公开(公告)号：US20080248565A1

公开(公告)日：2008-10-09

申请号：US12040798

申请日：2008-02-29

Applicant: Federico Katzen ,  Julia Fletcher ,  Wieslaw Kudlicki ,  Joseph Beechem ,  Lilin Wang

Inventor： Federico Katzen ,  Julia Fletcher ,  Wieslaw Kudlicki ,  Joseph Beechem ,  Lilin Wang

IPC: C12N5/06 ,  C07K14/00

CPC classification number: C12P21/02 ,  C07K1/02

Abstract: Systems and methods are provided for producing a protein of interest that is typically not amenable to expression in soluble form in in vitro expression systems. In some aspects, the invention provides methods of synthesizing proteins using in vitro protein synthesis systems that include a scaffold protein such as apolipoprotein or an amphipathic alpha helix containing (“AAHC”) protein, in which higher yields of soluble protein are produced than in the absence of the scaffold protein. The scaffold proteins may be provided in an in vitro protein synthesis system associated with lipid or not associated with lipid. The scaffold protein may be provided as a protein per se or may be encoded by a nucleic acid template and co-expressed with the protein of interest. The invention also provides compositions and kits for synthesis of proteins in soluble form, in which the compositions and kits include cell extracts for protein expression and isolation.

Abstract translation: 提供的系统和方法用于产生通常不适于在体外表达系统中以可溶形式表达的目的蛋白质。 在一些方面，本发明提供了使用体外蛋白质合成系统合成蛋白质的方法，所述体外蛋白质合成系统包括诸如载脂蛋白或含有（“AAHC”）蛋白质的两亲性α螺旋的支架蛋白，其中产生比在 缺少支架蛋白。 支架蛋白质可以提供在与脂质相关的体外蛋白质合成系统中或与脂质不相关。 支架蛋白质可以作为蛋白质本身提供，或者可以由核酸模板编码并与感兴趣的蛋白质共表达。 本发明还提供用于合成可溶形式的蛋白质的组合物和试剂盒，其中组合物和试剂盒包括用于蛋白质表达和分离的细胞提取物。

公开(公告)号：US07282339B2

公开(公告)日：2007-10-16

申请号：US10943463

申请日：2004-09-17

Applicant: Joseph Beechem ,  Kyle Richard Gee ,  David Carl Hagen ,  Iain D Johnson ,  Hee-Chol Kang ,  Christina Pastula

Inventor： Joseph Beechem ,  Kyle Richard Gee ,  David Carl Hagen ,  Iain D Johnson ,  Hee-Chol Kang ,  Christina Pastula

IPC: G01N33/53 ,  G01N33/532 ,  G01N33/533 ,  C12Q1/42 ,  C07K1/13

CPC classification number: G01N33/542 ,  C07F9/12 ,  C07F9/65318 ,  C07F9/65522 ,  C07F9/6596 ,  C07K16/46 ,  Y10S435/968 ,  Y10S436/80 ,  Y10S530/807 ,  Y10T436/13

Abstract: The present invention provides ligand-detection reagents, ligand analogs and methods for determining the presence of a ligand in a sample. The ligand-detection reagent comprises a ligand-binding antibody and a ligand analog to form an antibody-ligand analog complex wherein the ligand analog is covalently bonded to a reporter molecule. This complex may additionally comprise a labeling protein non-covalently bonded to the antibody to form a ternary complex wherein the labeling protein comprises a monovalent antibody fragment or a non-antibody protein that is covalently bonded to a label moiety. The reporter molecule is either quenched by the ligand-binding antibody or by the label moiety of the labeling protein, depending on the reporter molecule and the ligand-binding antibody, wherein the amount of quenching is directly related to the amount of ligand present in the sample. Alternatively, the ligand analog is fluorogenic wherein the ligand analog is essentially non-fluorescent in solution but when bound by the ligand-binding antibody the detectable signal increases. In this instance a decrease in signal, as opposed to the relieving of quenching, is measured for the presence of a target ligand.

Abstract translation: 本发明提供配体检测试剂，配体类似物和用于测定样品中配体存在的方法。 配体检测试剂包含配体结合抗体和配体类似物以形成抗体 - 配体类似物复合物，其中配体类似物共价键合到报告分子。 该复合物还可以包含与抗体非共价键合以形成三元复合物的标记蛋白质，其中标记蛋白质包含共价键合到标记部分的单价抗体片段或非抗体蛋白质。 取决于报道分子和配体结合抗体，报告分子被配体结合抗体或标记蛋白的标记部分淬灭，其中淬灭量与存在于 样品。 或者，配体类似物是荧光的，其中配体类似物在溶液中基本上是非荧光的，但当被配体结合抗体结合时，可检测信号增加。 在这种情况下，与目标配体的存在相比，测量了与消除淬灭相反的信号降低。

