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  • A schematic map of the pSFR1 Phagemid (Antherix, Pushchino, Russia) used for Phage display of L35Ae 10X.
    2017
    Co-Authors: Anna V. Lomonosova, Andrei B. Ulitin, Alexei S. Kazakov, Tajib A. Mirzabekov, Eugene A. Permyakov, Sergei E. Permyakov
    Abstract:

    The Phagemid is based on pSMART LC Amp vector (Lucigen®). Ampicillin resistance of a bacterial host is ensured by the β-lactamase gene, ‘bla-gene’. The gene of the 10X mutant of L35Ae from P. horikoshii was codon optimized for expression in E. coli [30], subjected to randomization of the regions coding the CDR-like loops 1–3 (Fig 1A, S1 Fig) and cloned between the NcoI and NotI restriction sites. The L35Ae gene is followed by a myc tag (‘Myc-tag’), a 6×His tag (‘His-tag’) and the gene of the attachment protein G3P from Enterobacteria Phage M13 (‘G3P’). The translated chimera of L35Ae 10X-myc (Fig 1B) and G3P is secreted due to the presence of a N-terminal pelB leader sequence (‘pLB-leader’).