Journal
NUCLEIC ACIDS RESEARCH
Volume 35, Issue 6, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkm047
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Funding
- MRC [G0400717] Funding Source: UKRI
- Medical Research Council [G0400717] Funding Source: researchfish
- Medical Research Council [G0400717] Funding Source: Medline
- Wellcome Trust Funding Source: Medline
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This article describes the construction of a set of versatile expression vectors based on the In-Fusion (TM) cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion (TM) has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His(6)-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.
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