Protein biochemistry methods

3.5.1. Purification of poly-Histidine tagged proteins from E. coli periplasm

A bacterial cell pellet from 3 l E. coli culture (section 3.3.2.) expressing scFvs was resuspended in 100 ml periplasm lysis buffer (30 mM Tris pH 8, 1 mM EDTA, 20% (w/v) sucrose) and incubated on ice for 10 min. Afterwards, the suspension was centrifuged at 4,500 rpm for 20 min at 4°C, the supernatant was separated and stored on ice. The pellet was resuspended in 50 ml of 5 mM MgSO4 and after 10 min incubation on ice it was again centrifuged at 4,500 rpm for 20 min at 4°C. Both supernatants were combined and centrifuged at 15,000 g for 20 min at 4°C using an SS-34 rotor (Thermo Scientific) to remove remaining cell debris. After overnight dialysis against 100 volumes

METHODS

46

of dialysis buffer (20 mM Tris pH 7, 300 mM NaCl), the His6 tagged scFvs were enriched by nickel affinity chromatography. 1 ml of Ni-NTA agarose beads (Qiagen) were added to the supernatant and imidazole was adjusted to a final concentration of 10 mM to hamper unspecific binding. After rotation at 4°C over night, the beads were collected by centrifugation at 3,500 rpm at 4°C for 10 min and applied to a Spin® chromatography column (Bio-Rad). Bead-bound impurities were removed by washing with one column volume of wash buffer (20 mM His (pH 6.5), 300 mM NaCl, 10 mM imidazole) and the protein was eluted in 5 elution steps of one column volume at a time using elution buffer (20 mM His (pH 6.5), 300 mM NaCl, 200 mM imidazole). All wash and elution fractions were collected and analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE, section 3.5.4.). Protein bands were visualized by Coomassie Brilliant Blue staining, which enabled the determination of the fractions containing the desired protein. These were pooled and further purified by analytical size exclusion chromatography (SEC) using a Superdex 75 10/300 column (GE Healthcare) in 20 mM His (pH 6.5), 300 mM NaCl (i.e. SEC buffer). Afterwards, the chromatography fractions were evaluated by SDS-PAGE and pure monomeric fractions were pooled and concentrated using an Amicon spin concentrator (Millipore, cutoff 10 kDa). Protein concentration was measured as absorption at 280 nm by Nanodrop ND-1000 (Peqlab Biotechnologies), proteins were shock-frozen in liquid nitrogen and stored at -80°C.

3.5.2. Purification of poly-Histidine tagged proteins from cell culture supernatant

All proteins expressed in HEK293-based expression systems were designed to carry an N- or C- terminal His6 tag and were secreted into the medium. Cell culture supernatants were harvested 5-7 days after transfection (section 3.4.3.) by centrifugation at 1,500 rpm. Afterwards, remaining cell fragments were removed in a second centrifugation step at 15,000 g using a SS-34 rotor (Thermo Scientific). Ni-NTA agarose beads (Qiagen) and 10 mM imidazole were added and the supernatant was incubated at 4°C for at least 2 h meanwhile rotating. Afterwards, nickel affinity chromatography was performed as described in 3.5.1. and protein fractions were analyzed by SDS- PAGE. Fractions containing the protein of interest in high purity were pooled and dialyzed over night against SEC buffer. The following day, the protein was concentrated and further purified using a Superdex 200 10/300 GL column (GE Healthcare), which was run in SEC buffer. The peak fractions were again evaluated by SDS-PAGE and the fractions containing the correct monomeric protein at high purity were pooled, concentrated, shock-frozen in liquid nitrogen and stored at -80°C until further use. Stability of the proteins after freezing was confirmed by analytical SEC

METHODS

47

using a Superdex 200 5/150 GL column (GE Healthcare) in SEC buffer. Prior to functional assays, all proteins were thawed on ice and centrifuged at 15,000 g for 10 min at 4°C.

3.5.3. Protein purification for analysis in murine NSG xenograft model

Proteins designated for injection into a murine non-obese diabetic (NOD) severe combined immunodeficiency (scid) (NSG) xenograft model were expressed and purified under endotoxin- free conditions. For this purpose, the proteins were expressed in HEK293-based suspension cells as described in 3.4.3. and purified according to 3.5.2. However, special attention was paid to obtain low endotoxin levels in the sample. Thus fresh, endotoxin-free Ni-NTA agarose beads, chromatography columns and plasticware were used. SEC columns as well as Äkta systems including tubings and adaptors were preincubated with 0.5 M NaOH for at least 4 h and rinsed with 3 column volumes of ddH2O. After nickel affinity chromatography, the protein was dialyzed against 1x DPBS, which was also used as running buffer for gel filtration chromatography. Before freezing in liquid nitrogen, the protein was sterile filtered using a 0.45 µM filter (Ultrafree-MC HV Centrifugal Filter units, Merck Millipore) and aliquoted under a laminar airflow cabinet. Low endotoxin levels were confirmed with the Pierce™ LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific) which was used according to manufacturer’s instructions.

3.5.4. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE)

The purity of protein samples was evaluated by denaturing polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) using precast 4-20% Bis-Tris gels of the RunBlue® _{SDS-PAGE Gel System (Expedeon).}247 _{Before loading the gel,} protein samples were mixed with Laemmli buffer and denatured at 95°C for 5 min. Protein separation was achieved by running the gels at 120 V in 1x RunBlue SDS Run TEO-Tricine buffer. Afterwards, proteins were either stained 30 min using Coomassie Brilliant Blue staining solution followed by destaining in water, or the gel was used for western blot analysis. PageRuler™ Unstained Protein Ladder or PageRuler™ Prestained Protein Ladder (both Thermo Fisher Scientific) served as size standards.

3.5.5. Western blot analysis

Protein samples of interest were separated according to their size by SDS-PAGE (section 3.5.4.) and transferred to a PVDF membrane by wet transfer using a Mini-Trans Blot® electrophoretic

METHODS

48

transfer cell (Bio-Rad). The PVDF membrane was activated in >99% EtOH, and all other components were presoaked in 1x transfer buffer. The gel and the PVDF membrane were sandwiched between a foam pad and two Whatman papers on each side, clamped tightly and transferred to the blotting chamber, which was filled with 1x transfer buffer and run at 100 V for 50 min. Afterwards, the membrane was washed in PBS-T for 5 min and subsequently incubated with a 1:10,000 dilution of His-HRP antibody (Miltenyi) in 3% milk powder/ PBS-T for 1 h at room temperature while agitating. The membrane was washed with 1x PBS-T three times for 20 min. For protein detection, it was incubated in 10 ml of 1x ECL solution for 1 min and subsequently placed in an exposure cassette. Either a light sensitive Hyperfilm™ ECL™ (GE Healthcare) was exposed for varying time intervals followed by film development in a Kodak X- Omat M35 developing machine, or the chemiluminescence was directly measured in a digital film developer (Amersham™ Imager 600, GE Healthcare). The buffer compositions are listed in Table 9.

3.5.6. Fluorescence-based thermal shift (ThermoFluor) assay

The thermal stability of proteins was determined in a fluorescence-based thermal shift (ThermoFluor) assay.248 Briefly, the proteins were diluted to a concentration of 100 ng/µl and mixed with a 1:500 dilution of CYPRO® Orange (Thermo Fisher Scientific). Protein unfolding over a temperature gradient from 10°C to 95°C was recorded using a CFX96 Touch Real-Time PCR Detection System (Bio- Rad, Munich, Germany) with a stepwise temperature increase of 0.5°C/ 10 sec and one scan after each cycle using FAM and SYBR Green I filter pairs.