A novel in vivo protein refolding technique

Proteins perform their functions in their native folded states and misfolding of proteins may cause severe diseases, including Alzheimer\u27s disease, Parkinson\u27s disease, prion disease and diabetes. Understanding protein folding is important for us to engineer proteins to treat these diseases. For protein therapeutics, large quantities of properly folded and functional proteins are required. The current technology produces recombinant proteins using either eukaryotic or prokaryotic expression system, both of them have major problems that prevent production of large quantities of properly folded and functional human proteins for protein therapeutics. Although the eukaryotic cells have comprehensive folding machinery that contains chaperones and folding enzymes and a complex quality control (QC) system to ensure that only properly folded proteins will be generated to perform their functions, either intracellular or extracellular, the protein yield is usually very low. Protein production using this system is usually costly. In contrast, prokaryotic cells can be used to produce large quantities of recombinant human proteins at a low cost. However, the produced human proteins using prokaryotic cells usually misfold and are not functional due to the much simpler protein folding machinery and QC system of these prokaryotic cells. To solve this problem, the in vitro protein refolding technique has been developed that either mimics the intracellular redox conditions to promote protein folding at a diluted concentration or uses column chromatography to refold the misfolded recombinant proteins. Although this in vitro protein folding technique has some success for small proteins with simple folds, the refolding efficiency is generally very low. For large proteins of complex folds of multiple domains, this in vitro protein refolding technique is usually not working. To solve these challenges, our lab recently developed an in vivo protein refolding technique that uses the intracellular folding machinery and QC system of the Endoplasmic reticulum (ER) of mammalian cells to refold the misfolded recombinant proteins produced using bacterial expression system. This novel technique uses the QQ-protein delivery technology developed in our lab to directly deliver bacterially expressed proteins into the ER for refolding. We showed that the intracellular folding machinery of mammalian cells had a large capacity to properly refold large quantities of misfolded bacterially expressed proteins and the QC system of the mammalian cells ensured that only properly folded proteins followed the normal intracellular trafficking pathway as their endogenous counterparts. Since the refolded proteins contain an affinity tag, we can purify the properly refolded proteins. This in vivo refolded technique takes the advantage of the high yield prokaryotic expression system and the comprehensive protein folding machinery/QC system of mammalian cells to efficiently produce large quantities of properly folded and biologically functional proteins. I optimized this in vivo protein refolding technique for the beta-propeller/EGF domain I of LDL receptor-related protein 6 (BP1-LRP6) and the ligand-binding domain of apolipoprotein E receptor 2 (LBD-ApoER2). These two proteins contain a large number of cysteines that form intracellular disulfide bonds. The folding of these two proteins is very challenging. I performed optimizations of experimental conditions that allow me to produce large quantities of properly folded and functional BP1-LRP6 and LBD-apoER2. The yield of refolding is about 20-60%, depending on different proteins, allowing me to produce milligram quantity of properly refolded and functional BP1-LRP6 and LBD-apoER2. The Far-UV Circular Dichroism (CD) Spectrum of refolded BP1-LRP6 showed a high percentage of beta-sheet which is consistent with the x-ray crystal structure of the beta-propeller/EGF domain of low-density lipoprotein receptor (LDLR). Refolded LBD-apoER2 showed the biological function of active binding the chaperone receptor-associated protein (RAP) in the ligand-blotting assay. My results suggested that, as a new tool, this protein refolding technique can be used to produce large quantities of properly folded and biologically functional proteins for many applications including protein therapeutics to treat human disease, structural biology and protein folding studies