Protein Purification Core

蛋白质纯化核心

• 批准号: 10262740
• 负责人: Joseph Tropea
• 金额: $ 112.94万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262740
• 关键词: Affinity Chromatography; Amino Acids; Asparagine; Bacteria; Baculovirus Expression System; Baculoviruses; Baltimore; Beta-N-Acetylglucosaminidase; Binding Proteins; Biotin; C-terminal; CCR; Catalytic Domain; Cell Membrane Permeability; Cells; Chimeric Proteins; Chromatography; Circular Dichroism Spectroscopy; Cleaved cell; Cloning; Collaborations; Collection; Complement; Crystallization; Crystallography; DNA cassette; Drosophila genus; Dyes; Endoglycosidase F; Enzymes; Formulation; Germany; Glycoproteins; Goals; Hand; Heteronuclear NMR; Histidine; Human; Human Resources; Hydrolase; Hydrophobic Interactions; Hydroxyapatites; In Vitro; Insecta; Institutes; Ion Exchange; Isoelectric Focusing; Isotopes; Laboratories; Lectin; Link; Maryland; Mass Spectrum Analysis; Membrane; Methods; Mission; Molecular Chaperones; Molecular Sieve Chromatography; Mutation; N-terminal; Oligosaccharides; Oxidation-Reduction; Peptide Hydrolases; Peptides; Plasmid Cloning Vector; Plasmids; Positioning Attribute; Potyvirus; Preparation; Procedures; Process; Production; Productivity; Protein Disulfide Isomerase; Protein Engineering; Proteins; Proteolysis; Reagent; Recombinant Proteins; Recombinants; Research Personnel; Research Support; Rhinovirus; Scheme; Selenomethionine; Series; Side; Site; Structure; System; TEV protease; TXN gene; Techniques; Technology; Testing; Time; Tobacco; Transfer RNA; Universities; Veins; Virus; Work; amidase; analytical ultracentrifugation; biophysical chemistry; design; experience; experimental study; expression vector; gel electrophoresis; light scattering; mRNA Stability; maltose-binding protein; medical schools; new technology; protein expression; protein purification; structural biology; tool; vector

The staff of the Protein Purification Core (PPC) use a number of techniques for successful protein production. The PPC has access to a wide variety of tools for the expression of recombinant protein in bacteria (our primary expression system), including many types of plasmid expression vectors and specialized bacterial strains. There is a rather large collection of strains to choose from, with genetic defects that influence proteolytic activity, mRNA stability, membrane permeability, and intracellular redox potential. In addition, there are strains that overproduce protein disulfide isomerase, molecular chaperones, transfer RNAs, and redox enzymes for co-expression with target proteins. There is an equally large and diverse collection of bacterial plasmid vectors for recombinant protein expression. Many of these use Gateway cloning technology (Thermo Fisher Scientific), making them quick and easy to use. The PPC staff has experience with all of the major regulatory systems (e.g., T7, tac, pBAD, trc, lambda PL, etc.) and various formats for the production of recombinant proteins (untagged or fused to MBP, GST, NusA, thioredoxin, Sumo, His-tag, Arg-tag, FLAG-tag, biotin acceptor peptide, Strep Tag II, etc.) to make full use of these reagents. The PPC has also established an insect cell protein production facility to compliment its bacterial production capability, using both the Bac-to-Bac baculovirus expression system (Invitrogen) and the Drosophila expression system (Thermo Fisher Scientific). Like most bacterial production, the insect cell facility utilize Gateway cloning technology to maximize productivity. The PPC plans to evaluate other expression systems as time permits. One of these, the flashBAC baculovirus expression system of Oxford Expression Technologies, is on the list. Indications are that this system may be valuable for secretory and membrane-bound protein production, and may be a good complement to our current insect cell expression systems. Similarly, in collaboration with the Protein Engineering Core of the Macromolecular Crystallography Laboratory (formally the Protein Engineering Section), the PPC has evaluated a new series of baculovirus expression cassettes designed to enhanced secretion of recombinant proteins from baculovirus-infected insect cells. This study is ongoing and demonstrates that the new expression cassettes work remarkably well. The PPC will incorporated this new technology into our insect cell protein production tool kit. The PPC personnel are experienced with all standard chromatography techniques required for protein purification. The core maintains a full array of supplies necessary for ion exchange, hydrophobic