Co- and Post-translational Co- and Post-translational Protein Modifications of Protein Modifications of

Therapeutics & BiopharmaceuticalsTherapeutics & BiopharmaceuticalsByBy

Dr. Mohamed Ramadan Hassan AttallaDr. Mohamed Ramadan Hassan AttallaDirector of Biotech. / Bio-similar Products & Director of Biotech. / Bio-similar Products &

Recombinant DNA / Protein Vaccines R&D Center – R&D SectorRecombinant DNA / Protein Vaccines R&D Center – R&D SectorVACSERA , EgyptVACSERA , Egypt

PTMs are covalent chemical modification PTMs are covalent chemical modification either during their ribosomal either during their ribosomal translationtranslation or often after or often after itsits synthesis is complete synthesis is complete..

Key role in functional Proteomics.Key role in functional Proteomics. Regulate activity, localization and Regulate activity, localization and

interaction with other cellular interaction with other cellular molecules, e.g., proteins, nucleic acids, molecules, e.g., proteins, nucleic acids, lipids and cofactors.lipids and cofactors.

IntroductionIntroduction

PhosphorylationPhosphorylation

Types of PTMsTypes of PTMs

GlycosylatioGlycosylationn

MethylationMethylation

ProteolysisProteolysis

UbiquitinationUbiquitination

AmidationAmidation

N-AcetylationN-Acetylation

S-NitrosylationS-Nitrosylation

SulfationSulfation

CarboxylationCarboxylation

LipidationLipidation PrenylatioPrenylationn

Pharmaceutical Proteins.Pharmaceutical Proteins. Pharmaceutical Enzymes.Pharmaceutical Enzymes. Monoclonal Antibodies.Monoclonal Antibodies. Recombinant Antibodies.Recombinant Antibodies.

Proteolytic Processing of ProinsulinProteolytic Processing of Proinsulin

ProteinProtein CompanyCompany DisorderDisorderFactor VIIIFactor VIII Baxter, BayerBaxter, Bayer Hemophilia AHemophilia AFactor IXFactor IX Genetics InstituteGenetics Institute Hemophilia BHemophilia BTissue plasminogen Tissue plasminogen activator (TPA)activator (TPA)

GenetechGenetech Acute myocardial Acute myocardial infarctioninfarction

InsulinInsulin Eli Lilly, Novo Eli Lilly, Novo NordiskNordisk

Diabetes mellitusDiabetes mellitus

Human growth Human growth hormonehormone

Eli Lilly, Genetech, Eli Lilly, Genetech, Upjohn, Novo Upjohn, Novo NordiskNordisk

GH deficiency in GH deficiency in children children (dwarfism)(dwarfism)

ErythropoietinErythropoietin Amgen, Ortho Amgen, Ortho BiotechBiotech

AnemiaAnemia

DNase IDNase I GenetechGenetech Cystic fibrosisCystic fibrosisVarious interferons Various interferons (IFN)(IFN)

Schering, Biogen, Schering, Biogen, Chiron, GenetechChiron, Genetech

Hepatitis B and C, Hepatitis B and C, multiple sclerosismultiple sclerosis

Example: A simple Example: A simple E. coliE. coli expression vector utilizing the expression vector utilizing the laclac promoter promoter. (a) The expression vector plasmid contains a . (a) The expression vector plasmid contains a fragment of the fragment of the E. coliE. coli chromosome containing the chromosome containing the laclac promoter and the neighboring promoter and the neighboring lacZlacZ gene. In the presence of the gene. In the presence of the lactose analog IPTG, RNA polymerase normally transcribes the lactose analog IPTG, RNA polymerase normally transcribes the lacZlacZ gene, producing gene, producing lacZlacZ mRNA, which is translated mRNA, which is translated into the encoded protein, b-galactosidase. (b) The into the encoded protein, b-galactosidase. (b) The lacZlacZ gene can be cut out of the expression vector with restriction gene can be cut out of the expression vector with restriction enzymes and replaced by the Granulocyte-Colony Stimulating Factor enzymes and replaced by the Granulocyte-Colony Stimulating Factor G-CSF cDNAG-CSF cDNA. When the resulting plasmid is . When the resulting plasmid is transformed into transformed into E. coliE. coli cells, addition of IPTG and subsequent transcription from the cells, addition of IPTG and subsequent transcription from the laclac promoter produces G-CSF promoter produces G-CSF mRNA, which is translated into G-CSF protein.mRNA, which is translated into G-CSF protein.

Expression systems are based on the insertion of a gene Expression systems are based on the insertion of a gene into a host cell for its translation and expression into into a host cell for its translation and expression into protein. Host cells include :protein. Host cells include :

Bacteria, e.g., Bacteria, e.g., Escherichia coli (E.coli), Bacillus subtilisEscherichia coli (E.coli), Bacillus subtilis ( (B. subtilisB. subtilis))Yeast Yeast Cultured insect cells Cultured insect cells Cultured mammalian cellsCultured mammalian cells

The choice of cell type used depends upon the protein to The choice of cell type used depends upon the protein to be expressed. All require DNA to be cloned into the an be expressed. All require DNA to be cloned into the an appropriate vector.appropriate vector.

