Development of a Fab binding protein domain

Royal Institute of Technology (KTH) Development of a Fab binding protein domain Master thesis 30 hp Sara Kanje 2011-‐12-‐13 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH Abstract The C2 domain of the immunoglobulin binding protein G has affinity for both the IgG fragment antigen binding (Fab) and the IgG fragment crystallisable (Fc). Mutating the C2 domain at specific positions could create an exclusively Fab binding protein domain. Potential areas of use for a Fab binding protein domain are site specific labelling of IgG, purification of antibody fragments or conjugation of other molecules to IgG. Previous work has found two mutations to partly delete Fc binding of the C2 domain while maintaining Fab-‐binding. Nine double mutants containing one of these mutations together with one new mutation were produced to entirely delete Fc binding. Two of the constructs are extra promising candidates. 2 Master thesis 30 hp Table of Contents Sara Kanje, Master of Science, Biotechnology, KTH Abstract ................................................................................................................................................... 2 1. Introduction ........................................................................................................................................ 5 1.1 Biotechnology ............................................................................................................................... 5 1.2 Antibodies ..................................................................................................................................... 5 1.3 Immunoglobulin-‐binding proteins ................................................................................................ 5 1.3.1 Protein A ................................................................................................................................ 6 1.3.2 M-‐type proteins ..................................................................................................................... 6 1.3.3 Protein L ................................................................................................................................. 7 1.3.4 Protein G ................................................................................................................................ 7 1.4 Protein engineering ....................................................................................................................... 8 2. Project outline ..................................................................................................................................... 8 3. Materials and methods ....................................................................................................................... 9 3.1 Rational design of constructs ........................................................................................................ 9 3.2 In vitro mutagenesis ...................................................................................................................... 9 3.2.1 Preparation of templates ....................................................................................................... 9 3.2.2 PCR ......................................................................................................................................... 9 3.3 Cleaving, ligation and transformation ......................................................................................... 10 3.3.1 Cleaving of DNA fragments .................................................................................................. 10 3.3.2 Cleaving of vector ................................................................................................................. 10 3.3.3 Ligation ................................................................................................................................. 11 3.3.4 Transformation .................................................................................................................... 11 3.4 Sequencing .................................................................................................................................. 11 3.5 Plasmid purification and transformation of the constructs ........................................................ 11 3.6 Batch cultivation and protein production ................................................................................... 11 3.6.1 Inoculation day 1 .................................................................................................................. 11 3.6.2 Cultivation day 2 .................................................................................................................. 12 3.6.3 Harvest and lysis day 3 ......................................................................................................... 12 3.7 ASPEC purification ....................................................................................................................... 12 3.8 Protein analysis ........................................................................................................................... 12 3.8.1 Protein concentration .......................................................................................................... 12 3.8.2 Protein purity ....................................................................................................................... 12 3.8.3 Protein mass ......................................................................................................................... 12 3.8.4 Protein ligand interaction .................................................................................................... 