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: 8536850
• 负责人: MICHAEL J BETENBAUGH
• 金额: $ 28.81万
• 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 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8536850
• 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，真核膜蛋白结构的阐明是有限的 蛋白质的部分原因是不能在异源宿主中表达足够数量的功能蛋白质。 哺乳动物膜蛋白(MMPs)在功能构象中特别难过度表达 由于其复杂的折叠和加工模式以及倾向于 以展开的和非功能性的形式积累。例如，四种生物医学上重要的基质金属蛋白酶： 5-羟色胺转运体(SERT)、神经降压素受体(NTR)、间充质-上皮转化因子(MET) 受体和电压敏感型钠通道(Nav1.7)都有复杂的翻译后加工 要求。当蛋白质在宿主中表达时，通常会出现这些MMPs的最佳表达 在进化上最接近靶标基质金属蛋白酶。事实上，NTR和SERT以其最高功能表达 哺乳动物宿主中每个细胞的水平。然而，哺乳动物和其他脊椎动物细胞系 由于其总的基质金属蛋白酶产量较低，仅为0.05 mg/L或更低，因此使用较少。如果MMP的收益率可以是 增加一个数量级或更多，将使用哺乳动物(和其他脊椎动物)培养系统 更广泛地归因于它们将复杂的基质金属蛋白酶结构靶点加工成功能性 形式。培养要求将从数百升减少到数十升。这一提议的一个假设是 哺乳动物膜蛋白序列的性质和当前宿主的处理效率 这使得在异源宿主中高水平表达功能蛋白成为问题。非常低级的自然 一些复杂的MMPs的丰富已经最小化了自然进化的表达压力和有限的 需要将其对宿主的毒性降至最低。因此，目前提议的项目的目标是开发 通过以下两个目标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改变结构或功能的情况下提高表达的氨基酸残基 重要的2)通过诱变来设计和进化哺乳动物宿主，以产生能够 更高效地处理复杂的MMP。将实施“就地”进化和选择技术， 允许用户在体细胞高度突变的B细胞宿主中突变靶MMPs的序列。同时， 哺乳动物生产宿主将被改造成抗凋亡蛋白、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 可通过抑制细胞的天然DNA修复机制来进行。这一方法将在四个方面进行测试 上述基质金属蛋白酶指标代表不同类别的基质金属蛋白酶，与多种疾病有关。这个 突变的蛋白质变体将在工程和进化的哺乳动物细胞系中表达，以便 将正在进行的结晶学项目的活性产品产量提高10到20倍。该项目将提供一个 基于蛋白质和细胞进化策略的表达高产量复杂MMPs的新范例。

项目成果

Thermostabilisation of the serotonin transporter in a cocaine-bound conformation.

• DOI: 10.1016/j.jmb.2013.03.025
• 发表时间: 2013-06-26
• 期刊: JOURNAL OF MOLECULAR BIOLOGY
• 影响因子: 5.6
• 作者: Abdul-Hussein, Saba;Andrell, Juni;Tate, Christopher G.
• 通讯作者: Tate, Christopher G.

MiRNA mimic screen for improved expression of functional neurotensin receptor from HEK293 cells.

• DOI: 10.1002/bit.25567
• 发表时间: 2015-08
• 期刊: BIOTECHNOLOGY AND BIOENGINEERING
• 影响因子: 3.8
• 作者: Xiao, Su;Chen, Yu-Chi;Betenbaugh, Michael J.;Martin, Scott E.;Shiloach, Joseph
• 通讯作者: Shiloach, Joseph

Directed evolution of mammalian anti-apoptosis proteins by somatic hypermutation.

通过体细胞超突变定向进化哺乳动物抗凋亡蛋白。

• DOI: 10.1093/protein/gzr052
• 发表时间: 2012
• 期刊: Protein engineering, design & selection : PEDS
• 作者: Majors,BrianS;Chiang,GiselaG;Pederson,NelsE;Betenbaugh,MichaelJ
• 通讯作者: Betenbaugh,MichaelJ

Engineering cells to improve protein expression.

改造细胞以改善蛋白质表达。

• DOI: 10.1016/j.sbi.2014.03.005
• 发表时间: 2014
• 期刊: Current opinion in structural biology
• 影响因子: 6.8
• 作者: Xiao,Su;Shiloach,Joseph;Betenbaugh,MichaelJ
• 通讯作者: Betenbaugh,MichaelJ