Advantages of bacterial cells :Advantages of bacterial cells : Simple physiology.Simple physiology. Short generation times, as bacteria grow and multiply rapidly.Short generation times, as bacteria grow and multiply rapidly. Large yields of product - up to 10 % of mass (low cost).Large yields of product - up to 10 % of mass (low cost).

With B. subtilis and some others, it is possible to induce secretion of a gene With B. subtilis and some others, it is possible to induce secretion of a gene product into the surrounding medium. This method is in use in the product into the surrounding medium. This method is in use in the pharmaceutical industry in the production of hormones such as insulin and pharmaceutical industry in the production of hormones such as insulin and human growth hormone.human growth hormone.Disadvantages of bacterial cells :Disadvantages of bacterial cells :

The expressed proteins often do not fold properly and so are biologically The expressed proteins often do not fold properly and so are biologically inactive.inactive.

The synthesised protein is often toxic to bacteria preventing the cell cultures The synthesised protein is often toxic to bacteria preventing the cell cultures from reaching high densities. A solution to this problem is to incorporate an from reaching high densities. A solution to this problem is to incorporate an inducible promoter, which may be turned on to transcribe the inserted gene inducible promoter, which may be turned on to transcribe the inserted gene after the culture has been grown after the culture has been grown

Lack of enzymes responsible for post-translational modifications (effect on Lack of enzymes responsible for post-translational modifications (effect on function of proteins), e.g., if the protein to be expressed is a glycoprotein, there function of proteins), e.g., if the protein to be expressed is a glycoprotein, there is not apparatus in the bacterium to 'stick on' the necessary sugar residues.is not apparatus in the bacterium to 'stick on' the necessary sugar residues.

Advantages of yeast cells :Advantages of yeast cells : Yeast is a simple eukaryote and performs many of the post-Yeast is a simple eukaryote and performs many of the post-

translational modifications required for human proteins.translational modifications required for human proteins. Can be induced to secrete certain proteins into the growth Can be induced to secrete certain proteins into the growth

medium for harvesting - e.g. Hepatitis B virus (HBV) vaccine.medium for harvesting - e.g. Hepatitis B virus (HBV) vaccine.Disadvantages of yeast cells :Disadvantages of yeast cells :

Presence of active proteases that degrade foreign (expressed) Presence of active proteases that degrade foreign (expressed) proteins, thereby reducing their yield (a solution to this problem proteins, thereby reducing their yield (a solution to this problem is the construction of yeast strains from which the protease genes is the construction of yeast strains from which the protease genes have been deleted).have been deleted).

Insect Cells :Insect Cells : Expression of foreign proteins in insect cells Expression of foreign proteins in insect cells through incorporation of their genes into baculovirus vectors through incorporation of their genes into baculovirus vectors

Advantages of insect cells :Advantages of insect cells : High level of expression.High level of expression. Correct folding.Correct folding. Post-translational modifications similar to those in mammalian cells.Post-translational modifications similar to those in mammalian cells. Cost, though more than for culturing bacteria and yeast, less than for Cost, though more than for culturing bacteria and yeast, less than for

mammalian cells e.g. potential vaccine for AIDS virus produced by mammalian cells e.g. potential vaccine for AIDS virus produced by expression of one of the HIV glycoproteins with this system.expression of one of the HIV glycoproteins with this system.

Disadvantages of insect cells :Disadvantages of insect cells : More difficult to work with.More difficult to work with. Expensive.Expensive. Slow generation time.Slow generation time. Not suitable for proteins with repetitive sequences.Not suitable for proteins with repetitive sequences.

Type of Type of antibodyantibody

CompanyCompany Therapeutic useTherapeutic use

Mouse, Mouse, HumanizedHumanized

Ortho Biotech, Protein Ortho Biotech, Protein Design, Hoffmann-Design, Hoffmann-LaRocheLaRoche

Prevents kidney Prevents kidney transplant rejectiontransplant rejection

ChimericChimeric CentocorCentocor Prevents blood clotsPrevents blood clotsChimericChimeric Genetech, Hoffmann-Genetech, Hoffmann-

LaRocheLaRocheNon-Hodgkin Non-Hodgkin lymphomalymphoma

Humanized Humanized (Herceptin)(Herceptin)

GenetechGenetech HER2-positive breast HER2-positive breast cancers cancers

HumanizedHumanized Am Home Prod, Celltech, Am Home Prod, Celltech, Schering, Millen. Pharm.Schering, Millen. Pharm.

Certain leukemiasCertain leukemias

HumanizedHumanized GenetechGenetech AsthmaAsthma

Genetically Engineered Genetically Engineered Humanized AntibodyHumanized Antibody

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