13 3 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH 4. Results ............................................................................................................................................... 13 4.1 Rational Design ........................................................................................................................... 13 4.2 Sequencing .................................................................................................................................. 13 4.3 Protein concentration, purity and verification ............................................................................ 13 4.3.1 BCA ....................................................................................................................................... 13 4.3.2 SDS-‐PAGE ............................................................................................................................. 14 4.3.3 MS ........................................................................................................................................ 14 4.4 Protein ligand interaction ........................................................................................................... 15 5. Discussion .......................................................................................................................................... 15 5.1 C2 double mutants ...................................................................................................................... 15 6. Future prospects ............................................................................................................................... 16 7. Acknowledgements ........................................................................................................................... 16 8. Appendix ........................................................................................................................................... 17 8.1 Appendix 1: Raw data ................................................................................................................. 17 8.1.1 MS Spectra ........................................................................................................................... 17 8.1.2 ProteOn™ data ..................................................................................................................... 19 9. References ........................................................................................................................................ 23 4 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH 1. Introduction 1.1 Biotechnology Biotechnology is a broad field including such different areas as agriculture, medicine and food processing. The advent of biotechnology stretches back to the time when people started making beer, fermenting bread and selectively breeding plants. Today the entire human genome has been sequenced, half the proteome has been mapped, and monoclonal antibodies are used to treat various diseases1. 1.2 Antibodies As a part of our immune system, B-‐cells in the blood express immunoglobulins on their surface. These immunoglobulins can bind to different pathogens with high specificity. Some B-‐cells can produce soluble immunoglobulins that are excreted, called antibodies. Antibodies consist of two identical heavy chains and two identical light chains. In humans there are five different classes of immunoglobulins: IgG, IgM, IgD, IgA and IgE. The classes differ in of what type of heavy chains they consist. IgG, the most common immunoglobulin, often serves as the model for describing antibody structure (figure 1). The antibody is often described as having two regions, a constant and a variable region. The variable region contains the antigen-‐
binding site and gives the antibody its specificity. The constant region interacts with other parts of the immune system, or foreign Figure 1Schematic picture of an immunoglobulin. The heavy chains (H) are showed in green, the light chains (L) in blue. proteins, in a non-‐immune way. Digestion of Domains are denoted constant (C) and variable (V). immunoglobulin with papain, a protease, results in three fragments. Two identical Fragment antigen-‐binding (Fab) parts and one Fragment crystallisable (Fc) part (figure 1). The region between the Fab and Fc fragment is quite flexible, called the hinge region.2 Antibodies recognise foreign antigens and present the antigens to other parts of the immune system2. In biotechnology and medicine, antibodies are used in various assays and as drugs. Examples where antibodies are used include different diagnostic procedures, e.g. enzyme-‐linked immunosorbent assay (ELISA), imaging, e.g. visualizing various cell and cell components with labelled antibodies1, affinity chromatography and immunoprecipitation3. Humanised monoclonal antibodies are used in the treatment of various diseases1. 1.3 Immunoglobulin-‐binding proteins A number of proteins located on the cell surface of certain bacteria have the ability to bind immunoglobulins in a non-‐immune way. These proteins are called immunoglobulin-‐binding proteins (IBP). IBP is a group of proteins with different size, structure and binding properties. They interact with various proteins from human or animal blood serum where many of the IBPs have binding sites for more than one protein4. The natural function of IBPs is believed to be to increase the virulence of 5 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH the bacteria expressing these proteins on their surface5.If the bacterial surface is coated with the host’s IgGs, and therefore undetectable from the immune system, surface antigens of the bacteria can be hidden from the host’s immune system6,7.There are three main types of IBPs, classified according to their IgG-‐binding abilities. All three types bind to the Fc fragment of IgG. Type I is protein A of Staphylococcus aureus, type II is M-‐proteins from group A streptococci and type III protein G of streptococci group C and G4. These three types of IBPs and their binding patterns to IgG from various species are presented in table 18. 8
