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Amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase.

Borg, Jörgen LU ; Nevsten, Pernilla LU ; Wallenberg, Reine LU ; Stenström, Martin LU ; Cardell, Susanna LU ; Falkenberg, Cecilia LU and Holm, Cecilia LU (2004) In Journal of Biotechnology 114(1-2). p.21-30
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Neuraminidase, Surface display, Baculovirus, Class II transmembrane protein, Amino-terminal anchor, Phage display
in
Journal of Biotechnology
volume
114
issue
1-2
pages
21 - 30
publisher
Elsevier
external identifiers
  • pmid:15464595
  • wos:000224598000003
  • scopus:4644343154
ISSN
1873-4863
DOI
10.1016/j.jbiotec.2004.05.014
language
English
LU publication?
yes
id
665a2bb9-9e80-4710-a0b1-2a9b2aedffbd (old id 129933)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15464595&dopt=Abstract
date added to LUP
2007-07-10 14:46:48
date last changed
2017-01-01 05:06:11
@article{665a2bb9-9e80-4710-a0b1-2a9b2aedffbd,
  abstract     = {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.<br/><br>
<br/><br>
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.},
  author       = {Borg, Jörgen and Nevsten, Pernilla and Wallenberg, Reine and Stenström, Martin and Cardell, Susanna and Falkenberg, Cecilia and Holm, Cecilia},
  issn         = {1873-4863},
  keyword      = {Neuraminidase,Surface display,Baculovirus,Class II transmembrane protein,Amino-terminal anchor,Phage display},
  language     = {eng},
  number       = {1-2},
  pages        = {21--30},
  publisher    = {Elsevier},
  series       = {Journal of Biotechnology},
  title        = {Amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase.},
  url          = {http://dx.doi.org/10.1016/j.jbiotec.2004.05.014},
  volume       = {114},
  year         = {2004},
}