EXPRESSION OF ERYTHROPOIETIN IN pET-28a VECTOR

BY AYESHA KHALIQ

ERYTHROPOIETIN

a glycoprotein - primary regulator of RBCs in mammals

stimulates bone marrow stem cells to differentiate into RBCs

controls hemoglobin synthesis

RBCs concentration

Human EPO is a 30,400-dalton molecule

contains 165 amino acids and four carbohydrate chains that

incorporate sialic acid residues

several forms of EPO - differ only in the carbohydrate content

In infants, EPO is produced mostly in the liver.

EPO synthesis from kidneys - shortly after birth

Its production is stimulated by reduced oxygen

content in arterial blood in the kidneys.

Circulating EPO binds to receptors on the

surface of erythroid progenitor cells that in turn

mature into RBCs.

Figure 1: Synthesis of red blood cells.

FUNCTIONS

RBCs production

The primary role of erythropoietin is an essential

hormone for red blood cell production. Without it,

definitive erythropoiesis does not take place.

Nonhematopoietic rolesErythropoietin has a range of actions including vasoconstriction-dependent hypertension stimulating angiogenesis inducing proliferation of smooth

muscle fibers increase iron absorption improves memory  may have effects on mood

COMMERICAL IMPORTANCE

Human EPO was first isolated and later purified

from urine in the 1970s.

Devised recombinant DNA methods to produce

EPO by the mid-1980s.

To treat anemia, primarily kidney failure, HIV

infection in patients treated with AZT, and

cancer chemotherapy.

COMMERCIAL BRANDS

5 types of erythropoiesis-stimulating agents currently

available; epoetin-alpha, epoetin-beta, epoetin-

omega, epoetin-delta, and darbepoetin-alpha.

All have the same amino-acid sequence, but glycosylation

varies as a result of type- and host cell specific differences

in the production process.

Darbepoetin-alpha is an erythropoietin analogue, carrying

two additional glycosylation sites, which produces a longer

half-life and potency.

Epoetin alfa

In 1983, American genetic research

corporation, Amgen,synthesized epoetin-alfa 

under the name Epogen.

They used the E. coli, baker's yeast, and a

number of mammalian cell lines, including the

Chinese Hamster Ovary cell line to produce

EPOGEN®.

Epoetin-alfa is formulated as a colorless liquid.

Epoetin beta

In 1988 a German pharmaceutical company

produced its own recombinant

erythropoietin; epoetin-beta, marketed

as NeoRecormon.

The clinical efficacy of both epoetin-alfa and

epoetin-beta is similar.

Darbepoetin alfa

In 2005, Amgen patented a new

erythropoietic, darbepoetin alfa, under

the brand name Aranesp® 

Although very similar to EPO, Aranesp®,

when administered, has a longer active

life than EPO.

Epoetin delta

This is one of the newest agents currently available.

Called DYNEPO®, - produced from human cell lines

Currently marketed by Shire.

DYNEPO® acts like other epoetins.

It has received considerable attention in the sports

world because DYNEPO® resembles human EPO

and may not be detected by standard urine tests.

SOURCES

Initially EPO was isolated and purified from aplastic anemia patients' urine, in the end of the 70s.

Isolation and characterization of the DNA region were achieved by obtaining the sequencing of physiologic EPO's amino acids, following a path reverse from its protein synthesis.

METHODOLOGY

messenger RNA (m-RNA) was isolated from

fetal liver cells

cDNA generated by using reverse

transcriptase

cDNA was incorporated to

bacterial plasmids

placed to a host E.

coli bacterium

contained the full genetic

information for EPO production

FIRST CLONED HEMATOPOIETI

C GROWTH FACTOR

SEQUENCE RETRIVAL FROM NCBI

Homo sapiens erythropoietin (EPO),

mRNA

1340 bp mRNA linear

Accession no. NM_000799

Source: Homo sapiens (human)

Exons: 5

For the expression of native protein, firstly restriction sites are introduced

in the EPO gene through primers.

EXPRESSION OF NATIVE PROTEIN

PRIMER DESIGNING

These restriction sites will be NcoI and

NdeI.

By using NEBcutter it is made sure that

these sites do not occur in the gene

sequence.

FORWARD PRIMER

5’- CCACCATGGGAGATGGGTGTGCAT -3’

NcoI site

REVERSE PRIMER

5’- GGACATATGTGGTCATCTGTC -3’

NdeI site

The above mentioned set of primers was used for the amplification of the EPO gene. As a result the amplified PCR product had the

restriction sites, NcoI and NdeI, incorporated in the flanking regions of the gene.

POLYMERASE CHAIN REACTION

PCR PRODUCT

The amplified gene (PCR product) and vector pET-28a were restricted with NcoI and NdeI restriction enzymes.

•After restriction the vector and gene were ligated by using ligase enzyme.

NATURALLY OCCURRING PROTEIN

>gi|219518752|gb|AAI43226.1| EPO protein [Homo

sapiens]

MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEA

ENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLAL

LSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQEA

ISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

NATIVE PROTEIN EXPRESSED IN BL-21 CELLS

RESULTS

The comparison of naturally expressing

protein and native protein expressed in

BL-21 show that the protein produced in

vitro is functional.

For the expression of fusion protein, another two restriction sites are

introduced in the EPO gene through primers.

EXPRESSION OF FUSION PROTEIN

PRIMER DESIGNING

These restriction sites will be NdeI and

XhoI.

By using NEBcutter it is made sure that

these sites do not occur in the gene

sequence.

FORWARD PRIMER

5’- CCACATATGGAGATGGGTGTGCAT -3’

NdeI site

REVERSE PRIMER

5’- GGACTCGAGTGGTCATCTGTC -3’

XhoI site

The above mentioned set of primers was used for the amplification of the EPO gene. As a result the amplified PCR product had the

restriction sites, NdeI and XhoI, incorporated in the flanking regions of the gene.

POLYMERASE CHAIN REACTION

PCR PRODUCT

The amplified gene (PCR product) and vector pET-28a were restricted with NdeI and XhoI restriction enzymes.

•After restriction the vector and gene were ligated by using ligase enzyme.

NATURALLY OCCURRING PROTEIN

>gi|219518752|gb|AAI43226.1| EPO protein [Homo

sapiens]

MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEA

ENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLAL

LSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQEA

ISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

FUSION PROTEIN EXPRESSED IN BL-21 CELLS

RESULTS

The comparison of naturally expressing

protein and fusion protein expressed in

BL-21 shows that the protein produced in

vitro is not only functional but is also

fused with a N-terminal histidine tag

which can be used for the purification of

the EPO protein.

THANK YOU