Many studies examining the biological function of recombinant proteins and their effects on the physiology of mammalian cells stipulate that the proteins be purified before being used as therapeutic agents. In this study, we explored the possibility of using unpurified recombinant proteins to treat mammalian cells. The recombinant protein was used directly from the expression source and the biological function was compared to purified commercially available, equivalent protein. The model for this purpose was recombinant FGF-2, expressed by Pichia pastoris, which was used to treat the murine fibroblast cell line, NIH/3T3. We generated a P. pastoris strain (yHL11) that constitutively secreted a biologically active recombinant FGF-2 protein containing an N-terminal c-myc epitope (Myc-FGF-2). Myc-FGF-2 was then used without purification either a) in the form of conditioned mammalian cell culture medium or b) during co-cultures of yHL11 with NIH/3T3 to induce higher proliferation and motility of NIH/3T3 cells. The effects of Myc-FGF-2 on cell physiology were comparable to commercially available FGF-2. To our knowledge, this is the first time the physiology of cultured mammalian cells had been successfully altered with a recombinant protein secreted by P. pastoris while the two species shared the same medium and culture conditions. Our data demonstrated the biological activity of unpurified recombinant FGF-2 on NIH/3T3 cells and provided a foundation for directly using unpurified recombinant proteins expressed by P. pastoris with mammalian cells, potentially as wound-healing therapeutics.

许多研究重组蛋白的生物学功能及其对哺乳动物细胞生理学影响的研究都规定，在将蛋白用作治疗剂之前必须对其进行纯化。在这项研究中，我们探索了使用未纯化的重组蛋白治疗哺乳动物细胞的可能性。直接从表达源使用重组蛋白，并将其生物学功能与纯化的市售等效蛋白进行比较。为此目的的模型是由巴斯德毕赤酵母表达的重组FGF-2，其用于治疗鼠成纤维细胞系NIH / 3T3。我们产生了巴斯德毕赤酵母组成性分泌具有N末端c-myc表位（Myc-FGF-2）的具有生物活性的重组FGF-2蛋白的菌株（yHL11）。然后在不纯化的情况下使用Myc-FGF-2，或者a）以条件哺乳动物细胞培养基的形式使用，或者b）在yHL11与NIH / 3T3共培养期间诱导NIH / 3T3细胞更高的增殖和运动性。Myc-FGF-2对细胞生理的影响与市售FGF-2相当。据我们所知，这是首次通过巴斯德毕赤酵母分泌的重组蛋白成功地改变了培养的哺乳动物细胞的生理。而两个物种具有相同的培养基和培养条件。我们的数据证明了未纯化的重组FGF-2在NIH / 3T3细胞上的生物学活性，并为将巴斯德毕赤酵母表达的未纯化的重组蛋白与哺乳动物细胞直接结合使用提供了基础，可能将其用作伤口愈合疗法。