8/11/2019 Biotech Productc

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Cloned Genes and

Production of GrowthHormones, Vaccines and

Commercial Chemicals

Human Peptide HormoneGenesIn the human body, peptidehormones are secreted afterencoding by peptide hormone genesin the specialized cells, for instanceinsulin and other Human GrowthHormones (hGH).

Insulin

This peptide hormone i.e. insulin issecreted by the Islets of Langerhansof pancreas which catabolizesglucose in blood. Insulin is a boonfor the diabetics whose normalfunction for sugar metabolismgenerally fails.

Insulin consists of two polypeptidechains, chain A (21 amino acid long)and B (30 amino acid long). Its

precursor is proinsulin which alsocontains two polypeptide chains, Aand B, and is connected with a thirdpeptide chain-C (35 amino acidlong). However, the recentdiscoveries reveal that precursor ofinsulin is pre- pro insulin which isabout 109 amino acids long. Thepre-pro- insulin is synthesized inbeta cells of pancreas, the structureof which is given below :

NH2-(Peptide)--Chain- (peptide-c)-A chain-COOH

In the beginning, efforts were madeto isolate mRNA for pre- pro- insulinfrom rats Islets of Langerhans ofpancreas and to synthesize cDNA.Thereafter, it was inserted into aplasmid. The recombinant plasmidswere transferred into the E. coli cells

which secreted pro-insulin.

ContentCloned genes and production ofchemicalsHuman peptide hormone genesInsulines

SomatotropinSomatostatin

-endorphinHuman interferon genesGenes for vaccinesVaccine for hepatitis-B virusVaccines for Rabies virusVaccines for poliovirusVaccine for foot and mouthdisease virusVaccines for small pox virus

Malaria vaccinesDNA vaccines

Genes associated with geneticdiseasesPhenylketonuriaUrokinaseThalassaemiaHemophilia

Enzyme engineeringCommercial chemicals

Prevention, diagnosis and cure of

diseasesPrevention of diseases

Diagnosis of diseasesParasitic diseasesMonoclonal antibodiesAntenatal diagnosis

Gene therapyTypes of gene therapyMethods of gene therapySuccess of gene therapy

Potential of gene delivering

system

Future needs of gene therapy

in IndiaDNA profiling (fingerprinting)

Methods of DNA profiling

Application of DNA profiling

Genetic databank

Reuniting the lost children

Solving disputed problems of

parentage, identity of criminals,rapists, etc

Immigrant disputeHurdles of DNA profiling

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8/11/2019 Biotech Productc

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Fig, 5.2. Production of recombinantinsulin in E.coli.

Itakura et. al. (1977) chemicallysynthesized DNA sequence for twochains, A and B, of insulin andseparately inserted into two pBR322plasmids by the side of -galactosidase gene. Therecombinant plasmids wereseparately transferred into

coli cells which secreted fused -galactosidase - A chain and -galactosidase - B chain separately.These chains were isolated bydetaching from -glactosidase inpure form in a amount of about 10mg/24 g of healthy and transformedcells (Sasson, 1984). Production ofrecombinant insulin is shown in Fig.5.2.

Animal and plant improvement

Transgenic Farm Animals

Crop Improvements

Transgenic plants

Nif gene transfer

Phaseolin gene transferConversion of C3plants to

C4plantsHerbicide resistant plants

Insect pest resistant plants

Plant improvement through

genetic transformation

Crop Protection

Use of antagonists

Use of insecticides

Abatement of pollution

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Detachment of proinsulin couldbe possible when an extramethionine codon was added atthe N-terminus of each gene forA and B chains. The two chains

(A and B) were joined invitro to reconstitute the nativeinsulin by sulphonating the twopeptides with sodiumdisulphonate and sodiumsulphite. Gilbert andVillakomaroff (1980) isolatedmRNA for insulin from B cells ofrat's pancreas and inserted intopBR322 plasmid in the middleof a gene normally coding the

penicillinase, and incorporatedit into E. coli cells. E. coli cellsproduced a hybrid protein(penicillinase+proinsulin) fromwhich the proinsulin wasseparated by using trypsin. It isestimated that clones of E.coli are capable of producingabout one million molecules ofinsulin per bacterial cell. Humaninsulin (humulin) is the first

therapeutic product producedby means of recombinanttechnology by Eli Lilly & Co.

SomatotropinSomatotropin, the hGH, issecreted by the anterior lobe ofpituitary glands which consistsof 191 amino acid units. Itssecretion is regulated by twoother hormones (somatostatinand growth hormone releasinghormone) produced byhypothalamus. Deficiency ofsomatotropin in about 3%cases is of hereditary. It hasbeen estimated to about 1 childin 5,000.

