昆虫Sf9细胞广泛用于生产重组蛋白,包括肌球蛋白.预计Sf9细胞中的蛋白质折叠机制可以满足外源肌球蛋白适当折叠的要求。令人感兴趣的是并非所有II类肌球蛋白都在Sf9细胞中功能性表达。在脊椎动物II类肌球蛋白中,非肌肉肌球蛋白和平滑肌肌球蛋白,但不是横纹肌肌球蛋白,在Sf9细胞中功能性表达,可能是由于Sf9细胞中缺乏脊椎动物横纹肌肌球蛋白特异性伴侣Unc45b。昆虫只表达一种通用的肌球蛋白特异性伴侣Unc45,预计它将负责所有昆虫肌球蛋白的折叠,包括横纹肌肌球蛋白.这一基本原理促使我们研究重组昆虫横纹肌肌球蛋白在Sf9细胞中的折叠。我们表达了来自三种昆虫物种(Locustamigratoria,果蝇和小菜蛾)在Sf9细胞中。类似于脊椎动物平滑肌肌球蛋白,但与脊椎动物横纹肌肌球蛋白不同,在Sf9细胞中表达的昆虫横纹肌肌球蛋白是可溶性的。纯化的重组昆虫横纹肌肌球蛋白显示正常的肌球蛋白功能,包括ATP依赖性肌动蛋白相互作用,肌动蛋白激活的ATP酶活性,和体外肌动蛋白滑动活性,表明Sf9细胞适合表达昆虫横纹肌肌球蛋白。因此,我们得出结论,与脊椎动物横纹肌肌球蛋白不同,需要横纹肌特异性伴侣(如Unc45b)进行折叠,昆虫横纹肌肌球蛋白可以通过昆虫细胞中的通用蛋白质折叠机制正确折叠。
Insect Sf9 cells are widely used for producing recombinant proteins, including myosin. It is expected that the protein folding machinery in Sf9 cells can meet the requirement for the proper folding of exogenous myosin. Of interest is that not all class II myosins are expressed functionally in Sf9 cells. Among vertebrate class II myosins, non-muscle myosin and smooth muscle myosin, but not striated muscle myosin, are functionally expressed in Sf9 cells, presumably due to lacking vertebrate striated muscle myosin-specific chaperone Unc45b in Sf9 cells. Insects only express a generic myosin-specific chaperone Unc45, which is expected to be responsible for the folding of all insect myosins, including striated muscle myosin. This rationale promotes us to investigate the folding of recombinant insect striated muscle myosins in Sf9 cells. We expressed the heavy meromyosin version of the striated muscle myosins from three insect species (Locusta migratoria, Drosophila melanogaster and Plutella xylostella) in Sf9 cells. Similar to vertebrate smooth muscle myosin, but unlike vertebrate striated muscle myosin, the insect striated muscle myosin expressed in Sf9 cells are soluble. The purified recombinant insect striated muscle myosins display normal myosin functions, including ATP-dependent actin interaction, actin-activated ATPase activity, and in vitro actin-gliding activity, indicating that Sf9 cells are suitable for expressing insect striated muscle myosin. We therefore conclude that, unlike vertebrate striated muscle myosin requiring striated muscle-specific chaperones (such as Unc45b) for its folding, insect striated muscle myosin can be properly folded by the generic protein folding machinery in insect cells.