Recombinant hemoglobin

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 O 2 and CO 2 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. Mechanism how hemoglobin form:-

10. 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 6.25 μ g/ml EDDA allow the espression of heme transpoter gene. After 8h ,40 μ g/ml IPTG was added to induse the expression of hemoglobin , and 15.2 μ m heme was added to increase imtracellular . Then observed the absorbance .

11. 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.

12. 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.

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