title: Overcoming inefficient secretion of recombinant VEGF-C in baculovirus expression vector system by simple purification of the protein from cell lysate.
abstract: The first reports about successfully expressed recombinant proteins with the use of a baculovirus vector were published over 30years ago. Despite the long time of refining this expression system, early problems with the production of baculovirus-derived secretory proteins are still not satisfactorily solved. The high expression level driven by baculoviral promoters often does not result in the desired yield of secreted recombinant proteins, which frequently accumulate inside insect cells and are only partially processed. During our attempts to produce vascular endothelial growth factor C (VEGF-C) with the use of a baculovirus vector we also faced an inefficient secretion of the recombinant protein to culture medium. We were not able to improve the outcome and obtain an acceptable concentration of VEGF-C in the medium by changing the culture conditions or utilizing different signal peptides. However, as a significant amount of native VEGF-C was detected inside the baculovirus-infected cells, we developed a simple method to purify recombinant, glycosylated VEGF-C from a lysate of the cells. The presented results indicate that the lack of a secretory protein in the insect cell culture medium after baculovirus infection does not necessarily signify failure in the production of the protein. As demonstrated by us and contrary to generally accepted views, the lysate of baculovirus-infected cells may constitute a valuable source of the biologically active, secretory protein.
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: tomasz.klaus@doctoral.uj.edu.pl.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Klaus
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: malgorzata.kulesza@doctoral.uj.edu.pl.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Kulesza
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: monika.bzowska@uj.edu.pl.
authors.forename: Monika
authors.initials: M
authors.lastname: Bzowska
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: barbara.wyroba@uj.edu.pl.
authors.forename: Barbara
authors.initials: B
authors.lastname: Wyroba
authors.affiliation: Institute of Bioengineering and Swiss Institute for Cancer Research (ISREC), School of Life Sciences, SV-IBI-LLCB, Station 15, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland. Electronic address: wkilarski@uchicago.edu.
authors.forename: Witold W
authors.initials: WW
authors.lastname: Kilarski
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: joanna.bereta@uj.edu.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Bereta
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D042783:Endothelial Cells / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D006031:Glycosylation; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D061987:Sf9 Cells / Q000737:chemistry*; D018411:Spodoptera; D042582:Vascular Endothelial Growth Factor C / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D042582:Vascular Endothelial Growth Factor C; C467483:vascular endothelial growth factor C, mouse; C000592875:Kdr protein, mouse; D040301:Vascular Endothelial Growth Factor Receptor-2; D040321:Vascular Endothelial Growth Factor Receptor-3
keywords: Baculovirus expression vector system; Secretion; VEGF-C; Vascular endothelial growth factor C