Materials and Methods
2. Materials and Methods 2.1 Materials 2.1.1 Commercial suppliers Unless otherwise stated, all chemicals were purchased from Merck, Sigma-Aldrich, Difco, Serva, Fluka, Hampton Research and Boehringer Mannheim. Enzymes were purchased from New England Biolabs (NEB), Roche, Stratagene and Sigma-Aldrich. Inhibitors were purchased from Serva. Antibiotics were purchased from Roche and Invitrogen. Consumed materials were purchased from BioRad, Fisher, Hampton Research, Millipore, NEB and Pharmacia Biotech. 2.1.2 Vectors The vectors in (Table 2.1) are ampicillin resistant (Ampr). For vector-maps refer to Figure 7.1, Appendix. Table 2.1. Vector characteristics of vectors used in the insect and E. coli expression systems. Baculovirus transfer vector pMage pAcG2T pVL1392 pVL1393 pVLSO Bacterial vector
Expression system Insect cells Insect cells Insect cells Insect cells Insect cells Expression system
E. coli E. coli
Promotor Polyhedrin Polyhedrin Polyhedrin Polyhedrin Polyhedrin
features GST-tagged, thrombin site GST-tagged, thrombin site None-tagged None-tagged GST-tagged, thrombin site
Created by Pharmingen Pharmingen Pharmingen Pharmingen me
Promotor Tac Tac
Created by in the lab me
GST-tagged, thrombin site, RBS, 2.MCS GST-tagged, thrombin site, RBS, 2.MCS
2.1.3 Bacterial and viral strains The Escherichia coli (E. coli) strain DH5α (Novagen) was used for vector amplification and E. coli strain BL21(D3) (Novagen) was utilized for recombinant protein expression. The BaculoGold Baculovirus DNA strain Autographa californica nuclear polyhedrosis virus (AcNPV) (Pharmingen) was used for generating the recombinant baculovirus.
Materials and Methods 2.1.4 Insect cell lines High Five (Hi5) cell line Hi5 cells were derived from Trichoplusia ni egg cell homogenates (Pharmingen). This cell line is highly susceptible to infection with AcNPV, and was used with several Baculovirus expression vectors (Table 2.1). The Hi5 cells were cultivated in suspension- and in monolayer cultures and were utilized for protein expression. Spodoptera frugiperda (SF9) cell line The SF9 cell line was cloned in 1983 from the parent line IPLB-Sf21 AE, which was derived from pupal ovarian tissue of the fall army-worm (Pharmingen). This cell line is highly susceptible to infection with AcNPV and was used with several Baculovirus transfer vectors (Table 2.1). SF9 cells were cultivated in monolayer culture and were used for virus production. 2.1.5 Media for E. coli strains LB-Agar:
10 g/L Trypton/Peptone, 10 g/L NaCl, 5 g/L yeast extract, 15 g/L agar
LB-Medium: 10 g/L Trypton/Peptone, 10 g/L NaCl, 5 g/L yeast extract for insect cell lines Monolayer culture media: 500 ml Graces insect media, 50 ml Fetal bovine serum, 2.25 ml Penicillin/Streptomycin/L-glutamine-Mix Suspension culture media:1 L SF900 II SFM media, 10 ml Penicillin/Streptomycin-Mix Cryo preservation media: SF900 II SFM media, 10 ml Penicillin/StreptomycinMix, 10 % sterile DMSO 2.1.6 Buffers and solutions Unless otherwise stated, all buffers and solutions were made using water of MilliQ quality. Buffers and solutions not listed below are described together with the method for which they were used.
Materials and Methods Acrylamide stock solution 30 % (v/v) acrylamide, acrylamide:bis-acrylamide ratio 37.5:1 Agarose gel loading buffer (6 x) 0.25 % (w/v) BromoPhenol Blue, 0.25 % (w/v) Xylene Cyanol Blue, 50 % (w/v) Glycerol Ammonium persulphate (APS) 10 % (w/v) APS Coomassie destain 40 % (v/v) methanol, 10 % (v/v) acetic acid Coomassie stain 40 % (v/v) methanol, 10 % (v/v) acetic acid, 0.1 % (w/v) Coomassie Brilliant Blue R-250 Cryoprotection solution I 1.4 M LiSO4, 0.1 M BTP pH 8.5, 0.2 M NaCl Cryoprotection solution II 1.8 M LiSO4, 0.1 M BTP pH 8.5, 0.2 M NaCl Cryoprotection solution III, 2.2 M LiSO4, 0.1 M BTP pH 8.5, 0.2 M NaCl Dialysis buffer 100 mM BTP pH 6.8, 0.2 M NaCl, 2 mM DTT Elution buffer 50 mM Tris pH 8.0, 0.2 M NaCl, 5 mM DTT, 20 mM reduced Glutathione HPLC Buffer Buffer A: 0.1 % TFA Buffer B: 100 % Acetonitrile, 0.1 % TFA Lysis buffer 50 mM Tris pH 8.0, 0.2 M NaCl, 5 mM DTT, 0.5 mM PMSF, 1 μM Leupeptin, 1 μM Pepstatin A, 0.3 μM Aprotinin SDS-PAGE loading buffer (5 x) 625 mM Tris/HCl pH 6.8, 25 % (w/v) sucrose, 10 % (w/v) SDS, 0.1 % (w/v) bromophenol blue, 10 % (v/v) β-mercaptoethanol SDS-PAGE running buffer 25 mM Tris/HCl pH 8.3, 0.1 % (w/v) SDS, 192 mM glycine
Materials and Methods TAE (50 x) 242 g Tris Base, 57.1 ml Acetic Acid (glacial), 37.2 g EDTA, add to 1 L H2O TFB1-buffer 30 mM KoAc, 100 mM RbCl2, 10 mM CaCl2, 50 mM MnCl2, 15 % (v/v) glycerol, adjust pH to 5.8 with acetic acid, sterile filtration TFB2-buffer 10 mM MOPS, 75 mM CaCl2, 10 mM RbCl2, 15 % (v/v) glycerol, adjust pH to 6.5 with HCl, sterile filtration Transfer buffer 10 mM CAPS pH 11, 10 % (v/v) methanol Wash buffer 50 mM Tris pH 8.0, 0.2 M NaCl, 5 mM DTT 2.1.7 Kits The Maxiprep-, the Miniprep-, the PCR-purification- and the DNA-extraction kits were all purchased through Qiagen. The BaculoGold Baculovirus DNA-transfection kit was purchased through Pharmingen. The crystallization screening kits, the Hampton Screen I/II, the MPD-Screen, the AmSO4-Screen, the PEG/Ion-Screen, the PEG-Screen, the Additive Screen I-III and the Detergent-Screen I-III were purchased through Hampton Research. Unless otherwise stated, all of the kits were used used strictly in accordance with the manufacturers instructions.
