Manipulation of gene expression in Prokaryotes and Eukaryotes.

SUB UNIT- 2.3. Promoters and gene expression in Eukaryotes.

2.3.3

Heterologous Protein Production In Yeast

SYNOPSIS

• Heterologous Proteins.• Yeast – a brief introduction. • Yeast Vectors. • Overview of production process.• Protein expression & secretion.• Factors affecting intracellular expression.• Other yeast species used for production.• Commonly manufactured proteins.• Conclusion.• References.

HETEROLOGOUS PROTEINS

• As the name suggests, they are composed of monomeric or multimeric forms of protein.

• They are a class of highly complex , folded & of high molecular weight proteins.

• They are synthesized through complex cycles and in limited quantities.

• They may be synthesized and secreted in specific organs.

• When they are produced synthetically, they are referred as Recombinant proteins.

• Examples – Insulin, Somatostatin , Growth factors , Virus antigens, albumin, Heparin , etc.

Insulin

Somatostatin

YEASTS• Eukaryotic , Unicellular micro-organism classified

in Kingdom Fungi.• Size is about 3 to 4 µm in diameter.• Chemoorganotrophs.• Reproduce Asexually(budding) & sexually(under

stress).• Saccharomyces cerevisae – most commonly used

species. • It was the first eukaryote whose genome was fully

sequenced.• Fermentation of sugars by yeast is used for

making alcoholic beverages ( wine, beer, rum, whiskey etc.) & as leavening agent for baking.

• Serves as a model organism for research in modern cell biology, genetics, biotechnology etc.

DAIGRAMATIC STRUCTURE

LIFE CYCLE OF YEAST

LIVE FORMS OF YEAST

DRIED YEAST

WHY IS YEAST PREFERRED ?

• Can be cultured easily in small vessels or large bioreactors.

• Well known genetically & physiologically.

• Can be easily manipulated.• Several promoters isolated. • Naturally occuring 2µm plasmid. • Capable of post -translational

modifications.• Product can be readily purified.• Recognized safe (GRAS) organism by

US Food & Drug Administration.

YEAST VECTORS • There are 3 types of yeast expression vectors .o Episomal or plasmid vectors (YEps)o Integrating vectors (YIps) o Yeast artificial chromosomes (YACs) • Of these Yeps are extensively used, as it

consists of high copy number 2µm plasmid. • Whereas contribution of YACs still needs to be

explored.• A typical yeast vector consists of : Ori ,

Promoter, Selection marker, Terminator & Polylinker.

• YACs in addition have Centromeric & Telomeric sequences which are host specific.

PLASMID VECTOR ARTIFICIAL CHROMOSOME

OVERVIEW OF PRODUCTION PROCESS

The production of heterologous proteins in S. cerevisae involves two major steps :

1. Introduction of foreign DNA into the host cells. This step has three major considerations:(a) Identification and isolation of the DNA to be

introduced(b) Identification of the vector and construction of

recombinant vector(c) Identification of the suitable expression system

to receive r DNA.

2. Factors affecting the expression of foreign DNA for

protein synthesis in the chosen expression system.

INTRACELLULAR PRODUCTION

• The initial step is production of protein strain.

• This is done by inserting the gene of interest between the promoter and termination sequence in the yeast vector .

• For eg. cDNA for human Cu\Zn – SOD was cloned between the promoter (GAPDp) & termination –polyadenylation sequence (GAPDt) of S.cerevisae glceraldehyde phosphate dehydrogenase gene.

INSERTION OF GENE OF INTEREST

gene ofinterest

knock-out stain

out-recombination

amdS

PROTEIN EXPRESSION STRAIN

gene of interest

amdS

homologous integration

LAC4 locus

gene of interest

gene of interest

MULTI COPY INTEGRATION gene of

interest

multi-copy integration

gene conversion

SECRETION OF PROTEINS

• The heterologous protein that are synthesized in yeast cells are usually secreted in the form of glycosylated proteins .

• Each protein must have a leader sequence along with O-linked or N-linked sugars.

• At first a correct Di-sulfide bond is formed leading to removal of leader sequences .

• Correct post translational modifications make the protein active .

• Thus an active recombinant protein is secreted.

Diagrammatic representation of secretion

of protein from cell

FACTORS AFFECTING INTRACELLULAR EXPRESSION

• Initiation of Transcription • RNA elongation • Incorporation of target gene into plasmid• Initiation of translation • Translational elongation • Polypeptide folding • Post translational processing • Amino terminal modifications • Glycosylation • Modification of amino acid within proteins• Stability of plasmids • Stability of proteins

PROBLEMS ASSOCIATED WITH S. cerevisae

• Low expression & modest yield.• Proteins are often hyperglycosylated.• Excess mannose residues alters the function

& makes the secreted protein antigenic. • Sometimes proteins are retained in the

periplasmic space & this increases the cost of purification.

• It also produces ethanol at high cell densities, which is toxic to the cells.

OTHER EXAMPLES OF YEAST USED FOR PRODUCTION OF HETEROLOGOUS PROTEINS

• Researchers examined many species of yeast and introduced some non saccharomyces species for production of heterologous proteins.

• The two most widely used yeasts species are Pichia pastoris & Kluveromyces lactis.

• Other yeast which are used include Hansela polymorpha, Candida albicans, etc.

• It has a highly efficient & tightly regulated promoter.

• It does not synthesize ethanol.

• It secretes very few protein, thus simplifying the purification of secreted recombinant proteins.

• It uses the integrating plasmid expression vector.

Pischia pastoris

3ᶦAOX 1

HIS 4

Ori

Amp r gene

Gene of interest

P. pastoris integrating expression vector

Kluyveromyces lactis

It has been used since 1950’s in preparation of several foodstuffs.1st heterologous enzyme bovine chymosin (milk clotting factor) was prepared from it. Today over 40 heterologous protein are produced from K. lactis.It can integrate YIP’s & YEP’s.

COMMONLY MANUFACTURED PROTEINS

Recombinant proteins produced by S. cerevisae expression system are as follows :

VACCINES• Hepatitis B virus surface antigen • Malaria circumsporozoite protein • HIV-1 envelope protein DIAGNOSTICS • Hepatitis C virus protiens• HIV-1 antigens

HUMAN THERAPEUTIC AGENTS

• Epidermal growth factor • Insulin • Insulin- like growth factor • Platelet- derived growth factor • Proinsulin • Fibroblast growth factor • Granulocyte- macrophage colony

stimulating factor• Antitrypsin • Blood coagulation factor XIIIa• Hirudin • Human growth factor • Human serum albumin

CONCLUSION• Production of heterologous proteins from

micro- organisms should be considered as an art .

• Researchers have begun to understand mechanisms involved in protein production and thus are willing to develop steps to increase protein production .

• Apart from production, stabilization & formulation of proteins also should be studied carefully for therapeutic & industrial purposes.

REFERENCESBOOKS• Molecular Biotechnology – Glick & Pasternak• Microbial Biotechnology – Glazer & Hiroshi• Basic Biotechnology – Ratledge & Kristiansen• Biotechnology –Satyanarayana• Molecular biotechnology- Channarayappa

WEBSITES• http://www.ias.ac.in/currsci/

may102001/1121.pdf• http://en.wikipedia.org/wiki/

List_of_recombinant_protein

THANK YOU