Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

Abstract

BackgroundThe rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut8 gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat fut8 gene.ResultsThe cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat fut8 gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of fut8 in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 fut8 gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.ConclusionAltogether, these data contribute to a better knowledge of fut8 expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.

Recent approaches in designing bioadhesive materials inspired by mussel adhesive protein

“…The polymeric form of dopamine or polydopamine has the capability of attaching to various types of surfaces ranging from noble metals to inert polymers and ceramics80 with different geometry and sizes 81, 82, 83. The amine group found in the polydopamine film has been found to contribute to the cohesive force formed between polydopamine films 84.…”

“…Additionally, polydopamine coating remains reactive and can be further modified with functional groups (i.e., —NH 2 , —SH, etc.) or metal ions 80, 82, 85. Antifouling polymers such as PEG and alginate can graft on polydopamine‐coated surfaces for designing antifouling surfaces that prevent non‐specific attachment of cells and bacteria 80, 86…”

Selectin Ligands Sialyl-Lewis a and Sialyl-Lewis x in Gastrointestinal Cancers

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An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

“…Endogenous proteases are involved in many biological processes, and hundreds of genes coding for these enzymes have been identified in plant genomes [12,13]. Protease activities in plant protein biofactories may lead to partial or complete hydrolysis of recombinant antibody chains in leaf cells or in the leaf apoplast [14, 15], typically leading to the concomitant isolation of full-size antibodies and stable fragments from crude protein extracts following purification [16]. Despite numerous reports on antibody degradation (e.g.…”