2.2 Methods 2.2.1 Polymerase chain reaction (PCR) 126.96.36.199 General PCR-protocol PCR enables the rapid and specific amplification of DNA-sequences. The oligonucleotides used for PCR were custom-made by Genelink. A typical PCR-reaction contained 10 μl of 10 x polymerase buffer, 3 μl of 10 mM dNTPs, 1 μM of the 5’- and 3’primers, 5 to 100 ng of template, 1 to 5 U of polymerase. Distilled water was also added to the final volume of 100 μl. The PCR-amplification program started with a 1 min denaturation step at 94 to 98 °C, followed by 10 to 30 sec primer annealing, which was performed 5 to 10 °C below the melting point of the primer. The next step in the cycle was primer extension at 72 to 75 °C for 1 to 10 min. After 30 cycles, a single primer extension of DNA-fragments 31
Materials and Methods was performed for 10 min at 72 to 75 °C. The quality of the amplified DNA-fragments was analyzed by agarose gel electrophoresis and purified using the PCR-purification kit (Material, 2.1.7). 188.8.131.52 PCR-screening PCR-screening was used to screen transformed bacterial colonies if they contained the vector with the desired insert by using vector- or gene-specific 5’- and 3’-primers. Single transformed bacterial colonies were selected from the LB-agar-plate and transferred into a PCR-tube containing 20 μl of the pre-pipetted PCR-reaction mixture. PCR was performed immediately and checked by agarose gel electrophoresis. 184.108.40.206 Overlapping-PCR Overlapping-PCR was performed to incorporate a point mutation into the 2ndphosphate-binding pocket of the scCdc4271-779 DNA-fragment as well as for internal DNAsequence deletions in the scAPC4 gene. The mutated 5’- and 3’-primers were derived from the position at the native template DNA and changed to the novel DNA-primer sequence bearing the desired mutation or internal deletion. Furthermore, native 5’- and 3’-primer were designed that comprised the whole native template DNA. At first, two DNA-fragments were amplified with the native template DNA, one with the primer pair native 5’- and mutated 3’primer and the other with the primer pair mutated 5’- and native 3’-primer. The result was two short DNA-fragments bearing the same introduced mutation/deletion, either at the 5’- or the 3’-end of the DNA-fragment. The next PCR was performed with the two mutated 5’- and 3’-DNA-fragments as a template, which overlapped (annealed) in the region where the mutation was introduced. As result of this PCR the amplified DNA bears the desired mutation/deletion inside the DNA-sequence. 220.127.116.11 Side-directed mutagenesis Side-directed mutagenesis PCR was performed for introducing a point mutation in the second multiple cloning site (MCS) of the pABLO vector. The mutated 5’- and 3’-primers were designed to cover approximately 20 base pair (bp) on each side of the respective point mutation to guarantee specific annealing. The whole vector-DNA was amplified by utilizing
Materials and Methods the pfu polymerase. On completion of the PCR, the methylated mother vector-DNA was digested for 2 h at 37 °C with the restriction enzyme Dam, which specific cuts methylatedDNA. PCR-purification of the newly synthesized mutated vector-DNA (Material, 2.1.7) and transformation then followed. 2.2.2 Cloning 18.104.22.168 Digestion of DNA DNA-digestion of the PCR-fragments and the vector-DNA was performed with appropriated restriction endonucleases using the buffer system, with time and temperature as recommended by the manufacturer, NEB. To prevent religation of the digested linearized vector-DNA, the 5’-phosphate groups were removed by treatment with Calf intestinal phosphatase (CIP). CIP is compatible with the NEB buffer 2 to 4 used by the restriction enzymes. One unit of CIP is directly added to 2.5 μg linearized vector-DNA digestion mix and incubated for 1 h at 37 °C. The analysis of the PCR-fragments and the vector-DNA digestion by agarose gel electrophoresis then followed and digested DNA-fragments were excised and purified with the DNA-extraction kit (Material, 2.1.7). 22.214.171.124 Ligation with T4 ligase For the ligation of digested DNA-fragments the molar ratio of insert-DNA to linearized vector-DNA was varied in a ratio of 1 to 1 up to 5 to 1, typically using 100 μg of vector. The DNA-fragments were ligated using 1 μl of T4 ligase in a total reaction volume of 20 μl following the instructions of the manufacturer (NEB). The ligated vector-DNA mix was used for transforming bacterial cells without further purification. 126.96.36.199 Preparation of competent E. coli cells A small amount ~10 μl of frozen competent E. coli cells were added to a flask containing 200 ml of LB-Medium with no antibiotic and incubated at 37 °C and 250 rpm. When the bacterial culture reached an OD600 value of approximately 0.5, the flask was removed from the shaker and cooled on ice. After 10 min, the bacteria culture was transferred to a pre-chilled 250 ml centrifuge tube and the cells were harvested by centrifugation at 3.5 K, 4 °C for 10 min. The cell pellet was re-suspended in 80 ml ice cold
