Cloning and Expression of recombinant Protein
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Protein G was cloned from streptococcal strains and expressed in E. coli BL21 cells. Various expression and purification conditions were optimized, including inducer type, temperature, media, and heat treatment. Protein G was purified using immobilized metal affinity chromatography and its ability to bind IgG subclasses was confirmed using a VersaFLo system. Heat treatment at 80°C removed contaminants and was an efficient step in downstream processing. Overall, the study successfully cloned and expressed recombinant protein G and analyzed its IgG binding properties.
![Bacterial Stains used for cloning and Expression
Strain Characteristics Company
BL21 (DE3)
(+) T7 polymerase IPTG induction, (-) lon and omp-t
proteases expression of non-toxic genes (+).
Novagen
Novablue
High transformation efficiency, blue/white screening of
recombinants by lacZ [alpha]-complementation and F
factor to support M13 growth.
Novagen
Plasmids constructs used for cloning and transformation
Strain Strain Characteristics Company
pET22b(+)
Expression vector with T7 promoter and terminator flanking MCS
(Multiple cloning sites), pelB leader sequence for subcellular targeting and
tag cds, ampicillin resistance; restriction enzyme cloning.
Novagen
PUC57 Vector length of 2,710 bp,isolated from E. coli DH5α, Genescript](https://image.slidesharecdn.com/proteingpresentation-150806090158-lva1-app6891/85/Cloning-and-Expression-of-recombinant-Protein-12-320.jpg)
Cloning and Expression of recombinant Protein
- 1. Cloning and expression of Streptococcal protein G
- 2. Monoclonal Antibody Market scenario * Leavy, O. 2010. Therapeutic Antibodies: Past, Present, and Future. Nat. Rev. Immunol. 10:297. # Journal of Chromatography B, Volume 848, Issue 1, 2007, 48 - 63 • US$15 billion*, • Highest of all biotherapeutics. 2008, Monoclonal antibodies (Mabs) • $62.3 billion. 2015,The world MAb market will reach #Projected annual production of monoclonal antibodies. Numbers are in metric tones and are a composite from market data
- 3. Antibodies or Immunoglobulins (Ig) • Immunoglobulins (antibodies) are glycoproteins. • Classified in five groups IgG, IgM, IgA, IgD and IgE. • Immunoglobulin G (IgG) constitutes approximately 75% of total serum Ig. • IgG is the principle antibody used in immunological research and clinical diagnostics. Structure of an antibody
- 4. Immunoglobulin binding proteins Bacterial surface protein, bind with the constant Fc region of an antibody Virulence factors, avoid the host immune response High degree of purity and selectivity Protein A Staphylococcus aureus Strong, Fc region Protein G Groups C and G streptococci Strong,Fc region Protein L Peptococcus magnus Binds to ƙ light chains Protein P Clostridium perfringens Binds to ƙ light chains Protein D Branhamella catarrhalis IgD. Binds small amounts of IgG Protein P Group A streptococci IgA. Binds weakly to IgG IgG-binding proteins from bacteria *P. Gagnon,Purification Tools for Monoclonal Antibodies,Validated Biosystems, Tucson, AZ (1996)
- 5. Function of Immunoglobulin binding proteins Adhesion, invasion, evasion. The many functions of Staphylococcus 5:15-5:55 PM aureus surface proteins. Timothy J. Foster BA, PhD, MRIA.
- 6. Protein G Immunoglobulin G- binding (IgG) protein G Group C and group G Streptococcal strains ~22 kDa migrates on SDS-PAGE gel ~32 kDa approximately 600 residues that bind to the Fab and Fc Considered a universal reagent in biochemistry and immunology Retain the IgG ability after proteolytic enzymatic treatment
- 7. Immunoglobulin binding specificities Immunoglobulin Type III receptor Type I receptor Protein G, FcRc Protein A Rabbit ++ ++ Goat ++ - - Mouse ++ ++ Rat + +/- Bovine ++ ++ Chicken - - Human IgG1 ++ ++ Human IgG2 ++ ++ Human IgG3 ++ - Human IgG4 ++ ++ Human IgA - + Human IgM - + Human IgD - - ++ Strong Binding;+ weak binding;- no binding;
- 8. Features of Protein G Binds to more IgG subclasses than protein A. Isolation of immune complexes. Specific purification of IgG. repetitive regions binds with Fc and Fab region of IgG, compared to Protein A. Specificity: 1. Greater affinity to most mammalian immunoglobulins than Protein A, including human IgG3 and rat IgG2a. 2. Does not bind to human IgM, IgD and IgA.
