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Promoter Choice Impacts the Efficiency of Plant Glyco‐Engineering
Author(s) -
Kallolimath Somanath,
Gruber Clemens,
Steinkellner Herta,
Castilho Alexandra
Publication year - 2018
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201700380
Subject(s) - glycosylation , nicotiana benthamiana , promoter , glycosyltransferase , golgi apparatus , glycoprotein , galactosyltransferase , biology , glycan , recombinant dna , n linked glycosylation , gene , microbiology and biotechnology , chemistry , gene expression , biochemistry , enzyme , cell
Glyco‐modulation of therapeutic proteins produced in plants has shown great success. Plant‐based expression platforms for tailored human‐like N‐glycosylation are based on the overexpression of foreign genes. However, drawbacks such as protein miss targeting, interference with endogenous glycosyltransferases, or with plant development hamper the widespread use of the technology. Here a technique that facilitates the generation of recombinant proteins with targeted N‐glycosylation at high homogeneity is described. It is focused on the synthesis of human‐type β1,4‐galactosylation by the overexpression of the human β1,4‐galactosyltransferase (GalT) in Nicotiana benthamiana . A GalT construct that targets the enzyme to the required late Golgi compartment ( ST GalT) is transiently co‐expressed with two pharmaceutically relevant glycoproteins. The impact of eight promoters driving the expression of ST GalT is evaluated by mass spectrometry (MS) ‐based analyses. It is shown that five promoters (amongst them high expressors) induce aberrant non‐human glycosylation. In contrast, three promoters, considered as moderately active, regulate gene expression to levels leading to an improved efficiency of di‐galactosylation (and subsequent sialylation) on the reporter proteins. The results point to the importance of promoter choice for optimizing glycan engineering processes.