Materials and Methods TFB1-buffer (Material, 2.1.6) and incubated on ice for 5 min. The cells were once again the cells were harvested by centrifugation at 3.5 K, 4 °C for 10 min. That new cell pellet was resuspended in 8 ml ice cold TFB2-buffer (Material, 2.1.6) and the cells were dispensed as 75 μl aliquots into micro-centrifugation tubes, shock-frozen in liquid nitrogen, and stored at -80 °C until further use. 188.8.131.52 Transformation of E. coli cells Competent E. coli cells were thawed in hand and 75 μl was mixed with 20 μl of the ligated vector-DNA mix or 100 ng vector-DNA. The mixtures were kept on ice for 30 min, followed by a heat shock for 1 min at 42 °C and then an immediately 2 min incubation on ice. Transformed E. coli cells were incubated for 1 h at 37 °C, with 0.3 ml LB-media containing no antibiotics. After this, the E. coli cells were spread on LB-Agar plates containing Ampr. Subsequent to over-night incubation at 37 °C, E. coli transformants were visible as colonies. To identify whether transformants contained the gene of interest, PCRscreening and vector digestion were performed from selected bacterial colonies. The PCR as well as the vector digestion were each analyzed using agarose gel electrophoresis. 184.108.40.206 Isolation of vector-DNA Transformed E. coli cultures were grown over-night at 37 °C and 240 rpm. The next day, 5 ml (200 ml) cell culture was harvested by centrifugation at 3.5 K for 10 min and DNA was purified with the DNA-Miniprep kit (DNA-Maxiprep kit) (Material, 2.1.7). Maxiprep purification was necessary when large DNA amounts were required, such as with the generation of recombinant Baculovirus. 220.127.116.11 Agarose gel electrophoresis Agarose gel electrophoresis was performed to separate DNA-fragments according to their size. The agarose concentration of the gels varied from 0.8 to 2.0 % depending on the size of DNA-fragments to be analyzed. The agarose was melted in 1 x TAE buffer (Material, 2.1.6) and 0.5 μg/ml Ethidium bromide was added before pouring the gel. Ethidium bromide is an organic dye with a plane structure that intercalates into DNA. It gets excited with UVlight (254 to 366 nm) and emits light of the orange-red spectrum (590 nm) that visualizes the
Materials and Methods DNA-fragments. Samples were mixed with 1/5vol of agarose gel loading buffer (Material, 2.1.6) before loading. Gels were run at 5 V/cm and photographed using the Mitsubishi P90 system. A 100 bp- or 1 kbp ladder from NEB was applied as a size standard. 18.104.22.168 Determination of DNA-concentration DNA has an UV-light absorption maximum at 260 nm due to the aromatic rings of its bases. Therefore, DNA-concentrations of the samples were determined by measuring the OD at 260 nm using a Perkin Elmar spectrophotometer. An OD260 of one equals a concentration of 50 μg/ml DNA. 22.214.171.124 DNA-sequencing Determination of DNA-sequences was done using the dideoxyribonucleotides triphosphate (ddNTPs) chain termination method from F. Sanger (Sanger et al., 1977). Samples were prepared following the instructions of Genewiz. The DNA-sequencing was performed by Genewiz and the DNA-sequencing results were analyzed with the programs DNA Star and Chromas. 126.96.36.199 Vector design pABLOmut vector The pABLOmut vector (Figure 2.1) was derived from the pABLO vector which is capable of simultaneously expressing a GST-fusion and a none-tagged protein (Appendix, Figure 7.1). The purpose of the pABLOmut vector design was to mutate the first restriction site NdeI in pABLOs second MCS which contains the first start codon ATG. By mutating the start codon from ATG to ATC (Appendix, Table 7.1) it was possible to use restriction sites
5’-T ATG AAG CCT……. M K L……... pABLOmut 5’-T ATC AAG CCT……. ……... Figure 2.1. Composition of the second MCS of the pABLO and the pABLOmut vector.