公开(公告)号：US07102005B2

公开(公告)日：2006-09-05

申请号：US10428192

申请日：2003-05-02

Applicant: Brian Agnew ,  Joseph Beechem ,  Kyle Gee ,  Richard Haugland ,  Jixiang Liu ,  Vladimir Martin ,  Wayne Patton ,  Thomas Steinberg

Inventor： Brian Agnew ,  Joseph Beechem ,  Kyle Gee ,  Richard Haugland ,  Jixiang Liu ,  Vladimir Martin ,  Wayne Patton ,  Thomas Steinberg

IPC: C07D239/88 ,  C07D277/62 ,  C07D311/82 ,  C07D417/02 ,  C07D495/04

CPC classification number: G01N33/50 ,  G01N33/5008 ,  G01N33/502 ,  G01N33/5091 ,  G01N2333/4709

Abstract: The present invention relates to phosphate-binding compounds that find use in binding, detecting and isolating phosphorylated target molecules including the subsequent identification of target molecules that interact with phosphorylated target molecules or molecules capable of being phosphorylated. A binding solution is provide that comprises a phosphate-binding compound, an acid and a metal ion wherein the metal ion simultaneously interacts with an exposed phosphate group on a target molecule and the metal chelating moiety of the phosphate-binding compound forming a bridge between the phosphate-binding compound and a phosphorylated target molecule resulting in a ternary complex. The binding solution of the present invention finds use in binding and detecting immobilized and solubilized phosphorylated target molecules, isolation of phosphorylated target molecules from a complex mixture and aiding in proteomic analysis wherein kinase and phosphatase substrates and enzymes can be identified.

Abstract translation: 本发明涉及用于结合，检测和分离磷酸化靶分子的磷酸盐结合化合物，包括随后鉴定与磷酸化靶分子或能够被磷酸化的分子相互作用的靶分子。 提供的结合溶液包括磷酸盐结合化合物，酸和金属离子，其中金属离子同时与靶分子上暴露的磷酸基团相互作用，并且磷酸盐结合化合物的金属螯合部分形成在 磷酸化结合化合物和导致三元复合物的磷酸化靶分子。 本发明的结合溶液用于结合和检测固定和溶解的磷酸化靶分子，从复杂混合物中分离磷酸化靶分子，并辅助蛋白质组分析，其中可以鉴定激酶和磷酸酶底物和酶。

公开(公告)号：US08603792B2

公开(公告)日：2013-12-10

申请号：US12748355

申请日：2010-03-26

Applicant: Theo Nikiforov ,  Daniel Mazur ,  Xinzhan Peng ,  Tommie Lloyd Lincecum, Jr. ,  Yuri Belosludtsev ,  Howard Reese ,  Dmitriy Gremyachinskiy ,  Roman Rozhkov ,  John M. Mauro ,  Joseph Beechem ,  Eric Tulsky ,  Imad Naasani ,  Kari Haley ,  Joseph A. Treadway

Inventor： Theo Nikiforov ,  Daniel Mazur ,  Xinzhan Peng ,  Tommie Lloyd Lincecum, Jr. ,  Yuri Belosludtsev ,  Howard Reese ,  Dmitriy Gremyachinskiy ,  Roman Rozhkov ,  John M. Mauro ,  Joseph Beechem ,  Eric Tulsky ,  Imad Naasani ,  Kari Haley ,  Joseph A. Treadway

IPC: C12N9/12

CPC classification number: C12Q1/6869 ,  C07H19/20 ,  C12N9/1241 ,  C12N9/1252 ,  C12N9/96 ,  C12Q1/6818 ,  C12Y207/07 ,  C12Y207/07007 ,  G01N21/6428 ,  G01N33/582 ,  G01N2021/6432

Abstract: Disclosed herein are conjugates comprising a biomolecule linked to a label that have biological activity and are useful in a wide variety of biological applications. For example, provided herein are polymerase-nanoparticle conjugates including a polymerase linked to a nanoparticle, wherein the conjugate has polymerase activity. Such conjugates can exhibit reduced aggregation and improved stochiometries wherein the average biomolecule:nanoparticle ratio approaches or equals 1:1. Also disclosed herein are improved methods for preparing such conjugates, and methods and systems for using such conjugates in biological applications such as nucleotide incorporation, primer extension and single molecule sequencing.

