Amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase.
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/129933
- author
- Borg, Jörgen LU ; Nevsten, Pernilla LU ; Wallenberg, Reine LU ; Stenström, Martin LU ; Cardell, Susanna LU ; Falkenberg, Cecilia LU and Holm, Cecilia LU
- organization
- publishing date
- 2004
- 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
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Polymer and Materials Chemistry (LTH) (011001041), Department of Experimental Medical Science (013210000), Immunology (013212020), Molecular Endocrinology (013212018)
- 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
- 2016-04-01 12:23:30
- date last changed
- 2022-01-27 03:05:24
@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}},
keywords = {{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}},
doi = {{10.1016/j.jbiotec.2004.05.014}},
volume = {{114}},
year = {{2004}},
}