Materials and Methods downstream of NdeI in the cloning process and to choose the initiation of protein synthesis by incorporating an ATG in the 5’-primer. The vector mutation was performed as described in Method, 188.8.131.52. pVLSO vector The pVLSO vector was derived from the none-tagged vector pVL1392, in which the GST-tagged from the vector pMage was cloned in front of pVL1392 MCS. Both vectors were digested with the restriction enzymes EcoRV and Not1, which resulted in two DNAfragments of each vector on an agarose gel. The smaller DNA-fragment from pMage (5’EcoRV-GST-Not1-3’) and the bigger DNA-fragment from pVL1392 (5’-EcoRV-Not1-3’) were excised, purified, ligated and transformed in E. coli, and the purified novel vector-DNA was confirmed by DNA-sequencing. The created pVLSO vector contains an N-terminal GST-tagged and misses the first two restriction sites BglII and PstI from the MCS of pVL1392 (Appendix, Figure 7.1). The designing of the pVLSO vector was performed to simplify the cloning efforts of the APC genes. With the GST-tagged vector pVLSO and the none-tagged vector pVL1392 the same restriction enzymes were used to obtain each APC gene with a single doubledigested PCR-product. The pVLSO vector contains the GST-fusion and the pvL1392 vector contains the none-tagged version of the same APC subunit. 184.108.40.206 Cloning of the studied genes and DNA-fragments The herein studied genes and DNA-fragments were constructed with the methods described above in 2.2.1 to 2.2.2. The composition of the utilized primer pairs and final constructs are found in Table 7.1 and Table 7.2 in the Appendix. 2.2.3 Cell biology methods 220.127.116.11 Cultivation of insect cell lines Hi5- and SF9 insect cell lines were grown in suspension- and in monolayer cultures. Small-scale cultivation of insect cells can be maintained as monolayer cultures. However, for large-scale insect cell cultivation this is too time-consuming. Therefore suspension cultures are used. To maintain Hi5- (SF9) monolayer cultures, confluent monolayer cultures were
Materials and Methods split every 3 days in a ratio of 1 to 5 (1 to 3) into monolayer culture media (Material, 2.1.5). Suspension cultures were maintained by splitting at a cell density of 4 x 106 cells/ml back to a density of 5 x 105 cells/ml in fresh suspension culture media (Material, 2.1.5). 18.104.22.168 Thawing, freezing and storage of insect cells Thawing of insect cells The cell culture vial was thaw in hand and cells were washed twice with 25 ml of suspension culture media. Centrifugation was performed at 1500 rpm for 2 min. After this, the cell pellet was re-suspended in 100 ml suspension culture media and cells were cultivated at 27 °C and 200 rpm. The viability of the cell culture was analyzed every 2 to 3 days and culture was expanded depending on the cell density. Freezing and storage of insect cells For freezing insect cells, 300 ml of 2 x 106 cells/ml cell culture were centrifuged at 1500 rpm for 2 min and the cell pellet re-suspended in 30 ml cryo preservation media. Aliquots of the cell culture were slowly frozen in the blue-topped isopropanol container and cell culture vials were kept for a day at –80 °C before they were transferred them to the liquid nitrogen tank for long-term storage. 22.214.171.124 Generating and amplification of recombinant Baculovirus The mixing of a Baculovirus transfer vector (Table 2.1) with the Baculovirus DNA strain AcNPV, allows recombination between their homologous sites and transfers the heterologous gene from the Baculovirus transfer vector to the AcPNV, resulting in the recombinant Baculovirus. For generating a recombinant Baculovirus, approximately 2 x 106 SF9 cells were seeded onto a 60 mm tissue culture plate, followed by a 5 min incubation period at RT to allow the cells to attach firmly to the tissue culture plate (50 to 70 % confluent). The monolayer culture media was then replaced with 1 ml of Transfection Buffer A (Material, 2.1.7). In the meantime 2 to 5 μg Baculovirus transfer vector containing the gene of interest were mixed with 0.5 μg AcPNV, generating the recombinant Baculovirus. After a 5 min incubation period, the sample was mixed with 1 ml of Transfection Buffer B, and was added
Materials and Methods drop-by-drop to the tissue culture plate to mix with Buffer A. The SF9 plate was incubated for 4 h at 27 °C before removing the mixture. Cells were washed twice with 3 ml monolayer culture media before once again adding 3 ml monolayer culture media to the SF9 monolayer culture. The plates were then incubated for 4 to 5 days at 27 °C. The supernatant, which included the recombinant passage 1-Baculovirus (P1-virus), was then harvested by centrifugation at 1500 rpm for 5 min and stored in a dark place at 4 °C until further use. To amplify, recombinant Baculovirus SF9 cells were grown to a confluence of 60 to 80 % on 15 cm tissue culture plates. Up to 2 ml of the monolayer culture media was removed before 0.1 to 1 ml of recombinant P1-virus was added to each plate. Plates were rocked for 1 to 1.5 h at RT, before 20 ml of monolayer culture media was added and the cells were incubated at 27 °C for 3 days. The supernatant (recombinant P2-virus) was then harvested by centrifugation at 1500 rpm for 5 min and stored in a dark place at 4 °C until further use. The amplification process was repeated with the P2-virus and the P3-virus. The P4-virus obtained was used for large-scale recombinant protein production. 126.96.36.199 Protein expression In insect cells Hi5 monolayer culture Hi5 monolayer culture cells were used to investigate protein expression levels of proteins of interests with a low passage of recombinant Baculovirus (P2-virus), as amplifying viruses and expanding cells is a very time-consuming process and insoluble expressed proteins cannot be used for further studies. Therefore, Hi5 monolayer culture cells were grown in 15 cm tissue culture plates to a confluence of 70 to 80 %. Up to 2 ml of the monolayer culture media was removed and the plates were infected with 0.1 to 1 ml of P2-virus. The plates were then rocked for 1 to 1.5 h at RT before 20 ml of monolayer culture media was added to the cells and they were incubated at 27 °C for 3 days. After this, the Hi5 cells were harvested and the cell pellet was stored at –80 °C or used as source of protein purification. In insect cells Hi5 suspension culture Hi5 suspension culture cells were used for large-scale protein expression. For that purpose, the P4-virus was necessary.
