Baculovirus expression vectors with single strand capability.

Recombinant baculoviruses offer a useful way of expressing eucaryotic genes, often in high yield (1). Current procedures for the generation of a recombinant virus make use of a transfer vector that, via recombination in vivo, place the gene of interest under control of the powerful polyhedrin promotor. At present the expression of mutant forms of the cloned gene (to study structure and function), requires transfer to M13 or plasmids for mutagenesis followed by recloning to the transfer vector for each mutant made. To speed-up this process we have constructed and tested a set of baculovirus transfer vectors that have single stranded capability. A baculovirus expression vector, pAcYMI, in which the unique Bam HI cloning site has been positioned so as to maximise the expression of non-fused proteins has been previously described (2). An Xhol -EcoRI fragment of approx. 5Kb encoding all the signals necessary for efficient expression and recombination was removed from pAcYMI, blunt ended and cloned between the filled-in EcoRI and Hind III sites of the plasmid pUC118 (3). Upon superinfection with the helper phage M13 K07 (method as outlined in (3)) large amounts of single stranded transfer vector are produced. For reasons that are unclear to us only one orientation of recombinant produced ssDNA efficiently; its structure is shown in Fig. 1A. To further improve pAcCL29the pUCpolylinker was removed from pUC118, blunt ended, and placed into the filled in BAM HI site of pAcCL29. Thus, plasmids pAcCL29-1 and pAcCL29-8 have a variety of unique restriction sites available for the cloning of genes to be expressed. pAcCL29-8 contains an ATG prior to the unique cloning sites making this vector suitable for the expression of coding fragments lacking an initiation methionine. The structure of each vector across the insertion sites is shown in Fig. 1B in the sense of the DNA strand that is packaged into phage particles.