Variable expression of multi-component protein complexes is increasingly necessary as more sophisticated projects are attempted. But how can this be achieved? Most of our expression work is based on the baculovirus system, in particular our flashBAC™ vectors that permit easy recombinant virus generation in a one-step process. These insect-specific viruses offer an ideal platform for producing a wide range of recombinant proteins including nuclear, cytoplasmic, membrane-bound and secreted examples.
But the structure of the baculovirus genome and the virus particle also provides for the insertion of a large amount of foreign DNA. The virus genome comprises a covalently closed, circular molecular of about 130Kbp, which is packaged in a rod-shaped capsid. The nature of this structure means that it is not constrained by the size of the virus genome and can expand to accommodate more coding sequences. Up to 8 different genes have been inserted into the baculovirus genome using multiple copies of the two very late gene promoters (polyhedrin and p10)1.
The only problem with current vectors for multiple expressions of foreign genes is that they all strive to produce as much protein as possible. This may not be necessary for the assembly of many multi component protein complexes such as virus like particles or enzyme complexes.
One approach that is used to obtain differential protein production is to divide the genes of interest between different recombinant baculoviruses and coinfect cells using variable multiplicities of infection* (MOI). The rationale for this is that delivering different amounts of virus to a cell will result in different amounts of recombinant protein. The problem with this method is that as the MOI decreases there is an increasing likelihood that some cells will not become infected with one of the viruses. In this event those cells not receiving all the viruses and in consequence all of the foreign genes, will not coexpress all of the target proteins.
A solution to this problem is to insert all of your genes in the same virus genome but under the control of baculovirus gene promoters of different strengths. How can this be achieved? It is actually quite easy to do as the efficiency of the polyhedrin and p10 gene promoters is reduced as deletions are made in the sequence between the transcription start site (TAAG motif) and the native ATG codon2,3. If you place one of your target coding regions downstream of this truncated promoter sequence you will downregulate its expression.
Currently, there are no “off the shelf” vectors available for this purpose, but it is something we are working on. For the time being if you are interested in variable expression of multi-component protein complexes contact us to discuss your requirements further.
*Multiplicity of infection: the number of infectious virus particles per cell added to a culture of cells. E.g. MOI of 10 = 10 infectious units of virus per cell.
References
1Noad, R. et al. (2009). Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA. BMC Molecular Biology 10:87, https://doi.org/10.1186/1471-2199-10-87.
2Matsuura, Y., Possee, R.D., Overton, H.A. and Bishop, D.H.L. (1987). Baculovirus Expression Vectors: the Requirements for High Level Expression of Proteins, Including Glycoproteins. Journal of General Virology 68: 1233-1250, doi: 10.1099/0022-1317-68-5-1233.
3Weyer, U. and Possee, R.D. (1988). Functional analysis of the p10 gene 5’ leader sequence of the Autographa californica nuclear polyhedrosis virus. Nucleic Acids Research 16, 3635-3653, https://doi.org/10.1093/nar/16.9.3635.