Noteworthy progress on this project has been made recently in two areas: understanding the mechanism by which certain highly soluble proteins enhance the solubility and promote the folding of their fusion partners, and the development of baculovirus vectors for the secretion of N-terminally His-tagged proteins from insect cells. Building on our previous investigation of the mechanism by which Escherichia coli maltose binding protein (MBP) enhances the solubility of its fusion partners, we have now shown that E. coli NusA, a completely unrelated protein that is also a very effective solubility enhancer, operates by the same or a very similar mechanism, suggesting that diverse solubility-enhancing proteins work in a fundamentally similar manner. We have also shown that MBP must be joined to the N-terminus of its fusion partner (rather than to its C-terminus) in order to enhance its solubility. This suggests that MBP needs to emerge first from the active ribosome for it to be fully functional as a solubility enhancer and is consistent with a model in which folded MBP binds to partially folded passenger proteins to inhibit their intermolecular aggregation. Additionally, baculovirus expression vectors were designed to secrete recombinant proteins using either the honeybee melittin or gp67 signal peptides in such a manner that the secreted proteins retain an N-terminal polyhistidine tag that can be removed by digestion with TEV protease. Both signal peptides were equally effective at promoting the secretion of MBP and green fluorescent protein (GFP). Moreover, both secreted proteins were processed correctly and could be captured on a Ni-NTA column. The yield of secreted GFP was high enough that the protein could be observed on a Coomassie-stained gel without Western blotting. The yield of secreted MBP was exceptionally high, constituting far and away the most abundant protein in the conditioned medium. Hence, these vectors are a useful addition to the toolbox for protein expression in insect cells.
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