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Vectors etc

There is no explanation here for general readers. We assume you will have arrived at this page because you know what baculovirus expression is all about. Our high throughput system is based on the traditional method of making recombinant viruses by recombination in insect cells combined with a novel suite of vectors generated in house that add a host of current fusion tags. Cloning hassle is minimised by using universal rare cutting enzymes (differential SfiI sites) in the PCR primers and having all vectors use the same reading frames across those sites. A single PCR product (incorporating protease sites if you choose them) can be then cloned in parallel into the vectors you think most suitable for your purposes. The technology is traditional (ligation dependent) so can be done by any competent lab, and is fast and fairly trouble free (although Sfi1 digests can be temperamental, sometimes requiring a TOPO step for the original PCR band). The vectors currently in the suite are shown below. The vectors within the shaded box all secrete proteins into the medium.

Key: His=hexahistidine; Sig=gp64 signal peptide; MBP=maltose binding protein; GFP=green fluorescent protein; VSVGTM=transmembrane domain of VSV G protein (for baculovirus display); Fc=human Ig Fc domain; TAP=tandem affinity purification (originally made in David Barford's lab but adapted for baculo use by us). 

Once the vectors are made we follow a streamlined virus isolation procedure that results in a reasonable volume of high titre stock by the time the first readouts of expression level are obtained. After this the scale-up required for any target protein amount can be calculated. This high throughput concept is shown below:

Vectors are always in development but we have run the high throughput concept now for about 2 years and it has proved to be robust and productive. It also has the virtue of being simple, amenable to automation and free of commercial licences. Some of the work done has recently been published (Pengelley et al.,) Go via the reagents page if you want to make use of this technology.




School of Biological Sciences

University of Reading, Whiteknights

Reading RG6 6AH UK

Tel/Fax/Message: +44 118 378 8902