Table 1 Binding properties of the three main types of immunoglobulin-‐binding proteins IgG Type I Type II Type III Human IgG1 ++ ++ ++ Human IgG2 ++ ++ ++ Human IgG3 -‐ ++ ++ Human IgG4 ++ ++ ++ Mouse IgG1 + -‐ + Mouse IgG2a ++ -‐ ++ Mouse IgG2b ++ -‐ ++ Mouse IgG3 ++ -‐ ++ Rat IgG1 + -‐ + Rat IgG2a -‐ -‐ ++ Rat IgG2b -‐ -‐ + Rat IgG2c ++ -‐ ++ Rabbit IgG ++ + ++ Bovine IgG1 -‐ -‐ ++ Bovine IgG2 ++ -‐ ++ Sheep IgG1 -‐ -‐ ++ Sheep IgG2 ++ -‐ ++ Goat IgG1 + -‐ ++ Goat IgG2 ++ -‐ ++ Horse IgG (ab) + -‐ ++ Horse IgG(c) + -‐ ++ Horse IgG (T) -‐ (+) (+) The IBPs most studied come from gram positive bacteria. They have in common an N-‐terminal signalling peptide, a functional region with the protein binding activity and a C-‐terminal sorting signal that anchors the protein to the cell wall4. 1.3.1 Protein A The first protein discovered to bind IgG outside of its antigen binding site was protein A. Protein A is located on the cell surface of S. aureus. It consists of five IgG-‐binding domains in its protein binding region, each domain consisting of 58-‐62 amino acids. The IgG-‐binding domains of Protein A are made up of three tightly packed α-‐helices4. Two are responsible for IgG-‐binding via side-‐chain to side-‐chain interactions in the area between the CH2 and CH3 domains in the Fc-‐region of the antibody9. Besides IgG, protein A interacts with other immunoglobulins i.e. IgM, IgA and IgE4. 1.3.2 M-‐type proteins The type II IBPs come from group A streptococci, the pathogen causing human streptococcal infections. Most of these IBPs belong to a super family called M-‐proteins. There are three families of M-‐proteins, Emm, Mrp and Enn. Each family consists of similar proteins but the families differ in 6 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH function and structure. The capacity of binding different IgGs varies among the M-‐proteins and they can be divided into subclasses within the type II IBPs. M-‐proteins also bind to various other plasma proteins i.e. albumin, fibrinogen and kininogens, IgA and C4BP protein4. 1.3.3 Protein L Certain strains of the anaerobic bacterium Peptostreotococcus magnus express an IBP called protein L on their surface. Protein L binds to the VL domain on the immunoglobulin, without interacting with the antigen binding site. Also protein L consists of multiple domains in tandem repeats, where five homologous repeats provides the binding to IgG as well as IgA and IgM. These domains are 72-‐76 amino acids in size and has a structure of an α-‐helix packed on a β-‐sheet made of four β-‐strands. Protein L forms its complex with the κ-‐chain in the VL domain mainly through one of the β-‐strands, the C-‐terminal end of the α-‐helix and a loop10. 1.3.4 Protein G Protein G is found on the surface of Streptococci group C and G10and binds to IgG with high affinity9. It binds to IgG of a wider variety of species than other IBPs11 (see table 1) and also to human serum albumin and α2-‐macroglobulin, a proteinase inhibitor of human plasma12. Since protein G binds to IgG only, it is a more selective IBP than e.g. protein A, protein L and M-‐proteins8. Protein G also has a higher affinity for both monoclonal and polyclonal IgGs than protein A13,14. Protein G consists of a signalling peptide and alanine-‐rich region (E) and two or three, depending on what strain, small albumin-‐binding domains (B) at the N-‐terminal end. Two or three, depending on what strain, IgG-‐
binding domains (C) followed by domains anchoring the protein in the cell wall and membrane (W, M) (figure 2)10,12,15. Figure 2Schematic picture of the domain structure of protein G. The different IgG-‐binding domains, C1, C2 and C3 all have similar secondary and tertiary structures10,15. C1 and C2 differ in only two amino acids, C2 and C3 in four, whereas there is a six amino acid difference between C1 and C315. Protein G is used for purification of antibodies and antibody fragments as well as antibody labelling and ELISA9,16. 1.3.4.1 C2 domain The three dimensional structure of C2 can be seen in figure 317. It is a 55 residue domain, consisting of a four-‐stranded β-‐sheet with an α-‐helix on top. The α-‐helix and the β-‐sheet are tightly packed with a hydrophobic core in between10. It is a highly thermostable molecule9. The C2 domain, as well as C1 and C3, exhibits binding to both Fc and Fab fragments of IgG10. The binding to Fab is weaker than that to Fc and constitutes around 10-‐15 % of the total IgG binding8.The binding sites for Fc and Fab are located on two separate and different surfaces of the C2 molecule7,18. C2 binds to Fc in the region in between CH2 and CH3. Three regions on C2 are responsible for the interaction, situated on the α-‐helix and the N-‐terminal region of β-‐strand three. The first region, in the centre of the α-‐helix, consists of charged and polar residues, forming hydrogen bonds with the IgG-‐molecule. The second region, also on the α-‐helix, consists of two charged amino acids forming hydrogen bonds to IgG. The third region 7 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH includes polar and non-‐polar amino acids from both the α-‐helix and the third β-‐strand. Very few hydrophobic interactions are found in the binding site C2:Fc. This interaction surface is rather small (700 Å2) and differs from most protein-‐protein interactions in being of polar character instead of hydrophobic7.Even though both protein A and protein G bind to the same region of IgG with α-‐
