Ni nta column

The ORF of NlSEF1 was amplified by PCR using the primers listed in Supplemental Table S1. The PCR product was ligated into the pMD19-T vector, sequenced, and then cloned into vector pET-30a. The recombinant vector NlSEF1:pET-30a and empty vector pET-30a (used as a control) were transformed into E. coli BL21 (DE3) strain. Expression was induced by adding IPTG (1 mM final concentration). The products from recombinant and empty vector were purified by using Ni-NTA columns (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. The purified products were concentrated with a YM-10 Centricon membrane (Millipore, Billerica, MA, USA) to remove imidazole. The final purified concentrated products from E. coli cells with the empty vector and recombinant vector were mixed with 2× SDS loading buffer, respectively, separated by SDS/PAGE in a 12.5% acrylamide gel (ISC Bioexpress, Kaysville, UT, USA), and stained with 0.025% Coomassie blue R-250 in water. The predicted mass of the mature recombinant protein NlSEF1 including 6 N-terminal His-tags is 31.63 kDa.

All AK2 variants as well as AK1 were heterologously expressed with a C‐terminal 6x‐His tag in Escherichia coli Rosetta 2 cells. The proteins were purified by Immobilized Metal Affinity Chromatography using Ni‐NTA columns (Qiagen), followed by size exclusion chromatography with a 16/600 Superdex 75 pg column (GE). Protein concentration was determined by measuring absorbance at 280 nm and using a calculated extinction coefficient (ProtParam).

The L28R mutation
in folA gene was constructed by using the Quick-Change Site-Directed
Mutagenesis kit (Stratagene). Six×HisTag was added at the C-termini
of the protein sequence for the WT and L28R constructs. The constructs
were cloned into expression plasmids pET24a-KanR. BL21 E.
coli cells were transformed with pET24a-folA–6×HisTag
and were grown overnight in selective media (LB + Kan) and then diluted
100 times into Terrific Broth (TB) media for further growth at 37
°C. Protein overexpression was induced when OD600 reached 0.6–0.8,
using 250 mM isopropyl β-d-1-thiogalactopyranoside
(IPTG) per 1 L of the medium, and the temperature was decreased to
18 °C for overnight growth, with 230 rpm shaking. Recombinant
protein was purified using Ni-NTA columns (Qiagen), dialyzed overnight
against 50 mM Tris-HCl, 300 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine
(TCEP), pH 8, and further purified using size-exclusion chromatography.

Gal4-VP16, GTFs, wild-type, and med6 ts mutant Pol II holoenzymes were prepared using several chromatographic steps as described previously [20 (link)]. Recombinant Med6p and mutant derivatives were expressed in E. coli strain BL21(DE) (Novagen) and purified through Ni⁺-NTA columns (Qiagen) under denaturing conditions. Purified proteins were renatured at 4°C through stepwise dialysis according to the protocol described by Thompson et al. [27 (link)] and stored at −80°C before use.

The monoclonal antibody D2B recognizing an external epitope of the PSMA antigen was produced by hybridoma technology and purified on protein G columns.21 (link) The scFv format was synthesized by DNA technology from the purified mRNA of the D2B hybridoma as previously described21 (link). The cDNA of scFv D2B was cloned in PHEN2 plasmid. The protein was produced in E.Coli HB2151 bacterial strain and purified on NiNTA columns (Qiagen, Courtaboeuf, France). The purification was checked by SDS-PAGE, binding capability and specificity were assessed by flow cytometry.
The control scFvD2BGF7.7, that is not able to bind PSMA antigen, was obtained replacing the sequence of the D2B variable light chain with the sequence of the surrogate light chain VpreB28 .
The labeling reaction was made using Xenolight CF770 antibody labeling kit (Perkin Elmer, Waltham, MA, USA) on batches of 500 μg of each scFv. Labeled scFv were stored in the dark at 4 °C. Quantification of the labeling was made on 2 μg of each antibody fragments by Fluorescence Molecular Tomography (FMT 4000, PerkinElmer, Waltham, MA, USA) using system software (TrueQuant) calibrated with the fluorophore Xenolight770 CF (X770). Fluorescence labeling of scFvD2 and scfvD2BGF7.7 were 0.415 pmol/μg and 0.285 pmol/μg respectively. Fluorescence level of scfvD2BGF7.7 was thus corrected by a 1.45 factor for comparison with scFv.

Ni-NTA columns (Qiagen) were loaded with HtrA1 conditioned medium containing 20 mM imidazole and 0.05% NP-40, washed three times (20 mM imidazole, 0.05% NP-40/PBS) and bound fractions were eluted (200 mM imidazole, 0.05% NP-40/PBS). Eluted fractions were subjected to centrifugal filter devices (Amicon) four times to remove excess imidazole and to concentrate the proteins (2000 g, 30 min, 4°C). Purified protein was reconstituted in PBS.
Samples were resolved on SDS-PAGE (4–20% gradient, Bio-rad) in the presence or absence of reducing agent (355 mM β-mercaptoethanol), transferred onto PVDF membrane (Bio-rad), blocked (5% non-fat dry milk/140 mM NaCl/10 mM Tris pH 8 (TBS-T)) and incubated in primary antibody (1∶1000 in 0.1% sodium azide/3% BSA/TBS-T) overnight (4°C). After washing, membranes were incubated in HRP-conjugated secondary antibodies (1∶5000 in 5% non-fat dry milk/TBS-T). Signal was detected using Western Lightning Plus (Perkin Elmer).