this presentation is about production of heterologous proteins.

1. HETEROLOGOUS PROTEIN
PRODUCTION IN YEASTS

2. PAPER- 6
Genetic 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

3. 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.

4. 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.

5. Insulin Somatostatin

6. 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.

7. DAIGRAMATIC STRUCTURE LIFE CYCLE OF YEAST
LIVE FORMS OF YEAST DRIED YEAST

8. 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.

9. 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.

10. PLASMID VECTOR ARTIFICIAL CHROMOSOME

11. 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.

12. 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 CuZn – SOD was cloned
between the promoter (GAPDp) & termination –
polyadenylation sequence (GAPDt) of S.cerevisae
glceraldehyde phosphate dehydrogenase gene.

13. INSERTION OF GENE OF INTEREST
amdS
gene of
interest
out-recombination
knock-out stain

14. PROTEIN EXPRESSION STRAIN
gene of
amdS
interest
LAC4 locus
gene of
homologous integration
interest
gene of
interest

15. MULTI COPY INTEGRATION
gene of
interest
multi-copy integration
gene conversion

16. 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.

17. Diagrammatic representation of secretion of
protein from cell

18. 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

19. 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.

20. 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.

21. Pischia pastoris
• 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.

22. 3ᶦ 1
AOX
Ori
HIS 4
Amp r
gene
P. pastoris integrating
expression vector Gene of
interest

23. 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.

24. 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

25. 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

26. 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.

27. REFERENCES
BOOKS
• 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