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Updated: Aug 14, 2026
High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension
Published on: December 28, 2015
High level expression of monomeric and dimeric human alpha1,3-fucosyltransferase V
Jan Münster1, Patrick Ziegelmüller, Edzard Spillner
1Institut für Biochemie und Lebensmittelchemie, Abteilung für Biochemie und Molekularbiologie, Universität Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany.
Abstract:
alpha3/4-Fucosyltransferases play a crucial role in inflammatory processes and tumor metastasis. While several human fucosyltransferases (FucTs) with different acceptor substrate specificities have been identified, the design of specific inhibitors for therapeutic approaches is hampered by the lack of structural information. In this study, we evaluated the expression of different constructs of human fucosyltransferase V to generate the large amounts required for structural studies. The truncated constructs lacking the transmembrane region and the cytosolic N-terminus, were expressed in baculovirus-infected Trichoplusia ni (Tn) insect cells and in two non-lytic expression systems, stably transfected human HEK 293 and T. ni cells. Since secretion of some glycosyltransferases is controlled by formation of dimeric molecules via disulfide bonds, one of the fucosyltransferase V constructs contained the N-terminal cysteine residue 64 for dimerization, whereas this residue was replaced in the other construct by serine. In both human and insect cells dimerization did not prove to be essential for efficient expression and secretion. On the basis of enzymatic activity, the yield of secreted fucosyltransferase V was approximately 10-fold higher in stably transfected insect cells than in HEK 293 cells. In particular the monomeric form of the enzyme provides a valuable tool for structural analyses to elucidate the fine specifity of fucosyltransferase V-mediated fucosylation of Lewis type glycans.
Insights
Human fucosyltransferase V was successfully expressed and secreted in insect cells, yielding 10-fold more enzyme than human cells. This provides a valuable tool for structural studies of fucosyltransferase V, aiding in the development of targeted cancer therapies.
Area of Science:
- Biochemistry
- Glycobiology
- Structural Biology
Background:
- Alpha3/4-fucosyltransferases are critical in inflammation and cancer metastasis.
- Lack of structural data hinders the development of specific fucosyltransferase inhibitors.
- Human fucosyltransferase V (FucTV) is a key enzyme in fucosylation.
Purpose of the Study:
- To express and purify human FucTV for structural and functional studies.
- To compare expression yields in insect versus human cell lines.
- To investigate the role of dimerization in FucTV secretion and activity.
Main Methods:
- Expressed truncated human FucTV constructs in baculovirus-infected insect cells (Trichoplusia ni) and HEK 293 cells.
- Utilized stably transfected insect and human cell lines for enhanced protein production.
- Compared expression and secretion of wild-type (cysteine at residue 64) and monomeric (serine at residue 64) FucTV constructs.
Main Results:
- Efficient expression and secretion of FucTV were achieved in both insect and human cells.
- Dimerization was not essential for FucTV secretion or expression.
- Stably transfected insect cells yielded approximately 10-fold more secreted FucTV than HEK 293 cells.
- The monomeric form of FucTV was successfully produced and is suitable for structural analysis.
Conclusions:
- Stably transfected insect cells are a highly effective system for producing large quantities of human FucTV.
- The monomeric form of FucTV is a valuable tool for elucidating its substrate specificity in fucosylation.
- This work facilitates the structure-based design of targeted inhibitors for cancer therapy.