helices, their interactions are different. Protein A binds to Fc mainly through hydrophobic interactions. Still many amino acids on Fc interacts with both proteins, explaining why they compete for binding to the Fc fragment on IgG15. The interaction between C2 and Fab is located exclusively on the antibody CH1 domain. This interaction is 2
accomplished through β-‐strand interactions. The second β-‐strand and the turn between the first and second β-‐strand of C2 (see 4 1
figure 1) forms a complex with the last β-‐strand on CH1 mainly 3
through “edge-‐to-‐edge” interactions stabilized by hydrogen bonds19,20, forming a continuous β-‐sheet over the protein-‐protein interaction surface7. The C-‐terminal region of the C2 α-‐helix interacts with the first β-‐strand of CH1. The interaction between Figure 3 Ribbon structure of the C2 domain of protein G (1qkz.pdb) with Fab and C2 is mainly between backbone atoms of Fab and β-‐strand numbering (1-‐4) starting backbone and side chain atoms of C2. The complex is stabilised by from the N-‐terminal the formation of a hydrophobic core when non-‐polar residues from the two proteins are buried in the interaction surface19. No larger changes in conformation upon binding of C2 to Fab have been observed9. CH1 is the most conserved region of IgG which, together with the fact that C2 mainly interacts with main chain atoms of CH1, may explain the wider specificity of protein G for IgGs from various species compared to other IBPs9,19. 1.4 Protein engineering With the help of genetic modifications, the amino acid sequence of a protein can be altered and thus its properties modified. Changing a protein’s properties with the help of rational design or directed evolution is called protein engineering. Examples of protein engineering are making an industrial enzyme more thermostable or altering the binding specificity of a protein interacting with another macromolecule1. 2. Project outline The aim of the project was to engineer a molecule to bind specifically to the Fab fragment of IgG from a wide range of species. C2 was chosen as it has a high affinity to Fab of many species, and does not interact with other immunoglobulin types. Wild type C2 binds not only to Fab but also Fc. Deleting Fc binding would result in a highly specific protein with multiple areas of use. Examples of such are, site specific labelling of antibodies, purification of antibodies and antibody fragments or a mean to conjugate e.g. toxins to an antibody used to fight disease. Another advantage of a Fab binder is that, after conjugation, Fc is free to interact with other molecules of interest. Previous work has found mutations A and B to be interesting to delete Fc binding, although not entirely. This project would therefore focus on producing double mutants with one new mutation and one of the two mutations mentioned above. The chosen mutants are presented in table 2. 8 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH Table 2 Chosen C2 double mutants C2 mutants C_A D_A E_A F_A A_B C_B E_B G_B H_B The first part of the project included in vitro mutagenesis of the desired constructs, sequencing and transformation of the plasmid containing the correctly mutated gene to an Escherichia coli strain suitable for protein production. The second part of the project included batch cultivation of the protein in E. coli, protein purification and protein analysis. The third part of the project was to analyse the purified C2 mutants to test their binding abilities to IgG, Fc and Fab. 3. Materials and methods 3.1 Rational design of constructs The 1FCC.pdb15 structure was used to rationally design nine double mutants. Both mutants with their mutations in the same binding region as well as different binding regions were chosen. Alanine and tryptophan were chosen as the inserted amino acids the reason being alanine is the amino acid with the smallest side chain and tryptophan has a big bulky side chain21. 3.2 In vitro mutagenesis 3.2.1 Preparation of templates A his-‐C2-‐vector with kanamycin (Km) resistance, T7 promoter and the original C2 constructs (wild type, A and B) were transformed into competent RR1ΔM15 cells. Purified plasmid was added to the competent cells that were incubated on ice for 30 min. The cells were then heat chocked at 40 °C for 4.5 min after which they were left on ice for an additional 5 min. This was followed by growth in 700 µl Tryptic Soy Broth (TSB) medium (30 g/l, Merck, Darmstadt, Germany) for 60 min. The cells were centrifuged at 2710 rpm for 2 min. 600 µl of medium was removed and the cells were resuspended in the remaining medium to be plated on agar-‐plates with Km (50 µg/ml, Duchefa, Haarlem, Netherlands). Cells with no added vector were used as negative control. 3.2.2 PCR To introduce the intended mutations in the C2 gene, polymerase chain reaction (PCR) was performed in either one or two steps. For constructs where primers could be designed so that they contained the Pst1 restriction site (located inside the C2 gene) one-‐step PCR was used. For the other constructs a two-‐step PCR reaction was performed. As template, DNA from E. coli containing one of the three 9 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH C2 constructs, was used. The reaction was performed in PCR-‐buffer (500 mM KCl, 20 mM MgCl2, 100 mM Tris HCl, 1 % Tween20, pH 8.5) with dNTPs (Fermentas, Burlington, Canada) and Taq polymerase (1U, KTH, Stockholm, Sweden). 3.2.2.1 One-‐step PCR In the one-‐step PCR, a forward primer containing the Pst1 restriction site and introducing a mismatch at the desired site of mutation was used. As backwards primer a biotinylated primer complementary to the 3’ end of the C2 gene with an overhang containing the Asc1 restriction site was used. The PCR products were verified using agarose gel electrophoresis. 