GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system.

BACKGROUND: Recombinant protein production and purification of large protein complexes in eukaryotes requires efficient methods to generate multi-gene expression constructs, where each individual gene is under the control of its own promoter and terminator. Current methods are based either on serial rounds of combination of several vectors containing loxP sites via the Cre-lox technology, or on multiple rounds of gene combination via PCR or other methods. These methods are multi-step, have lower efficiencies than single gene cloning, and may require laborious processes to verify that all genes of interest are present in the final product. Here, we describe a rapid and simple Golden Gate-based system for the generation of multi-gene expression constructs compatible with baculovirus expression vector systems (BEVS) using either Tn7 transposition or KO1629-based homologous recombination, which we refer to as "GoldenBac". RESULTS: This method is based on the construction of a series of vectors containing a promoter-gene of interest-terminator cassette flanked by cleavage sites of the BsaI type IIS restriction enzyme. This series of vectors can be cut by BsaI to excise cassettes with unique overhangs. In the same reaction, the cassettes are then ligated in the correct sequence in a final destination vector to generate multi-gene expression constructs containing 2-15 genes. Individual expression constructs can therefore be combined into a single vector in a single reaction, with over 90% efficiency when combining up to 14 expression cassettes. We demonstrate successful construction and expression of three different co-expression systems, the proteosomal lid complex, the anaphase promoting complex/cyclosome (APC/C), and a series of constructs used to test the effect of chaperone co-expression on the solubility of the HOIP protein. CONCLUSIONS: This robust, single-step cloning system provides an easy-to-use method for generation of multi-gene expression constructs for both transposition and homologous recombination-based baculovirus systems, making this technology available across all laboratories using baculovirus expression systems. This highly efficient and simple method allows for rapid incorporation of multi-gene expression cloning into the standardized service portfolio of protein production facilities and can also easily be adopted by any laboratory for routine generation of multi-gene baculovirus constructs.

Zocor Forte (simvastatin) has a neuroprotective effect against LPS striatal dopaminergic terminals injury, whereas against MPP+ does not.

Due to their potential role in preventing further deterioration of Parkinson's disease, anti-inflammatory strategies have attracted great interest. In this context, some studies point out the possible protective effect of anti-inflammatory compounds against the in vivo degeneration of dopaminergic neurons produced by lipopolysaccharide (LPS)-induced inflammatory processes and others. We have investigated the effect of the treatment of Zocor Forte (simvastatin) in LPS and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurodegenerative models to identify neuroprotective drugs for Parkinson's disease. We have perfused different concentrations of LPS or 1 mM 1-methyl-4-phenylpyridinium ion (MPP+) in the rat's striatum, 24 h after implanting a brain microdialysis probe, both with and without Zocor Forte (simvastatin) treatment. Results show that LPS perfusion produced a decrease in the basal release of dopamine. Forty-eight hours after implanting the probe, we have perfused 1 mM MPP+ to check the integrity of the dopaminergic terminals present around the cannula. Our model to study toxicity in the striatal dopaminergic terminals suggests that Zocor Forte (simvastatin) could prevent the neurotoxic damage produced by LPS, but not that produced by MPP+.