RECOMBINANTRECOMBINANTPROTEINS PROTEINS

RECOMBINANTRECOMBINANTPROTEINS PROTEINS PRESENTED BY:PRESENTED BY:

ASH . KASH . K

INTRODUCTION• Recombinant

proteins are the proteins that are produced by genetically modified organisms following insertion of the relevant DNA into their genome.

• The science of recombinant technology took birth when Cohen & Boyer (1973) were able to introduce a piece of gene containing foreign DNA into plasmid of E.coli .

• TOOLS OF GENETIC ENGINEERING :

ENZYMES

VEHICLE DNA

PASSENGER DNA

RECOMBINANT DNA TECHNOLOGY

• It involves following steps :

1. Isolation of DNA segment

2. Formation of Recombinant DNA

3. Production of multiple copies of recombinant DNA

4. Introduction of recombinant DNA into host

5. Screening of the transformed cells

NEED & APPLICATION :• Recombinant protein expression is the

foundation of today’s biomolecular research and the thriving Biotech industry.

• GOAL: Overproduction of proteins for

Structuralstudies

Enzymaticstudies

Antigen productionCommercial/

Pharmaceuticalapplications

• Genetic engineering produces proteins that offer advantages over proteins isolated from other biological sources.

• These advantages include:

• High Purity• High Specific Activity• Steady Supply• Batch to Batch Consistency

Examples of Recombinant Protein Products

1. HORMONES• Insulin• Human Growth Hormones• Erythropoietin• 2. BLOOD CLOTTING FACTORS Coagulation factor VIII Coagulation factor IX

3. IMMUNIZATION AGENTS Hepatitis B vaccine

4. RESEARCH ENZYMES Restriction endonucleases

5. INTERFERONS

Protein Production- Expression Systems

1. E.coli2. Yeast3. Filamentous

Fungi4. Mammalian

Cells 5. Plant cells6. Insect cells

E.coliADVANTAGES DISADVANTAGES

Fast growing missing or incorrect post-translational modifications e.g. missing glycosylation

Easy to perform •endotoxin contamination

Easy & cheap culture •Difficult purification (inclusion bodies)

High yield producer • Low yield of functional protein

INSECT CELLSAdvantages Disadvantages

•easy to infect glycosylation not as complex as in human systems

• stable integration (due to virus)

•viral proteases degrade target protein

easy purification: cells lyse by themselves after 96 h (due to virus)

•optimization required to establish viral transfection

reliable protein folding

• high yields

MAMMALIAN CELLSADVANTAGES DISADVANTAGES

• (Almost) human glycosylation and phosphorylation pattern

•so far - transfection may be difficult

•highest functionality production rates may be comparably low

lowest immunogenicity and very high compatibility to humans

•selection of single clones is very time-consuming

if non-human cells - not susceptible for human pathogenes

higher costs for culture

• high safety profile, easy permission as a drug

SUMMARY: Protein production – expression systems

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Mammalian cell lines

Insect cell lines

Bacterial expression systems

Han

dlin

g &

costs

Qu

ality

of

pro

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Sp

eed

of

pro

cess

60-70% of all recombinant protein pharmaceuticals are produced in mammalian cells

WHY SYNTHESIZE HUMAN INSULIN ?

•Patients’ immune systems do not produce antibodies against human insulin as they do with bovine or porcine insulin

•Projected decline in the production of animal-derived insulin

•Need for a more reliable and sustainable method of obtaining the • product.

•Protein hormone produced by beta cells of islets of Langerhans in the pancreas

•Regulates blood sugar by allowing uptake of glucose from bloodstream into body cells

•Patients with diabetes have insufficient or impaired production of insulin

STRUCTURE OF INSULIN

• Two polypeptide chains; one with 21 amino acids and the second with 30 amino acids

• Chains are linked via a disulfide bond

• Gene encoding the insulin protein is found on chromosome 11

Production of recombinant insulin in E.coli

Human Insulin (Humulin) is the first therapeutic product produced by recombinant technology by Eli Lilly & Co in 1980.

