Institute for Biotechnology and Biomedicine, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain; Department of Genetics and Microbiology, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain.
Institute for Biotechnology and Biomedicine, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain; Department of Genetics and Microbiology, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain; Bioengineering, Biomaterials and Nanomedicine Networking Biomedical Research Centre (CIBER-BBN), Bellaterra, Barcelona, Spain.
N Biotechnol. 2020 Jul 25;57:11-19. doi: 10.1016/j.nbt.2020.02.001. Epub 2020 Feb 3.
Efficient protocols for the production of recombinant proteins are indispensable for the development of the biopharmaceutical sector. Accumulation of recombinant proteins in naturally-occurring protein aggregates is detrimental to biopharmaceutical development. In recent years, the view of protein aggregates has changed with the recognition that they are a valuable source of functional recombinant proteins. In this study, bovine interferon-gamma (rBoIFN-γ) was engineered to enhance the formation of protein aggregates, also known as protein nanoparticles (NPs), by the addition of aggregation-prone peptides (APPs) in the generally recognized as safe (GRAS) bacterial Lactococcus lactis expression system. The L6K2, HALRU and CYOB peptides were selected to assess their intrinsic aggregation capability to nucleate protein aggregation. These APPs enhanced the tendency of the resulting protein to aggregate at the expense of total protein yield. However, fine physico-chemical characterization of the resulting intracellular protein NPs, the protein released from them and the protein purified from the soluble cell fraction indicated that the compactability of protein conformations was directly related to the biological activity of variants of IFN-γ, used here as a model protein with therapeutic potential. APPs enhanced the aggregation tendency of fused rBoIFN-γ while increasing compactability of protein species. Biological activity of rBoIFN-γ was favored in more compacted conformations. Naturally-occurring protein aggregates can be produced in GRAS microorganisms as protein depots of releasable active protein. The addition of APPs to enhance the aggregation tendency has a positive impact in overall compactability and functionality of resulting protein conformers.
高效的重组蛋白生产方案对于生物制药行业的发展是不可或缺的。重组蛋白在天然蛋白聚集体中的积累不利于生物制药的发展。近年来，随着人们认识到蛋白聚集体是功能性重组蛋白的宝贵来源，对其的看法发生了变化。在这项研究中，通过在公认安全（GRAS）细菌乳球菌表达系统中添加聚集倾向肽（APPs），对牛干扰素-γ（rBoIFN-γ）进行了工程改造，以增强蛋白聚集体（也称为蛋白纳米颗粒（NPs））的形成。选择 L6K2、HALRU 和 CYOB 肽来评估它们内在的聚集能力以引发蛋白聚集。这些 APPs 增强了所产生蛋白的聚集趋势，而牺牲了总蛋白产量。然而，对细胞内蛋白 NPs、从其中释放的蛋白和从可溶性细胞部分纯化的蛋白的精细理化特性进行的表征表明，蛋白构象的紧凑性直接与 IFN-γ变体的生物学活性相关，此处 IFN-γ 变体被用作具有治疗潜力的模型蛋白。APPs 增强了融合 rBoIFN-γ 的聚集趋势，同时增加了蛋白种类的紧凑性。rBoIFN-γ 的生物学活性在更紧凑的构象中更有利。天然蛋白聚集体可以在 GRAS 微生物中作为可释放活性蛋白的蛋白库进行生产。添加 APPs 以增强聚集趋势对所得蛋白构象的总体紧凑性和功能性具有积极影响。