- 9. Recombinant Protein G • The native Protein G binds albumin. • Serum albumin is a major contaminant of antibody sources. • Recombinant Protein G performed modifications: • Detached albumin binding site.
- 10. Aim of the study Clone and express protein G in E.coli. Purification and optimization of produced protein. Analysis of the binding ability of the protein.
- 11. Cloning strategy of recombinant Protein G
- 12. Bacterial Stains used for cloning and Expression Strain Characteristics Company BL21 (DE3) (+) T7 polymerase IPTG induction, (-) lon and omp-t proteases expression of non-toxic genes (+). Novagen Novablue High transformation efficiency, blue/white screening of recombinants by lacZ [alpha]-complementation and F factor to support M13 growth. Novagen Plasmids constructs used for cloning and transformation Strain Strain Characteristics Company pET22b(+) Expression vector with T7 promoter and terminator flanking MCS (Multiple cloning sites), pelB leader sequence for subcellular targeting and tag cds, ampicillin resistance; restriction enzyme cloning. Novagen PUC57 Vector length of 2,710 bp,isolated from E. coli DH5α, Genescript
- 13. EcoR1& NdeIpUC 57 cloning vector pET-22b (+) expression vector Digestion of plasmid and expression vectors M 57 22 6000 bp 600 bp
- 14. Verification of ligation of protein G insert M:marker,1,5,7,15,20 are colony picked after ligation Post ligation cells grown on agar+Amp. t ligation cells grown on grown DNA extracted from the picked colony. Agarose gel electrophoresis.
- 15. Transformed colonies in Novablue cells Protein G was successfully transformed by electroporation. Verified by performing restriction digestion by EcoR1.
- 16. Expression of protein G Cells were grown typically until mid-log phase. Induction occurs by the lactose analog isopropyl-thiogalactoside (IPTG). Cells were harvested and prepared for purification.
- 17. Inducers tested for expression Lactose IPTG
- 19. Protein G purification by IMAC (a) (b)
- 20. Mass spectrometry of protein G
- 21. Protein G purification system
- 22. Diafiltration
- 23. Heat treated protein G SDS-PAGE gel showing the effect of heat treatment of with protein G at (60 °C and 80 ° C). Lane 1 shows the crude cell lysate, lane 2& 3 shows 1 and 2nd time heat treatment at 60°C. Lane 4 & 5 shows the heat treated protein G 1st and 2nd time at 80 °C. Protein G heat treated in two cycles at 60°C and 80°C. Found to be stable and removed most of the cell bound contaminant at 80°C. Quick and efficient step in downstream processing.
- 24. Media optimization Testing for optimal media for protein G expression. Evaluated following media: 1.LB 2.SOB 3.SOC 4.M9 M 1 2 3 4 SDS-PAGE showing cell lysates during media optimization 1:LB,2:SOB,3:SOC,4:M9
- 25. M 1 2 3 Temperature optimization Effect of temperature on protein G expression was tested. Cells were grown until OD reached 0.6. Temperatures tested : 25,30 and 37°C. SDS-PAGE showing cell lysates during temperature optimization 1:25°C, 2: 30°C,3:37°C
- 26. M 1 2 3 Protein presence in media fraction Protein G released in the media fraction. The maximum release found at 37°C and least at 25°C. SDS-PAGE showing cell free media during temperature optimization 1:25°C, 2: 30°C,3:37°C
- 27. Immobilization of protein G on POROS AL matrix
- 28. VersaFLo system Assay phase Carrier flow rate (mL/min) Time duration (min) Equilibration 0.25 1 IgG injection 0.1 15 Rinsing 0.25 1 Elution 0.1 10 Rinsing 0.25 1
- 29. Binding of Protein G with IgG(a) (b) (c) (d)
- 30. (a) (b) (c) (d) Binding of Heat treated Protein G with IgG
- 31. Conclusions Protein G was successfully cloned in E.coli BL21 cells. The expression was optimized and analyzed on SDS-PAGE. Expression at higher temperature resulted in protein leakage. The binding ability with the IgGs was confirmed by VersaFLo system. Heat treatment could be as a critical step in downstream processing after the protein production.
- 32. Acknowledgements • Prof Bo Mattiasson. • Dr. Gashaw Mamo. • Dr. Martin Hedstrom. • Maru,Jit ,Lesedi,Roya,Rawana and Tarek. • Department of Biotechnology, Lund University.