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: 8310048
• 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/8310048
• 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; receptor; serotonin transporter; structural biology; success; therapeutic development; therapeutic target; transcription factor; voltage

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 makes 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.

与非膜相关蛋白相比，真核细胞膜蛋白结构的阐明是有限的。 部分原因是在异源宿主中不能表达足够量的功能蛋白。 哺乳动物膜蛋白（MMPs）在功能构象中特别难以过表达 由于其复杂的折叠和加工模式以及 以未折叠和非功能性的形式积累。例如，四种生物医学上重要的MMP： 5-羟色胺转运体（SERT）、神经降压素受体（NTR）、间质-上皮转化因子（MET） 受体和电压敏感性钠通道（Nav1.7）都具有复杂的翻译后加工过程 要求.这些MMP的最佳表达通常发生在蛋白质在宿主中表达时， 进化上最接近目标MMP。事实上，NTR和SERT以其最高功能水平表达， 在哺乳动物宿主中以每个细胞为基础的水平。然而，哺乳动物和其他脊椎动物细胞系具有 由于其0.05 mg/L或更低的低总MMP产率，因此很少使用。如果基质金属蛋白酶的产量可以 增加一个数量级或更多时，将使用哺乳动物（和其它脊椎动物）培养系统 更广泛地是由于它们将复杂的MMP结构靶标加工成功能性的MMP结构靶标的上级能力 form.培养需求将从数百升减少到数十升。该提议的一个假设是 哺乳动物膜蛋白序列的性质和当前宿主的处理效率 使得在异源宿主中以高水平表达功能性蛋白质成为问题。极低的自然 一些复杂的MMPs的丰富使自然进化表达压力最小化， 需要尽量减少它们对宿主的毒性。因此，目前拟议项目的目标是开发 通过以下两个目的来改进功能表达水平的技术：1）进化蛋白质结构， 鉴定其诱变可以改善表达而不改变结构或功能那些氨基酸残基 显著地2）通过诱变工程化和进化哺乳动物宿主以产生能够 更有效地处理复杂的MMP。将实施“原地”进化和选择技术， 允许使用者在体细胞超突变B细胞宿主中突变靶MMP的序列。与此同时， 哺乳动物生产宿主将用抗凋亡蛋白、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 通过抑制细胞的天然DNA修复机制来进行。这种方法将在四个 上述MMP靶点代表不同类别的MMP，并与许多疾病相关。的 突变蛋白变体将在工程化和进化的哺乳动物细胞系中表达， 为正在进行的晶体学项目增加10至20倍的活性产物产量。该项目将提供一个 基于蛋白质和细胞进化策略表达高产量复杂MMPs的新范例。