3.2.2.2 Two-‐step PCR In the first reaction of the two-‐step PCR, a forward primer complementary to the 5’ end of the C2 gene with an overhang containing the Xho1 restriction site was used. The desired mutation was introduced via a mismatch at the intended site in the backwards primer. The PCR product was verified on an agarose gel. In the second step the PCR product from step one diluted five times was used as forward primer. The primer described in one-‐step PCR was used as backwards primer. The resulting product was verified with gel electrophoresis. 3.3 Cleaving, ligation and transformation 3.3.1 Cleaving of DNA fragments The fragments were cleaved using Dynabeads® M-‐270 streptavidin (NorDiag, Oslo, Norway) coated with streptavidin. To perform the cleaving, 20 µl magnetic beads per fragment were washed twice with an equal amount of washing buffer (w/b) (2 M NaCl, 10 mM Tris-‐HCl, 1 mM EDTA, 0.1 % Tween20). After the wash, the beads were resuspended in w/b buffer. 20 µl of bead suspension was incubated with 20 µl of PCR-‐product (fragment) for 30 min on a rotamixer. The supernatant was removed and the beads were washed twice with 40 µl of restriction enzyme compatible buffer (1xNEB3 for Pst1, 1xNEB4 for Xho1 and 1xAsc1 (New England Biolabs, Ipswich, MA, USA)). A volume of 20 µl 1xNEB3 with 100 µg/ml BSA and 0.1 µl of Pst1 (New England Biolabs) were added to the one-‐
step PCR fragments, whereas for the two-‐step PCR fragments 1xNEB4 with 100 µg/ml BSA and Xho1 (New England Biolabs) were added. The DNA fragments were incubated with their respective restriction enzyme in 37 °C for 1 h on a rotamixer. After the incubation the beads were washed with 2 x 40 µl 1xNEB4 after which 20 µl 1xNEB4 and 0.1 µl Asc1 (New England Biolabs) were added to each sample. The samples were incubated on a rotamixer for 1 h in 37 °C. After heat inactivation of the enzyme for 20 min at 65 °C, the supernatant containing the cleaved construct was saved and stored. 3.3.2 Cleaving of vector Cultivations (10 ml) of RR1ΔM15 cells containing the his-‐C2-‐vector with C2 wild type (C2wt) in TSB medium supplemented with Km (50 µg/ml) were grown overnight in 37 °C. The cells were harvested and the plasmids purified using the QIAprep spin miniprep kit (Qiagen, Valencia, CA, USA) according to instructions. The vector was cleaved with either Pst1 and Asc1 or Xho1 and Asc1 for insertion of fragments from one and two-‐step PCR products respectively. The cleavage reaction was performed for 2 h in 37 °C followed by heat inactivation for 20 min in either 80 °C (Pst1 and Asc1) or 65 °C (Xho1 and Asc1). After cleaving, the entire volume was loaded on an agarose with uncleaved vector as a control. After gel electrophoresis the cleaved vector was cut out of the gel and purified with the E.Z.N.A® gel extraction kit (Omega bio-‐tek, Norcross, GA, USA) according to instructions. 10 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH 3.3.3 Ligation The concentration of the cleaved PCR-‐product and vector was measured using a NanoDrop (Thermo Scientific, Waltham, MA, USA). The ligation was performed with a 3:1 ratio of fragment to vector, T4 DNA ligase (200 U, New England Biolabs) and 1xT4 DNA ligase buffer (New England Biolabs). The samples were incubated in 25 °C for 1 h 15 min followed by heat inactivation of the enzyme for 10 min at 65 °C. Cleaved vector with no added fragment was used as negative control. 3.3.4 Transformation The ligated constructs and negative control were transformed into RR1ΔM15 cells using the same protocol as described previously and were plated on agar plates containing Km (50µg/ml). 3.4 Sequencing Eight colonies from the transformation of each construct were picked for a PCR screen to amplify the gene segment that was going to be sequenced. A forward primer binding upstream of the C2 gene on the his-‐C2-‐vector was used together with a backwards primer binding downstream of the C2 gene. The reaction was performed as described above. The PCR products were verified using agarose gel electrophoresis. The products from the PCR screen were used in a sequencing reaction to be analysed using the ABI Prism® 3730 x I DNA Analyzer (AME Bioscience, Sharnbrook, UK). Each construct was used in two reactions, one with forward primer only and one with backwards primer only. The reaction was performed with product from the PCR-‐screen, Big Dye terminator mix (Applied Biosystems Inc., Ca, USA) and 1 x CS (26 mM Tris, 6.5 mM MgCl2, pH 9). After the PCR reactions the products were precipitated with ethanol (96 %) and NaAc (0,1M) and centrifuged at 4400 rpm for 30 min. After discarding the supernatant, ethanol (75 %) was added to the precipitates followed by an additional centrifugation at 4400 rpm for 10 min. The supernatant was discarded and after all additional ethanol had evaporated the precipitate was dissolved in MiliQ water and analysed on the ABI Prism® 3730 x I DNA Analyzer. 3.5 Plasmid purification and transformation of the constructs After sequence analysis, colonies containing the correctly mutated gene were cultivated over night in TSB medium supplemented with Km (50µg/ml). A volume of 2 ml from each culture was harvested in a micro centrifuge tube for 3 min at 8000 rpm. The plasmids containing the constructs were purified using the QIAprep spin miniprep kit. Each C2 double mutant construct was then transformed into Rosetta™(DE3) (Novagen®, Merck4Biosciences, Darmstadt, Germany) cells using the transformation protocol previously described. The C2 double mutants were plated on agar plates containing Km (50 µg/ml). Competent cells with no added plasmid were used as negative control. 3.6 Batch cultivation and protein production All constructs were cultivated using batch cultivation in Erlenmeyer flasks (E-‐flask). 3.6.1 Inoculation day 1 Inoculums of the double mutants were prepared by adding colonies containing the desired construct to 1 ml TSB+Y medium supplemented with Km (50 µg/ml) in a deep well plate. The plate was covered with a semi permeable membrane. 