Materials and Methods For a typical 8 L protein expression experiment, 4 x 1 L bottles and 8 x 2.8 L flasks were autoclaved the day before starting the experiment. The next day, 4 x 1 L Hi5 suspension cultures with a density of 4 x 106 Hi5 cells/ml were centrifuged at 1500 rpm for 5 min. Each Hi5 cell pellet was re-suspended with 40 ml P4-virus and combined into a 1 L bottle which was rocked for 1 to 1.5 h at RT. After this, the bottle of infected Hi5 cells was divided into eight spinner flasks and to each flask 1 L fresh suspension culture media was added. The infected Hi5 suspension cultures were incubated for 2 to 3 days by 200 rpm at 27 °C. The Hi5 cells were then harvested by centrifugation at 4000 rpm for 10 min. The Hi5 cell pellets were stored at –80 °C or used as source of protein purification. In E. coli For a typical 12 L E. coli protein expression experiment, 12 flasks, each containing 1 L LB-media were autoclaved the day before starting the experiment. In addition, 100 ml LBAmpr-media was inoculated with a single colony from a LB-Ampr-agar plate and incubated over-night at 37 °C by 240 rpm. The next day, the autoclaved LB-media was supplemented with Ampr and was inoculated with a ratio of 1 to 1000 of the over-night starting culture. The flasks were shaken at 37 °C and 240 rpm until the E. coli cell culture density reached an OD600 of 0.6 to 0.8 and protein expression was induced with 1 mM IPTG. After induction, the flasks were shaken at RT at 240 rpm for 12 to 18 h. The E. coli cell cultures were then centrifugated at 4000 rpm for 10 min. The cell pellets were stored at –80 °C or used as source of protein purification. 188.8.131.52 Cell lysis Cell lysis is a method that disrupts cells and leads to the release of their proteins into the lysis buffer. Recombinant cell pellets from bacteria- and insect cell cultures were resuspended in lysis buffer to 30 ml of each L of cell culture (Material, 2.1.6). Cells were lysed three times at 15.000 psi with the Cell disruptor, EmulsiFlex-C5, Avestin. After cell lysis, the homogenate was centrifuged at 40.000 rpm for 1 h (Sorvall, SS34-rotor). The supernatant contains the soluble proteins (the crude extract) and is the source of protein purification.
Materials and Methods 2.2.4 Chromatographic methods Purification of GST-fusion proteins In the glutathione-S-transferase (GST)-fusion system, the target protein is fused to GST-tagged (26 kDa). The GST-tagged has a high binding affinity to glutathione and is therefore a convenient tool to purify GST-fusion proteins from crude extracts. For this purpose the glutathione ligand is coupled via a 10-carbon linker to highly cross-linked 4 % agarose beads, Glutathione 4 B (G4B) and have a binding capacity of approximately 10 mg recombinant GST/ml G4B. The appropriated amount of G4B was added to a gravity column and equilibrated with 5 CV of wash buffer, before the crude extract was applied twice over. After this, the column was washed with 20 CV of wash buffer (Material, 2.1.6). The specific bound GST-fusion protein was eluted with 5 CV of elution buffer (Material, 2.1.6). The free glutathione of the elution buffer competes for the matrix bound GST, in which process the GST-fusion protein is eluted from the beads. Ion exchange chromatography (IEC) IEC separates proteins due to their reversible adsorption to a counter charged group immobilized to a matrix (Table 2.2). The extent of tightness to which a protein is bound to an ion exchange matrix depends upon the factors influencing the net charge of the protein such as pH and ionic strength. The adsorpt proteins were separated by gradually increasing the salt-concentration in the mobile phase through which the protein with a low net charge are desorpt and eluted first. After the GST pull down experiment, the ion strength of the protein solution was reduced from 200 mM NaCl to 50 mM NaCl by diluting it in the same buffer, which contains no NaCl. The diluted protein sample was then loaded onto the IEC, equilibrated with 50 mM NaCl, and the proteins were eluted with a NaCl-gradient (50 mM NaCl to1 M NaCl). Table 2.2. Characteristics of ion exchange matrices. Ion exchange matrix
Source Q Source S Heparin (glycosaminoglycan)
anion cation cation
Materials and Methods Size exclusion chromatography (SEC) SEC separates proteins according to their size and shape. The gel filtration matrix contains pores, which allow smaller proteins to distribute in a larger volume than bigger proteins. As a consequence, the smallest protein is the last to elute. In this thesis, the Superdex 200 gel filtration matrix was used. It contains dextran-agarose beads with a pore size of 34 μm and is used to separate proteins of 10-600 kDa (according Pharmacia). To achieve a good level of separation, the protein loading volume never exceeded 2 ml. Reversed phase Chromatography (RPC) In RPC, proteins or peptides are bound through hydrophobic interaction to the stationary phase. The stationary phase consists of silicon-based molecules with chlorine as the reactive group, to which a hydrocarbon group is attached. The linear aliphatic hydrocarbon groups of 8 carbons (C8) or 4 carbons (C4) form the hydrophobic phase. A C8column was used for peptide- and a C4-column for protein purification. After the column was equilibrated with HPLC Buffer A (Material, 2.1.6), the sample was loaded. The acetonitrile-gradient was then started with HPLC Buffer B (Material, 2.1.6) and led to the elution of the proteins, initially with a minimum level of hydrophopic interaction. RPC was used as a sample preparation for mass spec analysis and for peptide purification. 2.2.5 Biochemical methods 184.108.40.206 Thrombin cleavage All GST-fusion proteins used in this thesis contain the thrombin recognition site LeuVal-Pro-Arg-Gly-Ser, which links the GST-tag to the protein of interest. Thrombin binds to the thrombin recognition site and cleaves the GST-fusion protein in the GST-protein and the target protein starting with the amino acid sequence Gly-Ser. The amount of thrombin necessary to cleave the GST-fusion protein was dependent on the accessibility of the thrombin recognition site. 220.127.116.11 SDS polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was used to separate proteins according to their molecular weight (MW). SDS is an anionic detergent and binds to proteins through hydrophobic interaction. It
Materials and Methods complexes proteins in a constant weight ratio and masks the protein charge with SDS own strongly negative charge, resulting in identical charge densities on the surface. Protein samples were prepared by boiling with SDS and DTT containing SDS-PAGE loading buffer, which destroys structure elements as well as it cleaves disulphide bridges of proteins. The MW of the separated proteins was determined through comparison with a SDS-PAGE MW standard (BioRad). The SDS-PAGE was carried out in a Mini ProteanII Cell (BioRad). The percentage of acrylamide used in the SDS-PAGE was dependent on the MW of the investigated protein. After this, protein gels were boiled for 1 min in coomassie staining solution (Material, 2.1.6) and then transferred into the coomassie destaining solution (Material, 2.1.6) until protein bands were clearly visible. For the purpose of record keeping, destained gels were dried with Slab Gel Dryer Model SE II 60 (Pharmacia Biotech). SDSgels were prepared as outlined (Table 2.3). Table 2.3. Buffers and solutions for 10 SDS-PAGEs. 50 ml SDS running PAGE 15 % 30 % Acrylamide 18.75 ml 2 M Tris pH 8.8 9.4 ml 20 % SDS 0.25 ml distilled H2O 21.05 ml TEMED 0.05 ml APS 10 % 0.5 ml
18 % 22.5 ml 9.4 ml 0.25 ml 17.3 ml 0.05 ml 0.5 ml
50 ml SDS stacking PAGE 4.3 % 30 % Acrylamide 5.4 ml 1 M Tris pH 6.8 6.8 ml 20 % SDS 0.25 ml distilled H2O 37.6 ml TEMED 0.05 ml APS 10 % 0.5 ml
18.104.22.168 Limited proteolysis Limited proteolysis is a powerful method, which determines flexible and unstructured regions in a protein of interest. The limited proteolysis of a protein can be controlled by the amount, time and the specificity of the enzyme used in the experiment. Under constant conditions, the fragmenting pattern of a protein is very characteristic and reproducible. To further analyze the fragmentation pattern, the protein fragments were separated and visualized on an SDS-PAGE. N-terminal sequencing- and mass spec analysis of the protein fragments then followed, which can lead to the identification of the more stable core domains of a protein of interest. Subsequent cloning of the protein, excluding the determined flexible regions, leads to more stable proteins that might be easier to crystallize.
