Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
Microb Cell Fact. 2022 Sep 5;21(1):180. doi: 10.1186/s12934-022-01905-2.
Komagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly.
We determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein.
Our observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.
毕赤酵母是制造治疗性蛋白的常用替代宿主，部分原因是其能够将重组蛋白分泌到细胞外空间。然而，细胞对分泌蛋白的不正确加工会导致非功能性的与产品相关的变体，这些变体在纯化过程中去除成本高昂，并且会降低整体工艺收率。与重组蛋白 N 端相连的分泌信号肽是决定蛋白质序列质量和产量的主要因素。在毕赤酵母中，来自酿酒酵母α交配因子的信号肽通常可产生最高效的重组蛋白分泌效价，但分泌蛋白的质量可能差异很大。
我们确定，SARS-CoV-2 受体结合域的一种聚集的与产品相关的变体是由信号肽不完全切割导致的 N 端延伸引起的。通过将短的、功能肽片段或天然信号肽的 EAEA 残基延伸到 N 端，我们消除了这种变体，并将分泌蛋白的效价提高了 76%。然后，我们将该策略应用于另外三种重组亚单位疫苗抗原，观察到相同的聚集的与产品相关的变体被一致消除。最后，我们证明通过在重组蛋白的 N 端添加一个氨基酸可以实现质量和分泌效价的提高。
我们的观察结果表明，在高尔基体中切割信号肽的蛋白酶的空间位阻可能导致不必要的 N 端延伸和相关的产品变体。我们证明了这种现象发生在多种重组蛋白中，并且可以通过最小化 N 端的修饰来改善空间位阻，从而解决这个问题。该策略可以使具有高效性α交配因子分泌信号肽的广泛重组蛋白的一致分泌成为可能。
