Isopropyl β d 1 thiogalactopyranoside (iptg)

All chemicals and amino acids were purchased from Sigma-Aldrich (Merck Singapore) and Chemimpex (Wood Dale, IL, USA) unless otherwise indicated. All solvents and reagents were used as received without further purification. VyPAL2 was prepared in-house, as previously reported.
Materials used in the study include phosphate saline buffer (Gibco^TM, ThermoFisher Scientific, Singapore), nitrilotriacetic acid-nickel (Ni-NTA, Biobasic, Singapore), Dulbecco’s Modified Eagle Medium (DMEM, Gibco), fetal bovine serum (FBS, Gibco), ethylenediaminetetraacetic acid (EDTA), 4-Methylbenzhydrylamine resin (MBHA resin, Chemimpex), Fluorenylmethyloxycarbonyl-amino acids (Fmoc-amino acids), tert-butyloxycarbonyl-amino acids (Boc-amino acids), trifluoroacetic acid (TFA), triisopropylsilane (TIS), dimethylformamide (DMF), dichloromethane (DCM), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, Chemimpex), N,N-Diisopropyl- ethylamine (DIPEA, Chemimpex), isopropyl β-D-1-thiogalactopyranoside (IPTG, Biobasic, Singapore), fluorescein (Sigma), LB broth (Biobasic, Singapore).

All chemicals were of analytical grade. Ampicillin, isopropyl β-D-1-thiogalactopyranoside (IPTG) and imidazole were obtained from Bio Basic Inc. (Markhem, ON, Canada). Sarcosine, Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) and HRP were ordered from Sigma-Aldrich (Saint Louis, MO, ^USA). H₂O₂^and phenol were from MERCK (Darmstadt, Germany). Milli-Q system ultra-pure water was used in all experiments. Pfu DNA polymerase was obtained from Promega (Madison, WI, USA). Restriction endonucleases and T4 ligase were purchased from New England Biolabs, Inc. (Ipswich, MA, USA).

In this study, due to the high cost and less amount of commercial antigen, it was decided to produce recombinant antigen to advance the bio-panning steps by using enough PTPRN antigen. For this purpose, the protein sequence of the extracellular domain region of the PTPRN antigen was extracted from the NCBI database. The antigen nucleotide sequence was codon optimized and synthesized. The nucleotide sequence was cloned into the pET15b expression vector (Novagen, Germany). The PTPRN-pET15b construct was extracted and transformed in BL21(DE3) E. coli strain (Pasteur Institute, Iran) after multiplication and transformation in Top10 bacteria. Three colonies of the transformed BL21 bacteria were selected to adjust the expression conditions. For this purpose, each clone was grown in 5 ml of Terrific Broth (TB) Medium. After the Optical density (OD) of 0.6 was achieved, Isopropyl β- d-1-thiogalactopyranoside (IPTG) (BioBasic, Canada) in 0.1 mM concentration was employed to induce the expression of the target protein. Samples were taken before, 2 h after, and overnight after induction and consequently analyzed using 12% SDS-PAGE. Finally, the PTPRN antigen was purified with Ni–NTA agarose (QIAGEN, Germany).

Bacterial expression vector encoding PfUCHL3 was a generous gift from Dr. Hidde Ploegh, Boston Children’s Hospital, MA and Katerina Artavanis-Tsakonas, Department of Pathology, University of Cambridge, Cambridge, UK. The gene encoding HsUCHL3 was amplified from HEK-293T cDNA using 5′-GGT CAT ATG GAG GGT CAA CGC TGG CTG-3′ and 5′-CCC AAG CTT CTA TGC TGC AGA AAG AGC-3′ as forward and reverse primer, respectively. The amplified gene was cloned between NdeI and HindIII of pET28a(+) as a N-terminus 6 X His tag. The gene sequences were verified by DNA sequencing. Anti-ubiquitin and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).  Isopropyl β-D-1-thiogalactopyranoside (IPTG) was obtained from Bio Basic Inc. (Markham, Canada). SYBR Green I, penicillin and nickel nitrilotriacetic acid (Ni–NTA) beads were procured from Thermo Scientific (Rockford IL, USA). Dithiothreitol (DTT) and hypoxanthine were obtained from Sigma (St. Louis, MO, USA). Powdered RPMI-1640 and Albumax II were obtained from GIBCO (ThermoFisher Scientific, Waltham, MA, USA). DMEM and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). All other reagents were of analytical grade.