maam hooria

1. EXPRESSION OF
ERYTHROPOIETIN
IN pET-28a
VECTOR
BY AYESHA KHALIQ

2. 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

3.  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.

4. Figure 1: Synthesis of red blood cells.

5. 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.

6. Nonhematopoietic roles
Erythropoietin 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

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. METHODOLOGY

15. 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
HEMATOPOIETIC
GROWTH
FACTOR

16. SEQUENCE RETRIVAL FROM NCBI

20.  Homo sapiens erythropoietin (EPO), mRNA
 1340 bp mRNA linear
 Accession no. NM_000799
 Source: Homo sapiens (human)
 Exons: 5

22. For the expression of native protein, firstly
restriction sites are introduced in the EPO gene
through primers.
EXPRESSION OF NATIVE PROTEIN

23. 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.

25. FORWARD PRIMER
5’- CCACCATGGGAGATGGGTGTGCAT -3’
NcoI site

26. REVERSE PRIMER
5’- GGACATATGTGGTCATCTGTC -3’
NdeI site

27. 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

29. PCR PRODUCT

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

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

33. NATURALLY OCCURRING PROTEIN
>gi|219518752|gb|AAI43226.1| EPO protein [Homo sapiens]
MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCA
EHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVN
SSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQEAISPPDAASAAPLRTITADTFRKLF
RVYSNFLRGKLKLYTGEACRTGDR

34. NATIVE PROTEIN EXPRESSED IN BL-21
CELLS

35. RESULTS
 The comparison of naturally expressing protein
and native protein expressed in BL-21 show that
the protein produced in vitro is functional.

36. For the expression of fusion protein, another
two restriction sites are introduced in the EPO
gene through primers.
EXPRESSION OF FUSION PROTEIN

37. 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.

39. FORWARD PRIMER
5’- CCACATATGGAGATGGGTGTGCAT -3’
NdeI site

40. REVERSE PRIMER
5’- GGACTCGAGTGGTCATCTGTC -3’
XhoI site

41. 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

43. PCR PRODUCT

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

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

47. NATURALLY OCCURRING PROTEIN
>gi|219518752|gb|AAI43226.1| EPO protein [Homo sapiens]
MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCA
EHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVN
SSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQEAISPPDAASAAPLRTITADTFRKLF
RVYSNFLRGKLKLYTGEACRTGDR

48. FUSION PROTEIN EXPRESSED IN BL-21
CELLS

49. 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.

50. THANK YOU