Expressing cloned protein in animal cell &processing

Expressing cloned gene in Animal cell : 

Expressing cloned gene in Animal cell By-praveen kumar Lecturer virani science college

Introduction : 

The production of proteins in animal cells is an important tool in numerous scientific and commercial areas. The proteins expressed cell system are needed for life science research and development In the field of biomedicine. Introduction

Why animal cell in expression : 

Trans regulation Separate mode of transcription for m, t &r-RNA Have many transcription factor. DNA methylation:- > Extensive methylation of cytosine- inactivate the gene in animal cell > Methylation is checked by- ~By RE Msp-1(Morexella bacteria) Why animal cell in expression

Continued… : 

~Recognize CCGG & CmCCG ~Another RE Hpa-2(Haemo parainfluenzae) recognize only methylated one. post translational modification:- ~—s-s- bond formation, ~intein splicing, ~proteolytic cleavage, ~chemical modification, ~protein glycosylation. ~Eukaryotic gene has generally leader sequence or signal sequence . Continued…

Selection system for animal cell : 

(A)CAT(chloramphenicol acetyl transferase) gene: -resistant to chloramphenicol Bacterial Dihydrofolate reductase gene-resistant to Methotrexate Thymidine kinase gene-resistant to 2-deoxystreptamin(G-148) Selection system for animal cell

Continued… : 

Green –fluorescent protein-as quantitive and qualitative marker mcr-A gene-resistant to Mitomycin-C Continued…

Vector used for animal cell : 

Baculovirus SV40 virus Adenovirus Vaccinia virus Bovine papilloma virus Retrovirus Vector used for animal cell

BACULOVIRUS EXPRESSION : 

In insect Two types: (1) Autographa California nuclear polyhydrosis virus. (2)Bombex mori nuclear polyhydrosis virus. DS DNA Can handle 130 kb base sequence. Clone on other plasmid vector as vaccinia virus BACULOVIRUS EXPRESSION

Insect cell : 

Insect cell

Mammalian system : 

Mammalian system

Protein production and nucleofection : 

Nucleofection of mammalian cells transient protein production stable protein production (Tech note coming soon) Nucleofection of insect cells (Optimized Protocols coming soon) Protein production and nucleofection

Transient nucleofection of mammalian cells : 

Benefits of Nucleofection: Fast protein expression (within a few days) Transfection in serum-free suspension cultures Higher efficiencies than with competitor methods Suited for validation, optimization and characterization experiments Substantial cost and time saving! Transient nucleofection of mammalian cells

Example of change in the expression of chosen protein : 

T4 lysozyme Change in thermo stability. It has 2 cysteine(54-97),butbnot under the –s-s- bond formation. 3Dstructure show Ileucine -3 is more closer to cyst-97 If ileu ATA is change into cys TGT –s-s- bond formed. Half life change from 11 min to 28 min at 67 c. Example of change in the expression of chosen protein

Enhancing specific activity of Rec-IFN-β : 

Specific activity is 106 antiviral unit/mg 100 times less than fibroblast derived IFN-beta. Due to incorrect bond formation. When cys-17 change to serine its specific activity regain, its stability and prolonged storage also increase. Enhancing specific activity of Rec-IFN-β

Improved α-1 antitrypsin : 

394 aa long residue, glycosylated serum protein Cleavage between met-358 &ser-359 If met 358 is replaced with valine problem of oxidation is shorted out. Effective in Emphysema. Improved α-1 antitrypsin

Altering kinetic property of protein : 

Subtilisin a biological detergent have met-222(active site) If it being replaced with non oxidative proe such as ser,ala, leu they will become resistant to oxidative damage. Long acting t-PA can also be modified. Altering kinetic property of protein

Increasing the potency of hirudin : 

It is a antithrombin,65 aa chain . Asn -47 is if converted to lys&arg the potency of hirudin increase 44 time in vitro &20 time in vivo. Increasing the potency of hirudin

We can overproduce the protein by animal cell expression : 

(1)By transformed cell - naturally or spontaneous transformed cell line is used FLOW-7000- for IFN-β. HT-1080- for urokinase . BEB- cell line- plasminogen activator. (2)By retroviral transformed cell (3)By maximize gene expression. We can overproduce the protein by animal cell expression

Technology in protein purification expressed by animal cell : 

(1)Using β-galactosidase via collagen peptide linker-it can be purified on resin bound paraamino phenyl thio galactoside. Collagen has specific binding affinity with PAPTG Technology in protein purification expressed by animal cell

. : 

(2)Using protein-A linked for purificatioin. specific binding affinity for Ig. (3)Poly arginine tailing-we can use any ion exchange chromatography. later pass carboxypeptidase. .

GenWay's specialty : 

GenWay specializes in mammalian expression system. To meet different needs for protein expression, mammalian cell protein expression can be carried out via the following different approaches: Transient burst expression Adenovirus-assisted expression. Stable mammalian cell line expression (CHO and HEK293) GenWay's specialty

GenWay's Mammalian Expression System : 

GenWay's Mammalian Expression System

summary : 

summary

References : 

Protein Expression: A Practical ApproachSJ Higgins, BD Hames - 1999 - books.google.com Protein Expression: a Practical ApproachMJ Clemens, GJM Pruijn, SJ Higgins, BD Hames - 1999 - Oxford, Oxford University Press, UK www.genway.com Animal Biotechnology, Ranga References

Thanks : 

Thanks