Fig. 3.

Screening for the best affinity-tagged subunit through co-infection of insect cells (Scheme 3). (a) Diagrams of the ten types of transfer plasmids. The final transfer plasmids, ABCD (RFP) and EFGH (GFP), are generated using Scheme 2, and each will express four protein subunits without affinity labels. Each of the other eight transfer plasmids will express one subunit with an N-terminal Twin-Strep (TS) tag. Either the 4V1 or 5V1 vector can be used here. (b) Production of ten types of recombinant baculoviruses (RBVs). Transformation of the ten types of plasmids into DH10Bac competent cells generates 10 types of RBVs. (c) Screening baculovirus combinations to find the subunit that results in the best purification. The ten types of RBVs are divided into eight groups, and each group contains BV-ABCD (RFP), BV-EFGH (GFP) and one BV-TSn (where n corresponds to the subunit, A to H). Insect cells are co-infected with eight groups of RBVs and strep-affinity resin is used to pull down proteins bound to the TS-tagged subunit. The tagged subunit that allows the best purification of the whole complex is selected. In this example, subunit H is the best. (d) Production of the multiprotein complex. Based on the screening result in (c), a new final transfer plasmid EFG-TSH (GFP) is constructed, which expresses an N-terminal TS-tagged subunit H. The whole protein complex will be purified from insect cells co-infected with BV-ABCD (RFP) and BV-EFG-TSH (GFP).