Fig. 5.3. Expression of Human Growth Hormone(hGH) in E.coli; Lac P/O, lactose

promoter/operator.

Turner's syndrome is one of the most common chromosome disorders in girlsand it is characterized by short stature and non-functioning of ovaries affectingapproximately 1 in 2,500 live female birth. The extraction of somatotropinpharmaceutically from the pituitary glands could not meet annual demand of thishormone. Biosynthesis of somatotropin was achieved through gene cloningprocedures.

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Double stranded cDNAs were produced from mRNA precursor of hGH which wasthen incorporated into bacterial cells where it expressed in non-precursor form.Bacteria were unable to convert peptide into biologically active form. Arecombinant plasmid containing a full length hGH cDNA (which fails to express)is cleaved with restriction enzymes that release a fragment containing the

complete hGH coding sequence after codon 24 (Fig. 5.3). Several overlappingcomplementary oligonucleotides are ligated to form one synthetic strand of smallDNA fragment which contains the coding sequence for the first 24 aminoacids ofmature hGH (after removal of the N-terminal signal peptide). The synthetic DNAand cDNA molecules are ligated to yield a new fragment which contains thecomplete coding sequence of hGH. It is ligated into a restriction site just downstream of the lac promoter / oprater region cloned on a plasmid. The rDNAplasmids are allowed to transform E.coli. Synthesis of hGH is induced by aninducer of lac operon (i.e.isopropylthiogalactoside (IPTG)). The hGH issubsequently purified. hGH is being produced commercially by this method.

About 100,000 moleculesof hormone per cell of E.coli have been produced(Newmark, 1979). One ofthe major difficulties wasthat at the N-end ofpolypeptide an extramethionine, the met-hGW, was attached.Methods are developed toremove methionine from

the met-hGW molecules.

SomatostatinSomatostatin, a 14residue polypeptidehormone is synthesizedin the hypothalamus. It isthe first polypeptidewhich was expressedin E. colias part of thefusion peptide (Itakura eat, 1977) which inhibitedthe secretion of growthhormone, glucagon andinsulin. It does notcontain any internalmethionine. Eight singlestranded DNA segmentswere synthesizedchemically which wereannealed in anoverlapping manner toform a double strandedDNA (the syntheticgene). It had single

Fig. 5.4. Synthesis of somatostatin, Lac P/O, lactosepromoter/operator, ST, somatostatin; Amp, ampicillin

resistance.

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stranded projections atthe each end as the sameare formed by Eco RI.The synthesized genecontained 51 base pairs

which were terminated bytwo non-sense (stop)codon and preceded by amethioine codon asbelow:

ATG - (42 base pairsencoding somatostatin) -TGATAG.

Two plasmids, pSOMI and

pSOM

11-3, wereconstructed. Thesynthetic gene wasintroduced into E. coli -galactosidase gene atdifferent sites.

The chemically synthesized gene was inserted down stream fromthe lac promoter in such a way that the gene fusion should have specified apolypeptide in which the first 7 amino acids of -galactosidase were fused tosomatostatin. But the somatostatin was not detected in transformed bacteria;

possibly it was degraded in E. coli (Glover, 1994). An alternative plasmid,pSOMII-3, was constructed which had the both lac promoter /operater

and lacL regions. The lacZgene encodes peptide of -galactosidase. If theresulting frame of lacL is maintained after inserting a DNA, a fusion peptide isproduced. The plasmid was cleaved in the lacL segment by restriction enzyme(Fig. 5.4). The synthetic gene was inserted into the plasmid at Eco RI site nearC-terminus of -galactosidase. The plasmid in the transformed bacterium directs

the synthesis of a fused protein consisting of NH2- termined segment of -galactosidase fragment coupled by methionine to somatostatin. It was stabilizedfrom proteolytic degradation by -galactosidase moiety (Glover, 1984). Thisfused protein was purified and treated with cyanogen bromide (CNBr) whichcleaves only protein at the carboxyl side of the methionine. Thus, the methioninelinker remains attached to -galactosidase fragment, and somatostatin wasreleased. Now, it has become possible to inhibit the degradation of foreignprotein in E. coli by introduction of protease inhibition (PIN) gene of T4 phage.

endorphin-endorphin (30 amino acid long neuropeptide with opiate activity) is anothergrowth hormone which was expressed in genetically engineered E. coli cells.

Shine et al. (1980) integrated DNA sequences of -endorphin, obtained from

mRNA, adjacent to -glactosidase genes on plasmid. The mRNA contained largeprecursor of protein that consisted of, besides -endorphin, the hormones -

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