Single-vector three-frame expression systems for affinity-tagged proteins

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Fältnamn	Indikatorer	Metadata
000		03285naa a2200481 4500
001		oai:DiVA.org:kth-21990
003		SwePub
008		100810s2002 \| \|\|\|\|\|\|\|\|\|\|\|000 \|\|eng\|
009		oai:prod.swepub.kib.ki.se:1961075
024	7	a https://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-219902 URI
024	7	a https://doi.org/10.1111/j.1574-6968.2002.tb11383.x2 DOI
024	7	a http://kipublications.ki.se/Default.aspx?queryparsed=id:19610752 URI
040		a (SwePub)kthd (SwePub)ki
041		a engb eng
042		9 SwePub
072	7	a ref2 swepub-contenttype
072	7	a art2 swepub-publicationtype
100	1	a Graslund, S.4 aut
245	1 0	a Single-vector three-frame expression systems for affinity-tagged proteins
264	1	b Oxford University Press (OUP),c 2002
338		a print2 rdacarrier
500		a QC 20100525
520		a An effort is presented to create expression vectors which would allow expression of an inserted gene fragment in three reading frames in a single vector from a single promoter but with three separate ribosome binding sites (RBS). Each expression frame would generate an in-frame fusion with an affinity tag to allow efficient recovery of the produced fusion proteins. In the first generation vector, three identical polyhistidyl tags (His(6)) were used as affinity tags for the three expression frames. In the second generation vector, three different tags, an albumin binding domain derived from streptococcal protein G, an IgG binding Staphylococcus aureus protein A-derived domain (Z) and a His(6) tag, were employed to allow frame-specific affinity recovery. To evaluate the systems, model genes have been inserted in three different frames in both vectors. The first vector was demonstrated to produce fusion proteins in all three frames, whereas for the second, with a much wider spacing between the RBSs and affinity tags, expression could only be demonstrated from the first two translational start sites. For both systems, the first translation start was found to be significantly favored over the others. Nevertheless, we believe that the presented results represent the first successful attempt to create single-vector three-frame expression systems, a concept that could become valuable in future combined cloning-expression vectors.
653		a expression vector
653		a reading frame
653		a affinity tag
653		a fusion protein
653		a synaptonemal complex
653		a functional-analysis
653		a purification
653		a binding
653		a dna
700	1	a Larsson, M.4 aut
700	1	a Falk, R.4 aut
700	1	a Uhlén, Mathiasu KTH,Bioteknologi4 aut0 (Swepub:kth)u1dulvmw
700	1	a Hoog, C.4 aut
700	1	a Ståhl, Stefanu KTH,Bioteknologi4 aut0 (Swepub:kth)u1svy8i4
710	2	a KTHb Bioteknologi4 org
773	0	t FEMS Microbiology Lettersd : Oxford University Press (OUP)g 215:1, s. 139-147q 215:1<139-147x 0378-1097x 1574-6968
856	4	u https://academic.oup.com/femsle/article-pdf/215/1/139/19111510/215-1-139.pdf
856	4 8	u https://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-21990
856	4 8	u https://doi.org/10.1111/j.1574-6968.2002.tb11383.x
856	4 8	u http://kipublications.ki.se/Default.aspx?queryparsed=id:1961075

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Av författaren/redakt...: Graslund, S.; Larsson, M.; Falk, R.; Uhlén, Mathias; Hoog, C.; Ståhl, Stefan

Artiklar i publikationen: FEMS Microbiolog ...

Av lärosätet: Kungliga Tekniska Högskolan; Karolinska Institutet

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