WHAT IS RECOMBINANT HEMOGLOBIN, PREPARATION METHOD, ETC

1. RECOMBINANT HEMOGLOBIN
BY-
VIBHA SINHA
M.Sc. BIOTECHNOLOGY

2. BLOOD
• The Red liquid that circulates in the arteries and veins of humans and
other vertebrate animals, carrying oxygen to and carbon dioxide from
the tissues of the body.
• It comprises of the following type of cells:-
• Red Blood Cells
• White Blood Cells
• Blood Platelets

3. FUNCTIONS OF BLOOD

4. HEMOGLOBIN
• Hemoglobin (Hb) is a complex of proteins
found in red blood cells
• It is composed of four globin protein
tetramer where there are two α subunits,
and two β subunits
• Iron is present in ‘Heme’ form

5. ARTIFICIAL BLOOD
• Artificial blood is a product made to act as a
substitute for Red Blood Cells
• Derived specifically for the sole purpose of
transport of O2 and CO2
• Designed to meet emergency conditions of
blood scarcity
• Generally, two types of oxygen carriers used
as blood substitutes-
• Perfluorocarbons (PFC)
• Hemoglobin-based Oxygen Carriers (HbOC)

6. RECOMBINANT HEMOGLOBIN
• Artificially synthesized haemoglobin used as oxygen
carrier in HbOCs
• It can be synthesized by using an expression vector
containing two mutant globin genes in a bacteria like
E.coli, and in plant animal
• Recombinant hb expression technology was first
develop by NAGAI and THORGERSEN in 1984.
• SOMATOGEN work in between 1988-1991.
• Bacteria use mainly Plesiomonas shigelloids.

7. Pleisomonas shigellides
• Is an intestinal pathogen, that use heme iron.
• It contain heme transport system,
• Consist Hug A the outer mambrne heme transpoter
• Ton B ExbBD ,which allow HugA to move heme into
the periplasm
• HugB which moves heme across the periplasm
• HugC and HugD which move heme into cytoplasm
• Three additional proteins HugWXZ may be required
for the utilization of heme as an iron source but not
for heme transport.

8. Bacterial strains and plasmids used in this
study :-

9. Lab scle expression observed by taking
absorbance, then fermentation.
donwnstrem processing by
centrifugation, and other purification
method is used.
Host cell is grown into media which
contain iron source and EDDA( ethylene
diamine di( o hydroxy-phenyl acetic acid)
and IPTG ( isopropyl β-D ,
thiogalactopyarnoside
Gene of intrest is cloned in to vector,
Vector is then transfer into host cell,

10. Mechanism how hemoglobin form:-

11. BL21(DE3) containing hemoglobin genes and P.
shigelloides heme transport genes produces high
levels of hemoglobin
 To assay haemoglobin production
,BL21(DE3)/pHB0.0 with or without the
heme transpoter system was grown in
Lbroth with 6.25μg/ml EDDA allow the
espression of heme transpoter gene.
 After 8h ,40μg/ml IPTG was added to
induse the expression of hemoglobin, and
15.2μm heme was added to increase
imtracellular.
 Then observed the absorbance.

12. Enhancement of Recombinant Hemoglobin
Production in Escherichia coli BL21(DE3) Containing
the Plesiomonas shigelloides Heme Transport
System
 PHB0.0 Hug contain both hemoglobin and
heme transpoter genes.
 Hemoglobin production was assessed in
BL21(DE3) transformed with the one
plasmid system .(PHB0.0Hug ,a high copy
number plasmid)
 Or cotransformed with the two two
plasmid system .(PHB0.0 high copy number
plasmid and pHUG 21 a low copy number
plasmid )
 Ulike Bl21 with twp plasmid system the
strain with one plasmid system produce
high spectrum value.

13. ADVANTAGE OF RECOMBINANT
HEMOGLOBIN
Universal donar
Identity to native human
hemoglobin
Ability to engineer
Store at room tem for 2-3
year
Half life 42 days
DISADVANTAGE OF
RECOMBINANT HEMOGLOBIN
Production scale –up
Requirement of high level of
purification
Production expense
Not do all the function of natural
blood , only carry oxygen.