11 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH 3.6.2 Cultivation day 2 100 ml of TSB+Y medium supplemented with Km (50 µg/ml) was added to a one litre E-‐flask. Inoculums of 1 ml from day 1 were then added and the flasks were left on a shaking table in 37 °C. After 3 h (OD600approx. 0.9) IPTG (1 mM) was added to induce protein production. The cultivations were then left over night on a shaking table in 25 °C. 3.6.3 Harvest and lysis day 3 The cultivations were harvested 18-‐20 h after induction in GSA-‐tubes at 4000 rpm, 8 min, 4°C.After discarding the medium,5 ml denaturing lysis buffer (7M Guanidine hydrochloride, 47 mM Na2HPO4, 2,65 mM NaH2PO4, 10 mM Tris-‐HCl, 100 mM NaCl, 20 mM β-‐mercaptoethanol, pH 8.0) was added to each cell pellet and the samples were vortexed to dissolve the pellet. The mixtures were transferred into SS34-‐tubes and left on a shaking table in 37 °C for 2 h. The lysed cells were then centrifuged at 16 000 rpm, 20 min, 4 °C and the lysates were separated from the cell debris. 3.7 ASPEC purification The C2 mutants were purified with IMAC on an automated system: ASPEC GC-‐274 (Gilson, Middelton, WI, USA). A volume of 1 ml TALON® Metal Affinity Resin (Clontech, Mountain view, CA, USA) per sample was used. The columns were equilibrated with20 ml washing buffer (6 M guanidiumhydrochloride, 46.6 mM Na2HPO4, 3.4 mM NaH2PO4, NaCl 300 mM, pH 6.0) after which the 5 ml sample was loaded on the matrix. After washing the columns with 30 ml washing buffer the samples were eluted with 2.5 ml elution buffer (6 M Urea, 50 mM NaH2PO4, 100 mM NaCl, 30 mM HAc, 70 mM NaAc, pH 4.9-‐5.0) in two fractions, 0.7 and 1.8 ml. 3.8 Protein analysis 3.8.1 Protein concentration The concentration of the purified proteins was determined with a bicinchoninic acid assay (BCA) kit (Thermo Scientific) according to instructions. 3.8.2 Protein purity The purity of the proteins was checked with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-‐PAGE). Samples were diluted 1:10 (concentration >3 mg/ml) or 1:5 (concentration <3 mg/ml) in 1xPBS (0.15 M NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4, pH 6.8-‐6.9) and mixed 3:2 with 50 % 5 x RED (0.1 M Tris-‐HCl, 5 mM EDTA, 0.44 M SDS, 3.6 M β-‐mercaptoethanol, bromophenol blue)/50 % glycerol. The samples were heated at 95 °C for 5 min before 10 μl was loaded on the Criterion™ Precast gel 15 % Tris-‐HCl (Bio-‐Rad Laboratories). A low molecular weight marker (GE Healthcare, Little Chalfont, UK) was also loaded on the gel. The gel was run 60 min at 200 V followed by a 3 x 5 min wash in deionized water. The gel was then stained for 1 h in Gel code® Blue stain reagent (Thermo Scientific) and left in deionized water to destain over night. 3.8.3 Protein mass To check that the different C2 mutants had their correct mass, Electrospray ionization (ESI) mass spectroscopy (MS) on the 6520 Accurate-‐Mass Q-‐TOF LC/MS (Agilent technologies, Santa Clara, CA, USA) was used. 12 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH 3.8.4 Protein ligand interaction To determine whether the C2 double mutants had lost their Fc binding the ProteOn™ XPR36 Protein Interaction Array System (Bio-‐Rad Laboratories) was used. Six surfaces were coupled with various ligands. After coupling, six different concentrations of the C2 double mutants were flowed over these surfaces to measure protein-‐ligand interactions. Three surfaces were coated with polyclonal Fc fragments. One surface had a human monoclonal IgG and one surface a human polyclonal IgG. The last surface was used as a blank reference. The surfaces were all activated with EDAC and sulfo-‐NHS according to recommended solutions and protocol (Bio-‐Rad Laboratories) at a flow rate of 30 μl/min for 300 s. The ligands were then added to the surfaces stepwise until they reached a certain coupling level. The three Fc surfaces had a response corresponding to an Rmax of 560, 640 and 672 RU respectively. The IgG surfaces had a response corresponding to an Rmax of 133 RU. All six surfaces were then deactivated with ethanolamine. The analytes (the C2 double mutants) were flowed over these surfaces at 500, 250, 125, 62.5, 31.25 and 0 nM concentrations at a flow rate of 100 μl/min for 180 s and the response of interaction between ligand and analyte was measured. C2wt and the Fc binding protein domain Z were used as controls. In between analytes the chip was regenerated with 10 mM HCl, 100 μl/min for 18 s. 4. Results 4.1 Rational Design With rational design, nine double mutants of the C2 domain were chosen, to be produced with in vitro mutagenesis, to delete Fc binding of the C2 domain. The mutants are presented in table 2. 4.2 Sequencing After PCR-‐mutagenesis and cloning all variants were sequenced to confirm that the constructs had the right mutations. All constructs had colonies, with the correct sequence, that could be cultivated except H_B. After in vitro mutagenesis of that mutant, colonies that contained the H_I mutations were the only ones possible to cultivate. 4.3 Protein concentration, purity and verification 4.3.1 BCA After cultivation and IMAC purification of the C2 mutants, BCA was run to investigate the concentration of the mutants after purification. The measured protein concentrations of the C2 mutants, and the amount of protein per litre cultivation, are shown in table 4. 