Equi-MOI ratio for rapid baculovirus-mediated multiprotein co-expression in insect cells integrating selenomethionine for structural studies.

Proteins often co-exist as multicomponent assemblies, making their co-expression essential in recombinant production processes. The baculovirus expression vector system is commonly used to produce recombinant multiprotein complexes mostly for structural and functional studies. Although AI-enhanced tools, such as AlphaFold, have revolutionized protein structure prediction, solving the phase problem remains the most significant challenge in X-ray crystallography for determining entirely novel, dynamic, or complex protein structures. To address this challenge, the early incorporation of selenomethionine into native proteins during production is especially advantageous for facilitating experimental phasing. Here, we describe a fast, effective, and versatile research protocol that uniquely combines these two challenging features. The principle of this method is based on using co-infection of several recombinant baculoviruses in so-called equal multiplicity of infection (MOI) or equi-MOI ratio, while at the same time, the balanced selenomethionine incorporation takes place to allow for an accelerated workflow. The delicate balance between individual conditions for producing selenomethionine-incorporated multiprotein complexes with high efficiency has been developed over several years of studying protein complexes; therefore, many useful tips and tricks are provided as well. Moreover, this protocol is straightforward to implement in any wet lab.