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Single-vector three-frame expression systems for affinity-tagged proteins
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0001-8993-048X
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2002 (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 215, no 1, 139-147 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2002. Vol. 215, no 1, 139-147 p.
Keyword [en]
expression vector, reading frame, affinity tag, fusion protein, synaptonemal complex, functional-analysis, purification, binding, dna
URN: urn:nbn:se:kth:diva-21990ISI: 000178785600022OAI: diva2:340688
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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