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

BMC Biotechnology

Research Article

Christine Gaucher¹  Alexandre Fontayne¹  Sylvie Jorieux¹  Béatrice Teylaert²  Edwige Meurice²  Anne Harduin-Lepers³  Marie Bobowski³  Philippe Delannoy³ 

[1] Laboratoire Français du Fractionnement et des Biotechnologies, Lille, France;Laboratoire Français du Fractionnement et des Biotechnologies, Lille, France;CNRS, UMR 8576, F-59650, Villeneuve d'Ascq, France;Univ. Lille Nord de France, F-59000, Lille, France;USTL, UGSF, F-59650, Villeneuve d'Ascq, France;CNRS, UMR 8576, F-59650, Villeneuve d'Ascq, France;

关键词: Electrophoretic Mobility Shift Assay;    Fucose;    Transcriptional Start Site;    Minimal Promoter;    KLF15 Protein;   

DOI  :  10.1186/1472-6750-11-1

 received in 2010-02-24, accepted in 2011-01-05,  发布年份 2011

来源: Springer

【 摘 要 】

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.

【 授权许可】

Unknown   
© Teylaert et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.