13 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH Table 3 Protein concentration of the C2 mutants C2 mutant Concentration (mg/ml) Yield (mg/l cultivation) C_A 7.15 50.1 D_A 1.73 12.1 E_A 7.93 55.5 F_A 5.76 40.3 A_B 7.12 49.8 C_B 6.93 48.5 E_B 7.79 54.5 G_B 7.54 52.8 H_I 7.31 51.2 4.3.2 SDS-‐PAGE An SDS-‐PAGE was run to verify that the C2 mutant proteins were pure. The protein purity results from SDS-‐PAGE can be seen in figure 4. The marker is shown as L (97, 66, 45, 30, 20.1, 14.4 kDa) and the sample numbering is shown in table 5. All proteins were found to be purified to homogeneity. Table 4 Sample numbering for SDS-‐PAGE in figure 4 C2 mutant C_A D_A E_A F_A A_B C_B E_B G_B H_I 1 2 3 4 5 6 7 8 9 97 kDa 66 kDa 45 kDa 30 kDa 20.1 kDa 14.4 kDa L 1 2 3 4 5 6 7 8 9 L Figure 4SDS_PAGE of the C2 mutants to check purity. 4.3.3 MS All samples were run with ESI-‐MS to verify that they had the correct molecular weights i.e.it was the right protein mutant. Table 6 presents the measured molecular weights of the C2 mutants and the theoretical molecular weight calculated from their respective protein sequence. The peak spectra can be found in appendix 1. 14 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH Table 5 Measured molecular weight with ESI-‐Ms and theroetical molecular weight C2 mutant C_A D_A E_A F_A A_B C_B E_B G_B H_I MW ESI (Da) 8135.88 8250.88 8135.93 8251.00 8148.99 8148.99 8149.00 8162.99 8308.00 MW theoretical (Da) 8135.57 8250.71 8135.61 8250.75 8148.66 8148.66 8148.70 8162.73 8307.90 4.4 Protein ligand interaction After verifying that all proteins were pure and of the right molecular weight, experiments were run on the ProteOn™ to investigate if the C2 double mutants had lost their Fc binding. The analytes were flowed over three surfaces coupled with Fc fragments and two surfaces coupled with whole IgGs. The data from the ProteOn™ experiments are shown in appendix 1. C2wt was used as a reference and had a maximum signal of 100-‐120 RU for the Fc surfaces and 80 RU for the IgG surfaces. Two double mutants, E_A and H_I, showed the most promising results. They both had a low maximum signal from the Fc interaction while their maximum signal from IgG interaction was five times as high (appendix 1). 5. Discussion 5.1 C2 double mutants Nine double mutants of the C2 domain of protein G were designed and produced to delete Fc-‐
binding of the protein. This was done to create an exclusively Fab-‐binding protein domain that could be used for site-‐specific labelling, purification of antibody fragments or conjugation of other molecules to IgG of a broad range of species. After in vitro mutagenesis, cleaving, ligation and transformation, sequencing of the nine constructs was performed. Out of the nine double mutants eight constructs had several colonies with the correct sequence. The exception was H_B for which a point mutation resulted in the mutant H_I. Even though some sequenced colonies had the right sequence for H_B, these would not grow on the agar plates. The reason for that is unclear. The nine resulting constructs were transformed to E. coli Rosetta cells for protein production. Cultivation and purification of the nine proteins was straightforward and yielded high concentrations of protein after purification. All samples were pure, as shown on SDS-‐PAGE (figure 4) and with ESI-‐MS (appendix 1). Looking at the SDS-‐PAGE the C2 double mutants migrate unexpectedly different in the gel although they have approximately the same molecular weight. The reason for this is unknown, but the same behaviour has earlier been observed. To test if the mutants had lost Fc binding while maintaining Fab binding, interaction analysis of the C2 double mutants was performed on the ProteOn™. Several attempts were made to get the right amount of ligand molecules on the surfaces for the ProteOn™. As the activation of the surface made 15 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH it responsive to only one ligand injection, problems were met obtaining a high enough amount of ligand on the surface. This theory is strengthened by the fact that activated surfaces that had ddH2O flowed over them before ligand coupling proved harder to couple. The experiment set up was supposed to elucidate if the mutant was interacting with Fc fragments and whole IgG. Since it proved very difficult to couple Fab fragments to the surfaces no Fab surfaces was used in the experiment. The assumption was made that if a mutant had lost Fc binding but bound to whole IgG, that interaction would come from C2:Fab binding. It should be mentioned that the response from coupling whole IgGs was lower than that of the Fc fragments. The Rmax of the Fc surfaces was almost five times higher than that of the IgG surfaces. Also, the full IgG molecules are three times larger than Fc. Hence the maximum number of available binding sites would be (5 x 3) 15 times higher at the Fc surface as compared to the IgG surface. The signal from the mutant:IgG interaction should then perhaps be higher compared to the signal from mutant:Fc interaction, if the surfaces had the same amount of molecules coupled on them. It will therefore be hard to compare the signals from the surfaces. Instead the mutants are compared to the C2wt interactions. Two mutants, E_A and H_I did look promising with very low Fc binding compared to IgG binding (appendix 1) and it will be interesting to further look into these with more interaction and kinetic experiments. They both had almost completely lost Fc binding when comparing to the C2wt interaction and maintained roughly 25 % of IgG binding compared to the wild type protein (appendix 1). This seems reasonable considering C2 has a lower affinity for Fab than Fc. Both these double mutants have their mutations in the same binding region. Comparing these with mutants that did not seem to loose binding to Fc at all e.g. E_B and A_B, that both have their mutations in different binding regions, it seems as the approach to put both mutations in the same binding region is more successful for deleting Fc binding than having the mutations in different binding regions. This also points at, at least for the E position, that a double mutation is needed, as the E mutation in itself is not sufficient to delete Fc binding. 