The baculovirus expression system has been used to express many thousands of proteins in eukaryotic cells for structural and biochemical studies (14).  In addition to its ability to express single recombinant proteins the baculovirus system has also been used to co-express multiple proteins that form complexes (15-22).  This is important as genome-wide interaction screens are revealing that many critical functions of living cells are carried out by multi-subunit protein complexes (23).  Two main strategies have been used to achieve co-expression of proteins using the baculovirus system.  The coinfection of cells with two or more viruses each of which expresses one recombinant protein is by far the most common approach (19,24,25).  However, often yields of protein complexes from these studies are very variable and co-infection of the same cells by two different baculoviruses does not follow the poisson distribution (26).  Thus while the co-infection approach is technically straightforward, it is practically limited to recovery of relatively simple complexes for applications that do not require large amounts of purified proteins. 
For applications such as structural studies and large vaccine trials where more protein is required, and for more complicated complexes formed from many subunits, the alternative approach of co-expressing proteins from multiple similar expression cassettes inserted at the polyhedrin or P10 loci has been used (15-18,22,27).  This approach has the advantage that every cell in the culture that is infected with recombinant virus expresses all of the proteins required for formation of the protein complex in a reproducible manner.  Where the coinfection and single baculovirus approaches have been compared, the latter has demonstrated significantly better yield of recombinant complex (28).  However, expression of multiple proteins using a single recombinant virus is not without drawbacks.  Although rod shaped baculovirus appears quite tolerant of insertions in its genome, genes are transferred to the virus by homologous recombination and the transfer vector must be of a size that is easily manipulated in E. coli.  Therefore there is a limit to the number of genes that can be inserted into a transfer vector.  In practice this means that it is rarely possible to express more than four proteins from a single locus.  In addition, baculoviruses contain sequences and express proteins that promote homologous recombination (29-32).  Therefore viruses that contain large amounts of repeated sequence are prone to rearrangement and recombination (21,33,34). 
The single baculovirus coexpression approach has been modified by insertion of a loxP site at the chiA/cathepsin locus of a bacmid that already contains the Tn7 target at the polyhedrin locus (20).  This allows insertion of multiple expression cassettes at each of these loci using recombination in E. coli.  As might be expected given that this system relies on the iterative duplication of an expression cassette that has been modified to express different genes there is evidence that inserts into this system are somewhat unstable genetically (21). 
Recently baculovirus research has benefited from use of the ET recombination system which has allowed selective knockout of viral genes (35-49).  However, the potential for this technique to engineer and facilitate the expression of proteins at genetic loci other than p10 and polyhedrin has not been examined. 
The present invention relates to a method for the efficient expression of multiple recombinant proteins (i.e., non-baculoviral proteins) from a single baculovirus genome.  The method allows single protein expression cassettes to be inserted efficiently at different loci within the viral genome using the ET recombination system.  Furthermore, the method allows for the expression of complexes with numerous subunits. 
The expression of single proteins using baculovirus as an expression system is well established.  The basic methodology of the expression system has changed very little since its first production.  At 133 kb the baculovirus dsDNA genome is too large to manipulate directly.  Genes encoding foreign proteins are therefore cloned, in E. coli, into vectors containing an expression cassette from the AcMNPV insect virus and this is then introduced with viral DNA into insect cells where viral and cellular proteins mediate homologous recombination that results in the production of an infectious (with respect to insect cells) virus that expresses the foreign protein.  Some variations on this theme have already been generated, including viral genomes that only replicate if recombination occurs (1,2), and performing recombination in E. coli with a bacmid based on the use of transposon Tn7 (3) to speed up the selection of recombinant virus.  The expression of multi-protein complexes in insect cells has also been achieved using the baculovirus expression system as briefly discussed above.  Initially, multiple viruses each expressing a single protein were used to co-infect susceptible cells and the protein complex purified after expression.  However, this is at best inefficient and not reproducible enough to be feasible for scale-up.  As an alternative, vectors that incorporate multiple expression units but recombine with a single virus genetic locus were produced (4-6).  These vectors had the advantage that they produced all recombinant protein subunits in the every infected cell and resulted in significantly higher yields of recombinant protein complex.  In addition, because only a single virus expresses all the proteins the results of infections were much more reproducible.  The disadvantage of these vectors is twofold.  Firstly, because the vectors contain repeated sequences and the baculovirus expresses proteins that promote homologous recombination, expression, particularly from constructs containing three or four expression units, is not always very stable genetically through large numbers of viral passages (which are necessary during industrial scale-up for baculovirus protein expression).  Secondly, there is a practical limit to the size of insert that can be easily maintained and manipulated in E. coli which limits the number of proteins that can be expressed from these vectors.  Another approach that has been proposed for the production of multiple proteins is the insertion of foreign protein(s) at one genetic locus in baculovirus, then selection of virus expressing that protein and the use of this viral genome to recombine at a second genetic locus to express other proteins (7).  However, because of the high degree of technical skill and amount of time involved in this approach (16 days for the first locus and 25 days for each subsequent locus) this approach has hardly ever been used.  A recent development was to modify the BAC based Tn7 transposon system to insert a LoxP site at a second genetic locus in baculovirus to also allow the insertion of extra genes at this locus in E. coli (8).  However, this method still suffers from the problems that repeat sequences from duplication of promoters in the bacterial vectors will mean that the construct is genetically unstable for multiple viral passages and transfer vectors will quickly reach the maximum practical size for manipulation in E. coli.  
The method of the present invention overcomes at least some of the problems inherent in the prior methods, such as genetic instability and the requirement for multiple rounds of recombination.