Amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase.
Methods currently used for surface display on insect cells and budded baculovirus, all utilize the sequences from class I transmembrane proteins. This gives rise to some problems when handling unknown genes or cDNAs encoding full-length proteins. First, the stop codon from the cloned gene will be located upstream of the sequence for the transmembrane region. Second, the chance of getting the sequences encoding the signal peptide and the transmembrane region in frame with the cloned gene is small. To minimize these problems, we here present a method by which cDNAs or genes of interest can be cloned and fused to the codons for the signal peptide and transmembrane region of neuraminidase (NA), a class II transmembrane protein of the influenza virus. By placing both the signal peptide and transmembrane region at the amino-terminal, potential problems regarding stop codons are eliminated and errors in frame-shift minimized. To obtain proof of principle, the gene encoding enhanced green fluorescent protein, EGFP, was subcloned into a shuttle vector downstream of the neuraminidase sequence and the fusion product was then transferred to a baculovirus vector and transfected into insect cells (Sf9). Using this method, EGFP was found to be expressed on the surface of both infected cells and budded virus in an accessible manner.
Department of Cell and Molecular Biology, Section for Molecular Signalling, Lund University, BMC, C11, SE-221 84 Lund, Sweden. jorgen.borg@medkem.lu.se
Jorgen
J
Borg
Pernilla
P
Nevsten
Reine
R
Wallenberg
Martin
M
Stenstrom
Susanna
S
Cardell
Cecilia
C
Falkenberg
Cecilia
C
Holm
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods; D005822:Genetic Vectors; D007313:Insecta; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D008565:Membrane Proteins; D019151:Peptide Library; D009439:Neuraminidase