6. Future prospects Further interaction and kinetic experiments of the C2 double mutants need to be performed. Most importantly investigations of the C2 double mutants’ interactions with the Fab fragment should be performed. This can be followed by further investigations of the most promising mutants to be able to draw kinetic conclusions about the mutants. In a more distant future a library introducing larger diversity into the C2 gene could be used for selections using e.g. phage display, to increase Fab binding for mutants where Fc binding has been completely deleted. Also positions for insertion of a photo crosslinking amino acid could be investigated. 7. Acknowledgements First and foremost I would like to thank Anna Konrad, Tove Boström and Sophia Hober for supervising me and letting me be part of this project. It has been a great journey full of learning, hard work and fun. I would also like to thank the Protein Factory for raising me in the laboratory and letting me come work in their lab all the time. I would like to thank everyone at plan 3 who has helped me and answered one of my many questions during my work with this project. Last but not least I would like to thank my partner Viktor and my fantastic family for all their love and support during my education. 16 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH 8. Appendix 8.1 Appendix 1: Raw data 8.1.1 MS Spectra C_A D_A E_A F_A 17 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH A_B C_B E_B G_B 18 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH H_I 8.1.2 ProteOn™ data C_A Fc
Fc
Fc
IgG
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IgG
D_A Fc
Fc
IgG
Fc
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IgG
19 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH E_A Fc
Fc
Fc
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IgG
IgG
F_A Fc
Fc
Fc
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IgG
IgG
A_B Fc
Fc
Fc
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IgG
IgG
20 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH C_B Fc
Fc
Fc
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IgG
E_B Fc
Fc
Fc
IgG
Blank IgG
G_B Fc
Fc
Fc
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IgG
21 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH H_I Fc
Fc
IgG
Fc
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C2wt Fc
Fc
Fc
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IgG
Zwt Fc
Fc
Fc
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22 Master thesis 30 hp Sara Kanje, Master of Science, Biotechnology, KTH 9. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. Clark DP, Pazdernik NJ. Biotechnology : applying the genetic revolution. Amsterdam: Elsevier; 2009. xiii, 750 s. p. Parham P. The immune system. New York, NY: Garland; 2005. xv, 431 s. p. Wilson K, Walker JM. Principles and techniques of biochemistry and molecular biology. Cambridge: Cambridge University Press; 2005. xiv, 783 s. p. Sidorin EV, Solov'eva TF. IgG-‐binding proteins of bacteria. Biochemistry (Mosc) 2011;76(3):295-‐308. Cleary P, Retnoningrum D. Group A streptococcal immunoglobulin-‐binding proteins: adhesins, molecular mimicry or sensory proteins? Trends Microbiol 1994;2(4):131-‐6. Bouvet JP. Immunoglobulin Fab fragment-‐binding proteins. Int J Immunopharmacol 1994;16(5-‐6):419-‐24. Sloan DJ, Hellinga HW. Dissection of the protein G B1 domain binding site for human IgG Fc fragment. Protein Sci 1999;8(8):1643-‐8. Björck L, Kronvall G. Purification and some properties of streptococcal protein G, a novel IgG-‐
binding reagent. J Immunol 1984;133(2):969-‐74. Derrick JP, Wigley DB. The third IgG-‐binding domain from streptococcal protein G. An analysis by X-‐ray crystallography of the structure alone and in a complex with Fab. J Mol Biol 1994;243(5):906-‐18. Tashiro M, Montelione GT. Structures of bacterial immunoglobulin-‐binding domains and their complexes with immunoglobulins. Curr Opin Struct Biol 1995;5(4):471-‐81. Akerström B, Nilson BH, Hoogenboom HR, Björck L. On the interaction between single chain Fv antibodies and bacterial immunoglobulin-‐binding proteins. J Immunol Methods 1994;177(1-‐2):151-‐63. Sjöbring U, Björck L, Kastern W. Streptococcal protein G. Gene structure and protein binding properties. J Biol Chem 1991;266(1):399-‐405. Akerström B, Björck L. A physicochemical study of protein G, a molecule with unique immunoglobulin G-‐binding properties. J Biol Chem 1986;261(22):10240-‐7. Akerström B, Brodin T, Reis K, Björck L. Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J Immunol 1985;135(4):2589-‐92. Sauer-‐Eriksson AE, Kleywegt GJ, Uhlén M, Jones TA. Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG. Structure 1995;3(3):265-‐78. Ståhl S, Nygren PA, Sjölander A, Uhlén M. Engineered bacterial receptors in immunology. Curr Opin Immunol 1993;5(2):272-‐7. Derrick JP, Maiden MC, Feavers IM. Crystal structure of an Fab fragment in complex with a meningococcal serosubtype antigen and a protein G domain. J Mol Biol 1999;293(1):81-‐91. Björck L, Kastern W, Lindahl G, Widebäck K. Streptococcal protein G, expressed by streptococci or by Escherichia coli, has separate binding sites for human albumin and IgG. Mol Immunol 1987;24(10):1113-‐22. Derrick JP, Wigley DB. Crystal structure of a streptococcal protein G domain bound to an Fab fragment. Nature 1992;359(6397):752-‐4. Lian LY, Barsukov IL, Derrick JP, Roberts GC. Mapping the interactions between streptococcal protein G and the Fab fragment of IgG in solution. Nat Struct Biol 1994;1(6):355-‐7. Cooper GM, Hausman RE. The cell : a molecular approach. Washington, D.C.Sunderland, Mass.: ASM Press ;Sinauer Associates; 2004. xx, 713 s. p. 23

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