interaction, lectin, hydroxyapatite, dye, size exclusion, and affinity chromatography. Materials for IMAC and chromatofocusing are also on hand. In addition to purification technology, the staff is very knowledgeable of methods required to characterize recombinant protein products. Among those used are gel electrophoresis and isoelectric focusing, mass spectroscopy, western analysis, N-terminal sequencing, dynamic light scattering and analytical ultracentrifugation, and circular dichroism spectroscopy. For structural studies, the PPC has in place standard operating procedures for the production of isotopically enriched proteins for heteronuclear nuclear magnetic resonance experiments and selenomethionine-substituted proteins for crystallography. Methods have been established for bacteria that eliminate the need to change strain by manipulating the medium formulation and induction parameters, and produce recombinant protein at levels equivalent to wild-type expression. For those proteins that fail to crystallize, the core can perform limited proteolysis as a way to identify potential structural domains, providing the Macromolecular Crystallography Laboratory investigator additional avenues for structural studies. This method has been extensively used both analytically, and on a preparative scale to produce structural domains that can be purified using conventional chromatography. The core produces and maintains 5 different kinds of tobacco etch virus (TEV) protease (catalytic domain) that are used by the Macromolecular Crystallography Laboratory (and others in the CCR) for in vitro cleavage of fusion proteins that contain an intervening protease recognition sequence. Available are an N-terminal heptahistidine-tagged TEV protease, an untagged TEV protease, an N-terminal heptahistidine-tagged TEV protease with a mutation that relaxes the amino acid requirement at the P1' position in the protease recognition sequence, an N-terminal and C-terminal histidine-tagged TEV protease, and a maltose binding protein-TEV protease fusion protein. All contain a mutation that minimizes autoinactivation. Each has its advantage depending on the design of the protein purification scheme. Similarly, the core produces and maintains two types of tobacco vein mottling virus (TVMV) protease (catalytic domain) also used for in vitro cleavage of fusion proteins. These are available as an N-terminal hexahistidine-tagged TVMV protease and an untagged TVMV protease. The protease recognition site is different from the TEV protease site and allows the use of both recognition sequences in a single fusion protein. As an alternative to these potyvirus proteases, the PPC has purified the human rhinovirus 3C protease (i.e., PreScission protease) using a bacterial expression plasmid obtained from Arie Geerlof (Helmholtz Center Munich, Institute of Structural Biology, Neuherberg, Germany). This protease has good activity even at 4C and will be quite useful in cleaving fusion proteins produced in several commercial vectors such as the pGEX-P series, pTriEx-9 and pET-47b(+). In addition, the PPC has also purified the amidase Peptide-N-Glycosidase F and the hydrolases endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F1 using bacterial expression plasmids obtained from Daniel Leahy (Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland). These enzymes will be invaluable at removing asparagine-linked oligosaccharide side chains from glycoproteins produced by our insect cell production facility, which often impede the crystallization process. Plans to expand our repertoire to include other glycosylhydrolases is underway as time permits. Recently the PPC has obtained from the Protein Engineering Core a series of expression plasmids producing a number of DARPins in various formats. These particular DARPins are specific for MBP and have been shown to enhance crystallization of MBP fusion proteins. In addition, an expression plasmid producing an MBP-specific monobody has been obtained (also from Protein Engineering Core) which similarly enhances crystallization of MBP fusion proteins. We have completed the purification of three MBP-specific DARPins and are in the process of purifying the MBP-specific monobody. Once tested, the proteins will be available to all in the Macromolecular Crystallography Laboratory for use. For FY2020, as part of our research support to the Macromolecular Crystallography Laboratory, the PPC has completed 24 cloning projects and performed 41 protein purifications. In addition 18 insect cell protein productions at the pilot and preparative levels were completed.

