Fusion protein systems are commonly used for expression of small proteins and peptides. An important criterion for a fusion protein system to be useful is the ability to separate the protein of interest from the tag. Additionally, because no protease cleaves fusion proteins with 100% efficiency, the ability to separate the desired peptide from any remaining uncleaved protein is also necessary. This is likely to be the more difficult task as at least a portion of the sequence of the fusion protein is identical to that of the protein of interest. When a high level of purity is required, gradient elution reversed-phase HPLC is frequently used as a final purification step. Shallow gradients are often advantageous for maximizing both the purity and yield of the final product; however, the relationship between relative retention times at shallow gradients and those at steeper gradients typically used for analytical HPLC are not always straightforward. In this work, we report reversed-phase HPLC results for the fusion protein system consisting of the N-terminal domain of ribosomal protein L9 (NTL9) and the 36-residue villin headpiece subdomain (HP36) linked by a recognition sequence for the protease factor Xa. This system represents an excellent example of the difficulties in purification that may arise from this unexpected elution behavior at shallow gradients. Additionally, we report on the sensitivity of this elution behavior to the concentration of the additive trifluoroacetic acid in the mobile phase and present optimized conditions for separating HP36 from the full fusion protein by reversed-phase HPLC using a shallow gradient. Finally, we suggest that these findings are relevant to the purification of other fusion protein systems, for which similar problems may arise, and support this suggestion using insights from the linear solvent strength model of gradient elution liquid chromatography.

融合蛋白系统通常用于表达小蛋白和肽。融合蛋白系统有用的重要标准是将目标蛋白与标签分离的能力。另外，因为没有蛋白酶以100％的效率裂解融合蛋白，所以也有必要将所需的肽与任何剩余的未裂解的蛋白分离。这可能是更困难的任务，因为融合蛋白的至少一部分序列与目的蛋白的序列相同。当需要高纯度时，通常将梯度洗脱反相HPLC用作最终纯化步骤。浅梯度通常有利于最大程度地提高最终产品的纯度和收率。然而，通常用于分析型HPLC的浅梯度和较陡梯度的相对保留时间之间的关系并不总是很简单。在这项工作中，我们报告了融合蛋白系统的反相HPLC结果，该系统由核糖体蛋白L9（NTL9）的N端结构域和36个残基的维林头基亚结构域（HP36）（通过蛋白酶因子的识别序列连接）组成a 该系统代表了纯化困难的一个很好的例子，这种困难可能是由于在浅梯度下这种意外的洗脱行为引起的。此外，我们报告了这种洗脱行为对流动相中添加的三氟乙酸浓度的敏感性，并提出了使用浅梯度通过反相HPLC从完全融合蛋白中分离HP36的优化条件。最后，我们认为这些发现与其他融合蛋白系统的纯化有关，可能会出现类似的问题，并使用来自梯度洗脱液相色谱线性溶剂强度模型的见解来支持这一建议。