Synthesis of the l- and d-SH2 domain of the leukaemia oncogene Bcr-Abl

The d- and l-versions of the Bcr-Abl SH2 domain (12.7 kDa) were synthesized. Key optimizations included pseudoproline incorporation, N-terminal hydrophilic tail addition and mild N-acetoxy succinimide acetylation. Their folding and activity are as for the recombinant protein. Our results will enable engineering of mirror-image monobody antagonists of the central oncoprotein Bcr-Abl.


Final cleavage for both protocols (A and B):
The resin was treated with the cleavage cocktail (92.5% TFA, 2.5% H2O, 2.5% TIS, 2.5% EDT up to 2 mL of cocktail for ~30 mg of resin). The resulting suspension was shaken for 2.5 h and the resin was filtered off. The resulting filtrated solution was concentrated with nitrogen gas until less than 1 mL of cleavage cocktail was left, and added to ice-cold Et2O (10 mL of Et2O for each 1 mL of TFA). After 10 min, the mixture was centrifuged (8000 rpm, 10 min, 4 °C), decantated, and the solid was again suspended in 10 mL of fresh ice-cold Et2O, sonicated and centrifuged again. This procedure was repeated once and the solid residue was dried. Then, the Nterminal Bcr-Abl SH2 peptide was dissolved in H2O/MeCN (50:50,v/v) with addition of 0.1% TFA, whereas the C-terminal Bcr-Abl SH2 peptide was dissolved in H2O/MeCN (85:15,v/v) with addition of 0.1% TFA. The peptides were purified by semipreparative reverse-phase (RP)-HPLC.

Synthesis of the Bcr-Abl SH2 domain and Fmoc-Gly-D-Ser(psiMe,Mepro)-OH
The two peptide fragments for the synthesis of the Bcr-Abl SH2 domain were designed as depicted in Fig. S1. An overview of the synthesized sequences is listed in Tab. S1. Tab. S3: Sequences of the synthesized peptide fragments, which were ligated and subsequently desulfurized to yield biotinylated Bcr-Abl(138-241). The underlined residues indicate the incorporation of the Fmoc-Gly-Ser(psiMe,Mepro)-OH pseudoproline.

Crude N-terminal Bcr-Abl SH2 peptide (L-1c)
Peptide L-1c was synthesized according to SPPS Protocol A with the usage of the L-pseudoproline Fmoc-Gly-L-Ser(psiMe,Mepro)-OH and the Capping Protocol A1. The usage of this capping protocol led to acetylation of the MeDbz linker as seen in the MS-ESI + spectrum.  Fig. S4: A) Structure of peptide L-1c; B) Analytical RP-HPLC chromatogram of peptide crude (HPLC method A, Tab. S1); C) MS-ESI + spectrum of L-1c (HPLC Method A, Tab. S1), peaks marked with * correspond to the peptide with acetylated MeDbz linker (733. 5

13.00
A B C S12

Crude N-terminal Bcr-Abl SH2 peptide (L-1d)
Peptide L-1d was synthesized according to SPPS Protocol A with the usage of the L-pseudoproline Fmoc-Gly-L-Ser(psiMe,Mepro)-OH and the capping protocol A2. The usage of this capping protocol suppressed the acetylation of the MeDbz linker as observable in the MS-ESI + spectrum.

Activated N-terminal Bcr-Abl SH2 peptide (L-2)
Peptide L-2 was synthesized according to SPPS Protocol A with the usage of the L-pseudoproline Fmoc-Gly-L-Ser(psiMe,Mepro)-OH and the Capping Protocol A2. The MeDbz linker activation was performed on resin, which was treated with 4-nitrophenyl chloroformate (2 eq., 20 mM) dissolved in 1500 µL 1,2-dichloroethane (DCE) for 45 min. The reaction was repeated twice. After washing the resin (6 × DCE, 6 × anhydrous DMF), 1500 µL of DIPEA (50 eq., 0.5 M) in anhydrous DMF were added. The reaction proceeded for 15 min, repeated for 30 min and then conducted a third time for 15 min. After cleavage of the peptide from the resin, the precipitated peptide was dissolved in H2O/MeCN (50:50, v/v) with addition of 0.1% TFA and purified via semipreparative RP-HPLC using a linear gradient of 5-40% MeCN with 0.1% TFA over 30 min. After lyophilization, 13. A B C S14

Mirror-image of activated N-terminal Bcr-Abl SH2 peptide (D-2)
Peptide D-2 was synthesized according to SPPS Protocol A with the usage of the D-pseudoproline Fmoc-Gly-D-Ser(psiMe,Mepro)-OH and the Capping Protocol A2. The MeDbz linker activation was performed on resin, which was treated with 4-nitrophenyl chloroformate (2 eq., 20 mM) dissolved in 1500 µL of DCE for 45 min. The reaction was repeated twice. After washing the resin (6 × DCE, 6 × anhydrous DMF), 1500 µL of DIPEA (50 eq., 0.5 M) in anhydrous DMF were added. The reaction proceeded for 15 min, was repeated for 30 min and then conducted a third time for 15 min. After cleavage of the peptide from the resin, the precipitated peptide was dissolved in H2O/MeCN (50:

8.93
A B C S16

Mirror-image of C-terminal Bcr-Abl SH2 peptide (D-3)
Peptide    Peptides L-2 (1 eq.) and L-3 (1.2 eq.) were dissolved in 710 µL of freshly prepared buffer containing 6 M guanidine-HCl, 200 mM Na2HPO4, 100 mM MPAA and 20 mM TCEP-HCl. The pH was adjusted to 7.0 using 10 M NaOH and the ligation reaction was carried out at room temperature (RT) for 24 h. Then, a spatula tip of TCEP-HCl powder was added to the mixture. After 30 min, the crude product was purified via semipreparative RP-HPLC using a linear gradient of 5-35% MeCN containing 0.1% TFA over 30 min. After lyophilization, 11.2 mg (0.72 µmol, 51%) of the desired peptide were obtained as TFA salt.       Peptide D-4 (1.5 mM) was dissolved in 655 µL of freshly prepared reaction buffer containing 6 M guanidine-HCl, 300 mM TCEP-HCl, 60 mM Na2HPO4, 8 mM red. L-glutathione and 40 mM VA-044. In detail, 500 mM TCEP-HCl was added to 393 µL of a 6 M guanidine-HCl/ 200 mM Na2HPO4 (pH 8.5) stock solution. This stock solution was mixed with 131 µL of a 6 M guanidine-HCl/ 40 mM L-glutathione stock solution and 131 µL of a 6 M guanidine-HCl/ 200 mM VA-044 stock solution to obtain 655 µL of the reaction buffer. The stock solutions can be stored at -80 °C. The pH was adjusted to 7.0 using 10 M NaOH and the desulfurization reaction was carried out at 37 °C for 16 h. 2 The crude product was purified via semipreparative RP-HPLC using a linear gradient of 5-40% MeCN containing 0.1% TFA over 15 min. After lyophilization, 10.8 mg (0.70 µmol, 71%) of the desired peptide were obtained as TFA salt.

Expression and purification of recombinant Bcr-Abl SH2
Recombinant Bcr-Abl SH2 domain was expressed with an N-terminal tag containing His6, GST and a tobacco etch virus (TEV) protease recognition motif using a modified pET expression vector. BL21(DE3) cells containing the plasmid were grown to OD600 = 0.8 at 37 °C in LB medium containing 50 µg/mL kanamycin. Afterwards, the cells were shifted to 18 °C and expression was induced via addition of IPTG at a final concentration of 0.5 mM. After 16 h, the cells were harvested by centrifugation at 6000 rpm and 4 °C for 15 min (Beckman Coulter JA-10 rotor, Beckman Coulter J2-MC centrifuge). The cells were resuspended in 30 mL of Ni-NTA buffer A (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 mM imidazole) per 1 L of bacterial culture containing EDTA-free protease inhibitor cocktail (Roche) and a spatula tip of DNase I (Roche) and lysed with an Avestin Emulsiflex C3 homogenizer (4 rounds, 10000 -15000 psi). The suspension was centrifuged for 1 h at 4 °C and 15000 rpm (Beckman Coulter JA-17 rotor, Beckman Coulter J2-MC centrifuge) and the supernatant was filtered through a 5 µm filter (25 mm, Low Protein Binding Durapore Membrane, Merck Millipore).
Protein purification was carried out using the N-terminal His6 tag by nickel-affinity chromatography (column: 5 mL His-Trap FF crude, GE Healthcare) on an Äkta Avant system (GE Healthcare) according to the manufacturer's instructions. Bound proteins were eluted with a gradient from 10 to 500 mM imidazole over 10 column volumes. The His6-GST tag was cleaved by addition of TEV protease in a 1:40 dilution to the collected protein fractions and incubation at 4 °C over night. The protein solution was further purified by size exclusion chromatography with buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM TCEP-HCl to remove the His6-GST tag and TEV protease on the Äkta Avant system (column: HiLoad 16/600 Superdex 75 pg). The fractions were checked via SDS-PAGE and pooled. The protein was usually concentrated up to 610 µM (7.1 mg/mL), aliquoted, flash frozen in liquid nitrogen and stored at -80 °C. The obtained protein was verified by mass spectrometry.

Circular dichroism (CD) measurements
The recombinant and synthetic proteins originally in HEPES buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM TCEP-HCl) were dialyzed three times against a 200-fold volume of phosphate-buffered saline (PBS) at pH 7.4, twice for 2 h and then for 16 h at 4 °C. Dialysis was carried out in Slide-A-Lyzer G2 dialysis cassettes (cutoff 3.5 kDa, Thermo Scientific). CD spectra were recorded in a quartz cuvette (path length: 0.1 cm, Hellma Analytics) containing 20 µg of sample in 300 µL PBS buffer (pH 7.4) on a JASCO J-815 circular dichroism spectrometer at 20 °C and a data interval of 0.1 nm. Samples were also measured in PBS buffer (pH 7.4) containing 6 M guanidine-HCl to obtain CD spectra of the unfolded proteins. The data was plotted using the software GraphPad Prism 8.
Tab. S6: Secondary structure content of recombinantly expressed and synthetic Bcr-Abl SH2 domains in L-and D-configuration. The percentages were calculated with BeStSel based on the obtained CD spectra from two independent measurements. 4 Secondary structure element Abl SH2  D-5 Nano differential scanning fluorimetry (nanoDSF) measurements The thermal denaturation curves were determined by measurements of intrinsic tryptophan fluorescence. This analysis was performed using label-free, native differential scanning fluorimetry on a Prometheus NT.48 instrument (NanoTemper). Approximately 10 µL of the protein samples in PBS (pH 7.4) were loaded in Prometheus NT.48 capillaries. The tryptophan residues of the proteins were excited at 280 nm and the fluorescence intensity was recorded at 330 and 350 nm. Excitation power was set to 80 % and the temperature of the measurement compartment increased from 21 to 95 °C at a rate of 1 °C/min. Melting temperatures (Tm) were determined by the Prometheus software through calculation of the fluorescence ratio at 330 and 350 nm and of the first derivative. The data was plotted using the software GraphPad Prism 8.