Predicting protein refolding from a knowledge of protein interactions under renaturation conditions

重组蛋白通常以包涵体的形式产生，包涵体是由错误折叠的蛋白质密集堆积而成的聚集体。分离天然蛋白质需要在高度变性的条件下溶解聚集体，然后进行变性去除步骤以允许蛋白质重新折叠。在变性过程中，部分折叠的中间体聚集不可逆地捕获无活性蛋白。在工业水平上，克服这个问题需要在低浓度下重新折叠蛋白质，这需要大型储罐。可以通过使用某些添加剂来提高收率，这些添加剂可以增强再折叠或防止聚合。放大后的工艺只能在分批或补料-分批模式下运行，在这种模式下，蛋白质饲料浓度和饲料流量可以优化。通常，再折叠的产量仍然很低（大约10%），在某些情况下，蛋白质根本不会再折叠，而在其他情况下，再折叠所需的时间比放大过程所允许的要长得多。因此，拟议项目的总体目标是：1)了解溶剂如何影响重折叠行为，以便在选择复性条件时做出明智的决定；2)为设计大规模重折叠操作提供蛋白质聚集模型；3)将蛋白质-蛋白质相互作用的知识纳入蛋白质内在可重折叠性的预测。4)开发可靠的量化可折叠收率的方法。为了实现这些目标，需要确定蛋白质结构特性和还原条件（包括变性剂、盐、添加剂、pH值、蛋白质浓度等变量）对再折叠产率的影响。我们将通过确定蛋白质内部和分子间相互作用的分子起源，然后将相互作用与蛋白质重折叠行为相关联或拟合来建立这种联系。分子内相互作用将根据蛋白质稳定性荧光测定法进行测量，渗透第二病毒系数（SVC）将用于表征蛋白质-蛋白质相互作用。我们方法的一个优点是它们能够快速筛选一系列溶剂条件和蛋白质。研究将包括与治疗相关的蛋白质，可能包括重组生长集落刺激因子（rGCSF）、人类生长激素（HGH）、促红细胞生成素（EPO）、胰岛素和胰岛素样生长因子（ILGF）。所选择的蛋白质系统包含广泛的行为，以便分离结构-相互作用关系。筛选溶剂条件（pH值、离子强度、变性剂浓度、氧化条件）将使我们能够基于蛋白质的物理化学性质（蛋白质电荷、形状和大小、疏水性）建立简化的相互作用模型。这些知识将使我们能够改进现有的方法，从稳定性预测内在可折叠性。此外，我们将确定溶剂成分（变性剂，添加剂）如何与蛋白质相互作用和/或调节蛋白质之间的相互作用。了解相互作用的机制将指导复性条件的选择，重点是寻找与下游操作相容的溶剂。作为工作的一部分，我们将开发量化重折叠收率的方法。目前的选择是使用反相高效液相色谱法在重折叠步骤后分离蛋白质；天然折叠的蛋白质与不正确或聚集的蛋白质具有不同的疏水性。该方法将在我们的工作中进行评估，通过结合生物活性测定、替代HPLC步骤、光谱方法（FTIR、CD）、DSC和光散射来表征RP-HPLC步骤中的单体峰。对于选择的少数情况，我们还将使用NMR，它对任何错误折叠的蛋白质特别敏感。表征的结果将指导我们选择一个好的策略来评估再折叠产率使用少量的实验可及的技术。

Recombinant proteins are often produced as inclusion bodies, which are densely packed aggregates of mis-folded proteins. Isolating the native protein requires dissolving the aggregates under highly denaturing conditions followed by a denaturant removal step to allow for protein refolding. During renaturation, aggregation of partially folded intermediates irreversibly traps inactive proteins. At the industrial level, overcoming this problem requires refolding proteins at low concentrations requiring large tanks. Yields can be improved by using certain additives, which enhance refolding or prevent aggregation. The scaled-up process can only be run in a batch or fed-batch mode, in which protein feed concentration and feed flowrate can be optimized. Often refolding yields can still be low (on the order of 10 percent) and in some instances, proteins do not refold at all, whereas in other cases refolding takes much longer than permitted for a scaled-up process. Thus the overall aims of the proposed project are 1) Understand how solvent influences refolding behaviour so as to make informed decisions about choosing renaturation conditions 2) Provide models of protein aggregation for designing large-scale refolding operations 3) Incorporate knowledge of protein-protein interactions into predictions of protein intrinsic refoldability. 4) Develop robust methods for quantifying refolding yield. Meeting these aims requires determining how refolding yield depends upon protein structural properties and the renaturation conditions (variables including denaturants, salts, additives, pH, protein concentration). We will make this connection by determining the molecular origin of protein intra and intermolecular interactions and then either correlating or fitting the interactions to protein refolding behaviour. The intramolecular interactions will be measured in terms of a protein stability fluoresence assay and an osmotic second virial coefficient (SVC) will be used to characterize the protein-protein interactions. An advantage of our methods is their ability to rapidly screen a range of solvent conditions and proteins. Studies will include therapeutically relevant proteins, which might include recombinant growth colony stimulating factor (rGCSF), human growth hormone (HGH), erythropoietin (EPO), insulin, and insulin like growth factor (ILGF). The protein systems are chosen to encompass a broad range of behaviour so as to isolate structure-interaction relationships. Screening solvent conditions (pH, ionic strength, denaturant concentration, oxidation conditions) will allow us to build up simplified interaction models based upon protein physicochemical properties (protein charge, shape and size, hydrophobicity). This knowledge would then allow us to improve upon current methods for predicting intrinsic refoldability from stability. Also, we will determine how solvent components (denaturants, additives) interact with the protein and/or modulate protein-protein interactions. Understanding the mechanism of interaction will guide the choice of renaturation conditions, with emphasis on finding solvents compatible with the downstream operations. As part of the work we will develop methods for quantifying the refolding yield. The current choice is to use RP-HPLC to separate proteins after the refolding step; natively folded proteins are expected to have different hydrophobicities from incorrectly or aggregated proteins. This method will be assessed in our work by characterizing the monomer peak from the RP-HPLC step using a combination of bioactivity assays, alternative HPLC steps, spectroscopic approaches (FTIR, CD), DSC, and light scattering. For a select few cases, we will also use NMR, which is especially sensitive to any incorrectly folded proteins. The results of the characterization will guide us in choosing a good strategy for assessing refolding yield using a small number of experimentally accessible techniques.