Materials and Methods Subtilisin The enzyme Subtilisin belongs to the serine protease family and has no preference in catalyzing the cutting of specific amino acid bonds. Trypsin The enzyme Trypsin belongs to the serine protease family and preferably cleaves the peptide bonds C-terminal of the amino acids of arginine and lysine. 22.214.171.124 Phosphorylation of proteins The Sic11-100 protein fragment was phosphorylated by using 1 to 100 molar ratio of Cdk6 bound to the herpesvirus K-Cyclin, prepared as previously described (Jeffrey et al., 2000) in 100 mM HEPES pH 7.0, 200 mM NaCl, 10 mM ATP and 10 mM MgCl2 at RT for 30 min. The phosphorylation sites and levels were verified by electrospray ionization mass spectrometry of reversed phase HPLC-purified tryptic digest. 126.96.36.199 Western blot To blot proteins from a SDS-PAGE to a Polyvinylidine diflouride membrane (PVDF), a wet transfer was performed in a Mini Trans-Blot Cell (BioRad). The PVDF membrane was submerged in 100 % methanol and was then washed twice with distilled H2O. Before the western blot transfer stack was assembled as outlined in (Figure 2.2), foam pads, Whatman filter paper and PVDF membrane were soaked in transfer buffer (Material, 2.1.6). The transfer was carried out at 300 mA for 2 h at 4 °C. Black cathode (-) Foam pad Whatman filter paper SDS gel PVDF membrane Whatman filter paper Foam pad Red anode (+) Figure 2.2. Assembly of a western blot transfer stack.
188.8.131.52 Sample preparation for N-terminal protein sequencing The N-terminal protein sequencing was performed using the solid-phase-Edmanreduction method. In each cycle, the most N-terminal amino acid of the polypeptide chain is 43
Materials and Methods reduced and determined. For this technique proteins need to be bound to a solid phase. Therefore, protein samples were prepared by separation on an SDS-PAGE followed by a western blot. The PVDF membrane of the western blot was stained with Coomassie until blue protein bands were visible and the membrane was air dried. The blue protein bands of interest were excised and the MSKCC Protein Analysis lab performed the N-terminal protein sequencing analysis. 184.108.40.206 Sample preparation for mass spectrometry Mass spectrometry is a method to identify the exact MW of proteins and requires only ~20 ng of protein. For mass analysis, proteins with a purity of at least 90 % were used. Mass analyses of protein fragments from limited proteolysis were prior-purified by FPLC or HPLC. Protein samples were sent and analyzed by the mass spectrometry facility in Berkeley, California. 220.127.116.11 Dialysis Dialysis was performed to adjust the buffer conditions of the protein- and peptide solutions used in the ITC-experiments. Protein- and peptide solutions were each filled in a size-appropriated dialysis bag and dialyzed against the dialysis buffer (Material, 2.1.6). The buffer was exchanged for three times with a sample to buffer ratio of 1 to 500. 18.104.22.168 Concentrating of proteins Protein solutions were concentrated with the ultrafiltration device from Amicon (Millipore) in accordance with the instructions of the manufacturer. 22.214.171.124 Determination of protein concentration A rough estimation of protein concentrations was achieved via Bradford assay. One microliter of protein solution was diluted into 1 ml Bradford solution, mixed and incubated for 5 min before reading at OD595. It was assumed that a reading of 0.3 equals a protein concentration of 10 mg/ml, lab standard. More accurate measurements of protein concentrations were determined by the use of the proteins distinct coefficient factors and the reading obtained at OD280.