蛋白质纯化核心（PPC）的人员使用多种技术来成功生产蛋白质。 PPC可以访问细菌（我们的主要表达系统）中重组蛋白表达的多种工具，包括许多类型的质粒表达载体和专门的细菌菌株。有很多可供选择的菌株，具有影响蛋白水解活性，mRNA稳定性，膜渗透性和细胞内氧化还原电位的遗传缺陷。此外，还有一些菌株使蛋白质二硫化物异构酶，分子伴侣，转移RNA和氧化还原酶过量产生菌株，以与靶蛋白共表达。有同样大的细菌质粒载体收集，用于重组蛋白表达。其中许多使用网关克隆技术（Thermo Fisher Scientific），使其易于使用。 The PPC staff has experience with all of the major regulatory systems (e.g., T7, tac, pBAD, trc, lambda PL, etc.) and various formats for the production of recombinant proteins (untagged or fused to MBP, GST, NusA, thioredoxin, Sumo, His-tag, Arg-tag, FLAG-tag, biotin acceptor peptide, Strep Tag II, etc.) to make full use这些试剂。 PPC还使用BAC-TO-BAC杆状病毒表达系统（Invitrogen）和果蝇表达系统（Thermo Fisher Scientific）建立了一种昆虫细胞蛋白生产设施，以补充其细菌生产能力。像大多数细菌生产一样，昆虫细胞设施利用门户克隆技术来最大化生产率。 PPC计划根据时间允许评估其他表达系统。其中之一是牛津表达技术的Flashbac杆状病毒表达系统。迹象表明，该系统可能对分泌和膜结合的蛋白质产生很有价值，并且可能是我们当前的昆虫细胞表达系统的良好补充。同样，PPC与大分子晶体学实验室的蛋白质工程核心（正式的蛋白质工程部门）合作，评估了一系列新的杆状病毒表达盒，旨在增强细菌病毒感染昆虫细胞的重组蛋白的分泌。这项研究正在进行中，并表明新的表达盒效果非常好。 PPC将将这项新技术纳入我们的昆虫细胞蛋白生产工具套件。 PPC人员具有蛋白质纯化所需的所有标准色谱技术经验。核心保持离子交换，疏水相互作用，凝集素，羟基磷灰石，染料，大小排除和亲和色谱所需的全部供应。 iMac和色谱的材料也在手头上。除了纯化技术外，员工还非常了解重组蛋白质产品所需的方法。其中包括凝胶电泳和等电聚焦，质谱，西方分析，N末端测序，动态光散射和分析性超速离心以及循环二分法光谱。对于结构研究，PPC具有标准操作程序，用于生产用于异位核能核磁共振实验的同位素富集蛋白和用于晶体学的硒甲米甲氨基氨基氨基化蛋白。已经为细菌建立了方法，从而消除了通过操纵培养基制剂和诱导参数来改变应变的需求，并在等于野生型表达的水平下产生重组蛋白。对于那些无法结晶的蛋白质，核心可以执行有限的蛋白水解作为识别潜在结构域的一种方式，从而为大分子晶体学实验室研究者提供了其他用于结构研究的途径。该方法已被广泛地分析，并且在制备量表上可以产生可以使用常规色谱纯化的结构结构域。核心产生并维持5种不同类型的烟草蚀刻病毒（TEV）蛋白酶（催化结构域），这些蛋白酶（催化结构域）被大分子晶体学实验室（以及CCR中的其他）用于体外裂解融合蛋白，这些融合蛋白包含一个间间蛋白酶识别的融合蛋白。可用的是N端七热碱标记的TEV蛋白酶，一个未加入的TEV蛋白酶，N端七个末端的TEV TEV蛋白酶，其在蛋白酶的蛋白酶识别序列中的P1位置放宽了氨基酸的需求，是N-所有这些都包含一个最小化自动活化的突变。每个都有其优势，具体取决于蛋白质纯化方案的设计。同样，核心产生并维持两种类型的烟静脉斑点病毒（TVMV）蛋白酶（催化域）也用于融合蛋白的体外切割。这些可作为N端六个末期标记的TVMV蛋白酶和未标记的TVMV蛋白酶使用。蛋白酶识别位点与TEV蛋白酶位点不同，并允许在单个融合蛋白中使用两个识别序列。作为这些托管病毒蛋白酶的替代方法，PPC使用从Arie Geerlof获得的细菌表达质粒（Helmholtz Center Munich，Neuherberg，Neuherberg，Germany）纯化了人类鼻病毒3C蛋白酶（即预缩蛋白酶）。该蛋白酶的活性即使在4C时也具有良好的活性，并且在裂解几个商业载体（例如PGEX-P系列Ptriex-9和PET-47B（+））中产生的融合蛋白非常有用。 In addition, the PPC has also purified the amidase Peptide-N-Glycosidase F and the hydrolases endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F1 using bacterial expression plasmids obtained from Daniel Leahy (Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore,马里兰州）。这些酶从我们的昆虫细胞生产设施产生的糖蛋白中去除天冬酰胺连接的寡糖侧链方面将是无价的，这通常会阻碍结晶过程。在时间允许的情况下，正在进行扩展我们的曲目以包括其他糖基水解酶的计划。最近，PPC从蛋白质工程核心获得了一系列以各种形式产生多种DARPIN的表达质粒。这些特殊的DARPIN是针对MBP的，已被证明可以增强MBP融合蛋白的结晶。另外，已经获得了产生MBP特异性单体的表达质粒（也来自蛋白质工程核），该质粒类似地增强了MBP融合蛋白的结晶。我们已经完成了三个MBP特异性DARPIN的纯化，并正在纯化MBP特异性单体。一旦测试，蛋白质将用于大分子晶体学实验室中的所有蛋白质。对于2020财年，作为我们对大分子晶体学实验室的研究支持的一部分，PPC完成了24个克隆项目，并进行了41种蛋白质净化。另外，在飞行员和制备水平上的18种昆虫细胞蛋白产物完成。