Fig. 1
From: Robust expression of LINE-1 retrotransposon encoded proteins in oral squamous cell carcinoma
Generation of the antigen for the production of Human L1 ORF2 specific antibody: a Schematic diagram of full length active human L1 retrotransposon with two encoded proteins (L1ORF1p and L1ORF2p). L1ORF2p (1275 amino acids in length with predicted MW 150 kDa) has three partially characterized domains: endonuclease (EN) (AA:1–239), reverse transcriptase (RT) (AA: 453–883) and a cysteine-histidine-rich domain (CCHC) (AA: 1096–1275). The EcoRI-HindIII restriction fragment (AA: 479–558 fragment name hL1RTEH) from the RT region was used as an antigen to make antibody against hL1ORF2p (shaded by yellow). b Alignment of the selected eighty amino acids stretches of the RT domain (hL1RTEH) among human, mouse and rat L1. The selected RT stretch showed 76.25 and 73.75% identity at the protein level with the same stretch present in mice and rat L1ORF2, respectively. c Predicted structure of hL1RTEH fragment and complete human RT domain (generated using PyMOL) [38]. d Sub-cloning scheme of human hL1RTEH fragment in pET-28a bacterial expression vector. Bacterial expressed hL1RTEH fragment encodes 130 amino acid polypeptides (from N terminal to C terminal AA: 1–42 vector, 43–124 RT fragment, and 125–130 vector) with predicted MW around 14 kDa. e Whole-cell lysate SDS-PAGE of E.coli expressed pET-hL1RTEH. Induced protein with a molecular weight around 14 kDa is shown by the arrow. f Purification of hL1RTEH from inclusion bodies by dissolving the pellet fraction in 8 M urea buffer (details in materials and methods). The purified protein in elution 1, 2 and 3 is shown by an arrow. g Dialysed and concentrated hL1RTEH fragment protein (antigen) injected to mice for antibody generation. h Silver staining of purified hL1RTEH (antigen) show the purity of antigen used to generate the antibody. i Western blotting of hL1RTEH using an anti-His antibody. MW: Molecular Weight, FT: Flow-through