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. 2.3.3Heterologous 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.
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.
OVERVIEW OF PRODUCTION PROCESSThe 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 forprotein 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 CuZn – SOD was cloned between the promoter (GAPDp) & termination – polyadenylation sequence (GAPDt) of S.cerevisae glceraldehyde phosphate dehydrogenase gene.
INSERTION OF GENE OF INTEREST amdS gene of interest out-recombination knock-out stain
PROTEIN EXPRESSION STRAIN gene of amdS interest LAC4 locus gene of homologous integration 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.
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.
3ᶦ 1 AOX Ori HIS 4 Amp r geneP. pastoris integratingexpression vector Gene of interest
Kluyveromyces lactisIt has been used since 1950’s inpreparation of severalfoodstuffs.1st heterologous enzyme bovinechymosin (milk clotting factor)was prepared from it.Today over 40 heterologousprotein are produced from K.lactis.It can integrate YIP’s & YEP’s.
COMMONLY MANUFACTURED PROTEINSRecombinant proteins produced by S. cerevisae expression system are as follows :VACCINES• Hepatitis B virus surface antigen• Malaria circumsporozoite protein• HIV-1 envelope proteinDIAGNOSTICS• 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.