Evolving membrane proteins for high-level expression in modified mammalian cells

用于在修饰的哺乳动物细胞中高水平表达的进化膜蛋白

• 批准号: 8152106
• 负责人: MICHAEL J BETENBAUGH
• 金额: $ 29.7万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8152106
• 关键词: Amino Acid Sequence; Antibodies; Apoptosis; Apoptotic; B-Lymphocytes; Binding; Cell Culture Techniques; Cell Line; Cell Separation; Cells; Collaborations; Complex; Crystallization; Crystallography; DNA Repair; Detergents; Development; Disease; Drug abuse; Engineering; Ensure; Epithelial; Evolution; Fluorescence; Funding; Generations; Goals; In Situ; Ion Channel; Joints; Laboratories; Ligands; Lipids; Malignant Neoplasms; Mammalian Cell; Membrane Proteins; Mental Depression; Mesenchymal; Molecular Chaperones; Molecular Conformation; Mutagenesis; Mutate; Mutation; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurotensin Receptors; Neurotransmitters; Pain; Pattern; Peptide Sequence Determination; Pharmacologic Substance; Point Mutation; Process; Production; Property; Proteins; Proteolytic Processing; Publications; Receptor Protein-Tyrosine Kinases; Research; Resolution; Selection Criteria; Sodium Channel; Structure; System; Technology; Testing; Tissues; Toxic effect; Transcription factor genes; Variant; base; cellular engineering; design; directed evolution; glycosylation; improved; interest; milligram; overexpression; pressure; programs; protein expression; protein structure; public health relevance; receptor; serotonin transporter; structural biology; success; therapeutic development; therapeutic target; transcription factor; voltage

DESCRIPTION (provided by applicant): Elucidation of eukaryotic membrane protein structures is limited in comparison to non-membrane associated proteins in part due to the inability to express sufficient amounts of functional proteins in heterologous hosts. Mammalian membrane proteins (MMPs) are particularly difficult to overexpress in functional conformations useful for structural determination due to their complex folding and processing patterns and tendency to accumulate in unfolded and non-functional forms. For example, the four biomedically important MMPs: serotonin transporter (SERT), neurotensin receptor (NTR), mesenchymal-epithelial transition factor (MET) receptor, and voltage-sensitive sodium channel (Nav1.7) all have complex post-translational processing requirements. Optimal expression of these MMPs often occurs when the protein is expressed in a host that is evolutionarily closest to the target MMP. Indeed, NTR and SERT are expressed at their highest functional levels on a per cell basis in mammalian hosts. However, mammalian and other vertebrate cell lines have been used sparingly due to their low overall MMP yields of 0.05 mg/L or lower. If the yields of MMPs could be increased by an order of magnitude or more, mammalian (and other vertebrate) culture systems would be used more widely due to their superior capabilities for processing complex MMP structural targets into a functional form. Culture requirements would be reduced from hundreds to tens of liters. A hypothesis of this proposal is that the nature of mammalian membrane protein sequence and the processing efficiency of the current hosts make it problematic to express functional proteins at high levels in heterologous hosts. The very low natural abundance of some complex MMPs has minimized the natural evolutionary expression pressures and limited the need to minimize their toxicity on hosts. Therefore, the goal of the current proposed project is to develop technologies to improve functional expression levels by the following two aims: 1) evolving protein structure to identify those amino residues whose mutagenesis can improve expression without altering structure or function significantly 2) engineering and evolving mammalian hosts through mutagenesis to generate cell lines able to process complex MMPs more efficiently. "In situ" evolution and selection technologies will be implemented that allow users to mutate the sequence of target MMPs in a somatic hypermutating B cell host. In parallel, mammalian production hosts will be engineered with anti-apoptotic proteins, molecular chaperones, and transcription factors capable of improving protein processing. Furthermore, directed evolution of cell lines will be undertaken by inhibiting the cells natural DNA repair machinery. This approach will be tested on the four MMP targets above that represent different classes of MMPs and are related to a number of diseases. The mutated proteins variants will be expressed in engineered and evolved mammalian cell lines in order to increase active product yields by 10 to 20 fold for ongoing crystallography projects. This project will provide a new paradigm for expressing high yields of complex MMPs based on protein and cellular evolution strategies. PUBLIC HEALTH RELEVANCE: Many of the most important therapeutics are targeted towards mammalian membrane proteins in different tissues in the body. Our ability to develop improved pharmaceuticals for treating diseases including cancer, neurodegeneration, depression, drug abuse, and pain will be greatly accelerated by an improved understanding of the structural nature of complex mammalian membrane proteins. The goal of this project is to develop the technologies that enable mammalian cells to produce high yields of biomedically important membrane proteins needed for subsequent crystallization and structural analysis.

描述（由申请人提供）：与非膜相关蛋白相比，真核膜蛋白结构的阐明受到限制，部分原因是无法在异源宿主中表达足够数量的功能蛋白。哺乳动物膜蛋白（MMPs）由于其复杂的折叠和加工模式以及倾向于以未折叠和非功能形式积累，因此在用于结构确定的功能构象中特别难以过表达。例如，四种生物医学上重要的MMPs：血清素转运体（SERT）、神经紧张素受体（NTR）、间充质上皮转化因子受体（MET）和电压敏感钠通道（Nav1.7）都有复杂的翻译后加工要求。这些MMP的最佳表达通常发生在蛋白质在进化上最接近目标MMP的宿主中表达时。事实上，在哺乳动物宿主中，NTR和SERT在每个细胞的基础上以最高的功能水平表达。然而，哺乳动物和其他脊椎动物细胞系由于其总体MMP产量较低（0.05 mg/L或更低）而很少使用。如果MMP的产量可以增加一个数量级或更多，哺乳动物（和其他脊椎动物）培养系统将得到更广泛的应用，因为它们具有将复杂的MMP结构目标加工成功能形式的优越能力。培养需求将从数百升减少到数十升。本文的一个假设是，哺乳动物膜蛋白序列的性质和当前宿主的加工效率使得在异源宿主中高水平表达功能蛋白存在问题。一些复杂MMPs的天然丰度非常低，这使得自然进化表达压力最小化，并限制了将其对宿主的毒性最小化的需要。因此，目前提出的项目目标是通过以下两个目标来开发提高功能表达水平的技术：1)进化蛋白质结构，以识别那些突变可以在不显著改变结构或功能的情况下改善表达的氨基酸残基；2)通过诱变改造和进化哺乳动物宿主，以产生能够更有效地处理复杂MMPs的细胞系。将实施“原位”进化和选择技术，允许用户在体细胞超突变B细胞宿主中突变目标MMPs的序列。与此同时，哺乳动物生产宿主将被设计成抗凋亡蛋白、分子伴侣和能够改善蛋白质加工的转录因子。此外，细胞系的定向进化将通过抑制细胞的自然DNA修复机制来进行。这种方法将在上述四种MMP靶点上进行测试，这些靶点代表不同类别的MMP，并与许多疾病有关。突变蛋白变体将在工程和进化的哺乳动物细胞系中表达，以便将活性产物产量提高10至20倍，用于正在进行的晶体学项目。该项目将为基于蛋白质和细胞进化策略的高产量复杂MMPs的表达提供一个新的范例。