Materials and Methods 126.96.36.199 Purification of the studied proteins and protein complexes Purification of the Fbw7-Skp1 complex Due to its insolubility in the E. coli strain BL21(DE3), the Fbw7232-707-Skp1 complex was purified from Hi5 insect cells, whereas the Fbw7263-707-Skp1 complex and the Fbw7279707
-Skp1 complex were purified from the E. coli strain BL21(DE3). All Fbw7-Skp1
complexes were purified using the following protocol: Initially, cells were disrupted and the GST-fusion complex was isolated from the crude extract by GST-affinity chromatography. The eluted GST-fusion complex (50 mM Tris pH 8.0, 200 mM NaCl, 5 mM DTT and 20 mM Glutathione) was then digested with 1 % thrombin over-night. The next day, the protein complex was diluted to 50 mM NaCl, 5 mM DTT, 50 mM Tris pH 8.0, it was loaded onto the Heparin column equilibrated in the same buffer, and the complex was eluted with a linear NaCl-gradient. The peak fractions were pooled and concentrated by ultrafiltration before purification continued by size exclusion chromatography. A maximum of 2 ml of complex was loaded onto an equilibrated Superdex 200 column (20 mM BTP pH 6.8, 5 mM DTT, 200 mM NaCl) and was collected in 0.5 ml fractions. The peak fractions were pooled and stored at –80 ºC until further use. Purification of the scCdc4-Skp1 complex The scCdc4111-779-Skp1 complex, the scCdc4271-779-Skp1 complex as well as the mtscCdc4S464A/T465V-Skp1 complex and the mt-scCdc4R443M/S464A/T465V-Skp1 complex were purified from Hi5 insect cells. All scCdc4-Skp1 complexes were purified using the following protocol: Initially, cells were disrupted and the GST-fusion complex was isolated from the crude extract by GST-affinity chromatography. The eluted GST-fusion complex (50 mM Tris pH 8.0, 200 mM NaCl, 5 mM DTT and 20 mM Glutathione) was digested with 5 % thrombin over two nights. Then, the protein complex was diluted to 50 mM NaCl, 50 mM MES pH 6.0, 5 mM DTT and loaded onto a Source S column equilibrated with the same buffer. The elution of the complex then followed, with a linear NaCl-gradient. The peak fractions were pooled and concentrated by ultrafiltration before purification continued by size exclusion chromatography. A maximum of 2 ml of complex was loaded onto an equilibrated Superdex 200 column (100 mM BTP pH 6.8, 5 mM DTT, 200 mM NaCl) and was collected in 0.5 ml fractions. The peak fractions were pooled and stored at –80 ºC until further use.
Materials and Methods Purification of APC4 GST-fusion of full-length scAPC4, spAPC4 and hAPC4 were purified from Hi5 insect cells and the APC4 fragments were purified either from E. coli strain BL21(DE3) or from Hi5 insect cells. All APC4 subunits and APC4 protein fragments were purified using the following protocol: Initially, cells were disrupted and the GST-fusion complex was isolated from the crude extract by GST-affinity chromatography. The eluted GST-fusion complex (50 mM Tris pH 8.0, 200 mM NaCl, 5 mM DTT and 20 mM Glutathione) was digested with 0.1-1 % thrombin over-night. Then, the protein complex was diluted to 50 mM NaCl, 50 mM Tris pH 8.0, 5 mM DTT and loaded onto a Source Q column equilibrated with the same buffer. The elution of the complex with a linear NaCl-gradient then followed. The peak fractions were pooled and concentrated by ultrafiltration before purification continued by size exclusion chromatography. A maximum of 2 ml of complex was loaded onto an equilibrated Superdex 200 column (50 mM Tris pH 8.0, 5 mM DTT, 200 mM NaCl) and was collected in 0.5 ml fractions. The peak fractions were pooled and stored at –80 ºC until further use. Purification of all other APC subunits and fragments The purification of all other APC subunits is identical to the described purification protocol for APC4. However, the buffer composition of the final size exclusion chromatography was different for hAPC7 (10 mM BTP 6.8, 200 mM NaCl, 6 mM DTT) and hAPC2554-822-hAPC11 (20 mM Tris pH 7.8, 200 mM NaCl, 5 mM DTT). Purification of Sic1-100 The His-Sic11-100 protein fragment was purified by Bing Hao from the E. coli strain BL21(DE3).
Materials and Methods 2.2.6 Isothermal titration calorimetry (ITC) ITC is a method that measures the thermodynamic properties of an intermolecular protein-protein interaction in the equilibrium by directly measuring the heat evolved during the association of a ligand with its binding partner (Figure 2.3). In a single experiment, the values of the association constant (Ka), the stoichiometry (n) and the enthalpy of binding (ΔHb) are determined. The free energy and entropy of binding can be determined from (Ka) (Pierce et al., 1999).
Syringe for loading sample and for titrating peptide
Reference cell heater ΔT
Sample cell feedback heater Reference cell
Figure 2.3. ITC instrument. It consists of two identical cells (reference cell containing water, sample cell the probe) composed of a highly efficient thermal conducting material surrounded by a jacket in which water of constant temperature is circulating. During an injection of a titrant into the sample cell the feedback heat detector senses and adjusts the temperature difference between the cells through a time dependent input of power (µcal/sec).
Prior to the binding studies of the Fbw7-Skp1 and scCdc4-Skp1 complex with their phosphorylated peptides Cyclin E and Sic1, the complexes and peptides were dialyzed in the same dialysis buffer and beaker (Material, 2.1.6). The final dialysis buffer was used for concentration adjustments. Before the ITC experiments, all samples were centrifuged and degassed for 5 min. ITC was performed on the MCS ITC unit from Microcal. The sample cell was filled with a 17 to 50 µM solution of the complex and was titrated to saturation with the 0.8 to 1 mM peptide solution contained in the syringe. Each ITC experiment was performed with 25 injections of 5 µl peptide solution at 2 min intervals and 25 ºC as described (Min et al., 2002). The data was analyzed using the program MicroCal Origin
Materials and Methods version 7.0. All dissociation constants (Kd) represent a mean of two or three independent determinations. 2.2.7 Protein crystallization In effort to find conditions that allow a protein of interest to crystallize, a trail and error approach is applied with the screening of many different crystallization conditions. If a potential promising protein crystallization condition is found, then a screening around that condition is performed with varying parameters such as protein concentration, buffer and salt condition, temperature and drop size. 188.8.131.52 Hanging drop vapor diffusion method In the hanging drop vapor diffusion method, the hanging drop (2-10 µl) was equilibrated against a much larger reservoir volume of precipitant solution (0.5 ml) in an enclosed chamber (Figure 2.4). The hanging drop was performed on a cover slide by mixing protein and reservoir solution in a 1 to 1 volume ratio. After the setup, the concentration of both solutions in the hanging drop will be half. During the equilibration process, water vapor diffuses through the vapor phase from the hanging drop, which contains a high vapor pressure, to the reservoir solution, which contains a low vapor pressure.
