Cloning and expression of Streptococcal protein G

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

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

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)

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.

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

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; 

Features of Protein GBinds 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.

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.

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.

Cloning strategy of recombinant Protein G

Bacterial Stains used for cloning and ExpressionStrain                    Characteristics Company

BL21 (DE3)(+) T7 polymerase IPTG induction, (-) lon and omp-t

proteases expression of non-toxic genes (+). Novagen

NovablueHigh 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

EcoR1& NdeIpUC 57 cloning vector

pET-22b (+) expression vector

Digestion of plasmid and expression vectors  M        57            22

6000 bp

600 bp

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.

Transformed colonies in Novablue cells

Protein G was successfully transformed by electroporation.

Verified by performing restriction digestion by EcoR1.

Expression of protein GCells were grown typically until mid-log phase.

Induction occurs by the lactose analog isopropyl-thiogalactoside (IPTG).

Cells were harvested and prepared for purification.

Inducers tested for expression

Lactose IPTG

Lactose IPTG

1:10mM,2:20mM,3:30mM 1:0.1mM,2:0.5mM,3:1mM

Protein G purification by IMAC

(a)

(b)

Mass spectrometry of protein G

Protein G purification system

Diafiltration

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.

Media optimizationTesting for optimal media for protein G expression.Evaluated following media:1.LB2.SOB3.SOC4.M9

M 1 2 3 4

SDS-PAGE showing cell lysates during media optimization 1:LB,2:SOB,3:SOC,4:M9

   M           1           2          3

Temperature optimizationEffect 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

   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

Immobilization of protein G on POROS AL matrix

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

Binding of Protein G with IgG(a) (b)

(c) (d)

(a) (b)

(c) (d)

Binding of Heat treated Protein G with IgG

ConclusionsProtein 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.

Acknowledgements• Prof Bo Mattiasson.• Dr. Gashaw Mamo.• Dr. Martin Hedstrom.• Maru,Jit ,Lesedi,Roya,Rawana and Tarek.• Department of Biotechnology, Lund

University.