Metcalf Kevin James, Bevington James Lea, Rosales Sandy Lisette, Burdette Lisa Ann, Valdivia Elias, Tullman-Ercek Danielle
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, 94720, USA.
Department of Biomedical Engineering, Northwestern University, Evanston, IL, 60208, USA.
Microb Cell Fact. 2016 Dec 23;15(1):213. doi: 10.1186/s12934-016-0606-4.
Bacterial production of natively folded heterologous proteins by secretion to the extracellular space can improve protein production by simplifying purification and enabling continuous processing. In a typical bacterial protein production process, the protein of interest accumulates in the cytoplasm of the cell, requiring cellular lysis and extensive purification to separate the desired protein from other cellular constituents. The type III secretion system of Gram-negative bacteria is used to secrete proteins from the cytosol to the extracellular space in one step, but proteins must unfold during translocation, necessitating the folding of secreted proteins in the extracellular space for an efficient production process. We evaluated type III secretion as a protein production strategy by characterizing and quantifying the extent of correct folding after secretion.
We probed correct folding by assaying the function after secretion of two enzymes-beta-lactamase and alkaline phosphatase-and one single-chain variable fragment of an antibody. Secreted proteins are correctly folded and functional after unfolding, secretion, and refolding in the extracellular space. Furthermore, structural and chemical features required for protein function, such as multimerization and disulfide bond formation, are evident in the secreted protein samples. Finally, the concentration of NaCl in the culture media affects the folding efficiency of secreted proteins in a protein-specific manner.
In the extracellular space, secreted proteins are able to fold to active conformations, which entails post-translational modifications including: folding, multimerization, acquisition of metal ion cofactors, and formation of disulfide bonds. Further, different proteins have different propensities to refold in the extracellular space and are sensitive to the chemical environment in the extracellular space. Our results reveal strategies to control the secretion and correct folding of diverse target proteins during bacterial cell culture.
通过分泌到细胞外空间来实现天然折叠的异源蛋白的细菌生产，可通过简化纯化过程和实现连续加工来提高蛋白产量。在典型的细菌蛋白生产过程中，目标蛋白积累在细胞胞质中，需要进行细胞裂解和广泛纯化，以将所需蛋白与其他细胞成分分离。革兰氏阴性菌的III型分泌系统用于将蛋白从胞质溶胶一步分泌到细胞外空间，但蛋白在转运过程中必须解折叠，这就需要在细胞外空间对分泌的蛋白进行折叠才能实现高效生产过程。我们通过表征和量化分泌后正确折叠的程度，评估了III型分泌作为一种蛋白生产策略。
我们通过检测两种酶（β-内酰胺酶和碱性磷酸酶）以及一种抗体单链可变片段分泌后的功能，来探究正确折叠情况。分泌的蛋白在细胞外空间解折叠、分泌和重新折叠后能正确折叠并具有功能。此外，蛋白功能所需的结构和化学特征，如多聚化和二硫键形成，在分泌蛋白样品中很明显。最后，培养基中NaCl的浓度以蛋白特异性方式影响分泌蛋白的折叠效率。
在细胞外空间，分泌的蛋白能够折叠成活性构象，这需要进行翻译后修饰，包括：折叠、多聚化、获取金属离子辅因子以及形成二硫键。此外，不同蛋白在细胞外空间重新折叠的倾向不同，且对细胞外空间的化学环境敏感。我们的结果揭示了在细菌细胞培养过程中控制多种目标蛋白分泌和正确折叠的策略。