Reservoir solution Figure 2.4. Protein crystallization with the hanging drop vapor diffusion method.
The loss of water from the hanging drop decreases its volume and leads to an increase of all the concentrations of the nonvolatile components in the hanging drop, which drives the system towards supersaturation (metastable-, labile- and precipitation zone) (Figure 2.5). When supersaturation reaches the labile zone and micro protein crystals are formed, the macromolecule solution concentration decreases as the macromolecules become part of the growing crystal. Crystal growth stops when the hanging drop and reservoir solution have the 48
Materials and Methods
Precipitation Zone Labile Zone
Metastable Zone Saturation Undersaturation
Crystallizing agent concentration
Figure 2.5. Phase diagram of a protein solution under crystallization condition.
same vapor pressure (McPherson, 1998). For the protein crystallization experiments, screening kits from Hampton Research and home-made screening kits were used. For improving crystal quality Additive-Screen I-III and Detergent-Screen I-III were tested (Hampton Research). 184.108.40.206 Co-crystallization Co-crystallization is a method used to obtain crystals of protein-peptide complexes. For co-crystallization experiments of Fbw7-Skp1 with their specific phosphorylated Cyclin E or c-Myc peptides, the dimer was mixed with the peptides in a 1 to 2 molar ratio and used for setting up the co-crystallization experiments with the hanging drop vapor diffusion method. 2.2.8 X-ray crystallography A detailed description of the physical principles of X-ray crystallography is beyond the scope of this work. For an introduction on the theoretical background as well as on the theory of the applied methods, the following book is recommended (McPhearson, 1998). 220.127.116.11 Crystal-preparation and x-ray data collection To collect x-ray data in the -170 ºC nitrogen-stream, the protein crystals need to be cryoprotected to avoid damage to the crystal lattice due to the formation of ice. The transfer of protein crystals to a cryoprotectant was performed in three steps, where only the 49
Materials and Methods concentration of the cryoprotectant such as glycerol, PEG or LiSO4 is increased. The ability of a cryoprotectant to cryoprotect a protein crystal has to be determined for each crystallization condition separately using a trail and error approach. The cryoprotection of the Fbw7-Skp1 crystals in the presence and absence of peptides was performed as follows: A crystal was picked up with a loop from a hanging drop and placed for 30 sec in the cryoprotection solution I (Material, 2.1.6) before transferring the crystal to the cryoprotection solution II, which only increased the concentration of the cryoprotectant, LiSO4 to 1.8 M. After 30 sec, the crystal was transferred to a cryoprotection solution III, where only the concentration of the cryoprotectant LiSO4 was increased to 2.2 M. The cryoprotected crystal was then picked up by a loop, flash frozen and stored in liquid nitrogen until data collection was performed. X-ray data collection is the last experimental step in crystallography. It includes mounting the crystals on a goniometer head in an x-ray beam and measuring the intensity and pattern of the diffraction spots using an automated detector. The synchroton data of the Fbw7-Skp1 crystals in the presence and absence of peptides were collected at the National Synchroton Light Source (NSLS) at Brookhaven, NY, and at the Advanced Photon Source (APS) in Argonne, IL. 18.104.22.168 Structure determination To determine the protein structure from the reflection intensities of the collected data frames, the intensities need to be Fourier-transformed in an electron-density map. To achieve this, it is necessary to know the phase of each reflection. However, the phase information of each reflex is lost during data collection. Three major techniques have evolved to solve the phase problem: multiple isomorphous replacement (MIR), multiple anomalous dispersion (MAD) and molecular replacement (MR). In this thesis, MR solved the phase problem of Fbw7-Skp1. MR is the method of choice when the target protein has homology to a protein whose structure has already been determined. In the case of Fbw7 as the search model, the structures of scCdc4 (Orlicky et al., 2003), β-Trcp1 (Wu et al., 2003) and Skp1 (Schulman et al., 2000) were used. The patterson-search was performed with the program AmoRE, which
Materials and Methods searches for an orientation and position of the search model that can be fitted into the diffraction data. The patterson-solution was optimized by rigid body refinement. 22.214.171.124 Model building and refinement After the phase problem was solved, an electron-density map was calculated and visualized with the program O. Model building is an iterative process, comprising of the manual fitting of molecule parts in the electron-density map, as the map gradually becomes interpretable. After each fitting cycle, the new structure-factors are calculated and the new calculated phases are used with the original reflection intensities to calculate a better interpretable electron-density map. In principle, refinement involves the adjusting of the positions and temperature factors of all the atoms in the model, as well as the building of water molecules, ions and other ligands. The quality of the refinement is evaluated by the Rfactor, which is a value of agreement between the observed structure-factor amplitudes (Fobs) and those calculated from the model (Fcalc). To prevent the structure from being over-refined, an unbiased Rfree-factor was calculated during the refinement process using about 5 % of the data randomly omitted from the refinement. The electron-density maps of the four Fbw7Skp1 structures in the presence and absence of peptide were refined with the CNS program (Bruenger et al., 1998) until their R-factor yielded around 25 %.