• STEPS1. Synthesis of artificial genes coding for A & B chains of

insulin2. Construction of 2 recombinant plasmids 3. In each case artificial gene was ligated to a lacZ’ reading

frame present in a pBR322-type vector4. The recombinant plasmids were separately transferred into

E.coli cells which secreted fused B-galactosidase-A chain & B-galactosidase-Bchain separetely5. These chains were isolated by detaching from B-

galactosidase in pure form6. Detachment of proinsulin could be possible when an extra

methionine codon was added at the N’- terminus of each A & B chains

7. The 2 chains A & B were joined invitro to reconstitute the native insulin by sulphonating the 2 peptides with Na disulphonate & Na sulphite.

8. Final product is Humulin - chemically identical to human insulin

Synthesis of Human Growth hormones in E.coli

• Somatostatin & Somatotropin are the 2 proteins that act in conjugation to control growth processes in human body, their malfunction leads to painful and disabling disorders such as acromegaly ( uncontrolled bom growth and dwarfism)

• Early research showed that the condition could be treated w/ injections of human growth hormone

Growth hormone could only be obtained from human pituitary glands

These were obtained from cadavers Later studies showed that the cadaver supplied

GHs were often contaminated, so other methods needed to be developed to artificially produce human growth hormone

Production of recombinant Somatostatin

• Being a very short protein, only 14 a.a in length, it was ideally suited for artificial gene synthesis.

• Strategy involved Insertion of

artificial gene into lacZ’ vector

Synthesis of fusion protein

Cleavage with cyanogen bromide

Production of recombinant Somatotropin

• Somatotropin presented a more difficult problem

• This protein is 191 a.a in length, equivalent to almost 600 bp, a dificult prospect for today’s DNA synthesis capabilities

• Infact a combination of artificial gene synthesis and cDNA cloning was used to obtain a somatotropin-producing E.coli strain.

• mRNA was obtained from pituitary gland & a Cdna library prepared

• Somatotropin Cdna turned out to have a unique site for the restriction endonuclese HaeIII, which cuts the gene into 2 segments

• The longer segment, consisting of codons 24-191, was retained for use in construction of the recombinant plasmid

• The smaller segment was replaced by an artificial DNA molecule that reproduced the start of the somatotropin gene and provided the correct signals for translation in E.coli

• The modified gene was then ligated into an expression vector carrying the lac promoter

• Production of vaccines through recombinant DNA technology 1.INJECTABLE HEPATITIS B VACCINE

• 2. EDIBLE VACCINE

PROTEIN PURIFICATION

• Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture.

• Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest.

• The various steps in the purification process separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins.

• Separation steps exploit differences in protein size,

physico-chemical properties and binding affinity

Methods of Protein Purification

Precipitation and differential solubilization

Ultracentrifugation

Chromatographic Methods Affinity Chromatography Ion exchange chromatography HPLC

AFFINITY CHROMATOGRAPHY

• Affinity chromatography is a method of separating biochemical mixtures, based on a highly specific biologic interaction such as that b/w Ag & Ab, enzyme & substrate or receptor & ligand

List of Recombinant Proteins

• Human Recombinants that largely replaced animal or harvested from human types

Human growth hormone(rhGH) Humatrope from Lilly & Serostim from Serono replaced cadaver harvested hGH

Human insulin(rhI) Humulin from Lilly & Novo Nordisk among others; largely replaced bovine & porcine insulin for human therapy

Factor V111 Kogenate from Bayer replaced blood harvested factor V111

• Human Recombinants with Recombination as only source

Erythropoietin(EPO) Epogen from Amgen Granulocyte colony-stimulating factor ( G-CSF) sold as

Neupogen from Amgen DNAse by Genetech Tissue plasminogen activator (TPA) Activase by Genetech

• Animal Recombinants Bovine somatotropin (bST) Porcine somatotropin (pST) Bovine Chymosin

• Viral Recombinants Envelope protein of the hepatitisB virus marketed as

Energix-B by SmithKline Beecham