Abstract translation: 本文公开了包含连接到具有生物活性并可用于多种生物应用的标签的生物分子的缀合物。 例如，本文提供了聚合酶 - 纳米颗粒缀合物，其包括与纳米颗粒连接的聚合酶，其中缀合物具有聚合酶活性。 这样的缀合物可以表现出减少的聚集和改进的沉淀物，其中平均生物分子：纳米颗粒比接近或等于1:1。 本文还公开了制备这种缀合物的改进方法，以及在生物应用中使用这种缀合物的方法和系统，例如核苷酸掺入，引物延伸和单分子测序。

公开(公告)号：US08535894B2

公开(公告)日：2013-09-17

申请号：US13610009

申请日：2012-09-11

Applicant: Robert Archer ,  Richard Haugland ,  Rosaria Haugland ,  Joseph Beechem ,  David Hagen

Inventor： Robert Archer ,  Richard Haugland ,  Rosaria Haugland ,  Joseph Beechem ,  David Hagen

IPC: G01N33/53

CPC classification number: B82Y5/00 ,  B82Y10/00 ,  G01N33/53

Abstract: Provided are labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents. The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label. Discrete subsets of labeling reagent and immune-labeled complexes are provided that facilitate the simultaneous detection of multiple targets in a sample-complexes are distinguished by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody. This is particularly useful for fluorophore labels that can be attached to labeling reagents and subsequently immuno-labeled complexes in ratios for the detection of multiple targets.

Abstract translation: 提供标记试剂和用于标记一抗的标记试剂和用于使用包含靶结合抗体和一种或多种标记试剂的免疫标记复合物检测样品中的靶标的方法。 标记试剂包括单价抗体片段或非抗体单体蛋白质，由此标记的蛋白质对靶标结合抗体的特定区域具有亲和力，并且共价连接到标记物上。 提供标记试剂和免疫标记复合物的离散子集，其有利于同时检测样品中多个靶标的复合物，其特征在于i）标记与标记试剂的比例，或ii）所述标记的物理性质，或iii ）标记试剂与所述靶结合抗体的比例，或iv）所述靶结合抗体。 这对于可以连接到标记试剂和随后的用于检测多个靶的比例的免疫标记的复合物的荧光团标记物特别有用。

公开(公告)号：US20120329042A1

公开(公告)日：2012-12-27

申请号：US13562159

申请日：2012-07-30

Applicant: Joseph BEECHEM ,  Theo NIKIFOROV ,  Vi-En CHOONG ,  Xinzhan PENG ,  Guobin LUO ,  Cheng-Yao CHEN ,  Michael PREVITE

Inventor： Joseph BEECHEM ,  Theo NIKIFOROV ,  Vi-En CHOONG ,  Xinzhan PENG ,  Guobin LUO ,  Cheng-Yao CHEN ,  Michael PREVITE

IPC: G01N21/64

CPC classification number: C12Q1/6869 ,  C07H19/20 ,  C12N9/1241 ,  C12N9/1252 ,  C12N9/96 ,  C12Q1/6818 ,  C12Y207/07 ,  C12Y207/07007 ,  G01N21/6428 ,  G01N33/582 ,  G01N2021/6432

Abstract: Provided herein are systems and methods for nucleotide incorporation reactions. The systems comprise polymerases having altered nucleotide incorporation kinetics and are linked to an energy transfer donor moiety, and nucleotide molecules linked with at least one energy transfer acceptor moiety. The donor and acceptor moieties undergo energy transfer when the polymerase and nucleotide are proximal to each other during nucleotide binding and/or nucleotide incorporation. As the donor and acceptor moieties undergo energy transfer, they generate an energy transfer signal which can be associated with nucleotide binding or incorporation. Detecting a time sequence of the generated signals, or the change in the signals, can be used to determine the order of the incorporated nucleotides, and can therefore be used to deduce the sequence of the target molecule.

Abstract translation: 本文提供了用于核苷酸掺入反应的系统和方法。 该系统包含具有改变的核苷酸掺入动力学并且与能量转移供体部分连接的聚合酶，以及与至少一个能量转移受体部分连接的核苷酸分子。 当核苷酸结合和/或核苷酸掺入期间聚合酶和核苷酸彼此接近时，供体和受体部分进行能量转移。 当供体和受体部分进行能量转移时，它们产生可与核苷酸结合或掺入相关联的能量转移信号。 检测所产生的信号的时间序列或信号的变化可用于确定掺入的核苷酸的顺序，因此可用于推导靶分子的序列。

公开(公告)号：US20110311963A1

公开(公告)日：2011-12-22

申请号：US13049660

申请日：2011-03-16

Applicant: W. MICHAEL LAFFERTY ,  JOSEPH BEECHEM ,  HONGYE SUN ,  MICHAEL METZKER

Inventor： W. MICHAEL LAFFERTY ,  JOSEPH BEECHEM ,  HONGYE SUN ,  MICHAEL METZKER

IPC: C12Q1/68

CPC classification number: C12Q1/6874 ,  C12Q2523/319 ,  C12Q2525/186 ,  C12Q2565/513

Abstract: A method of sequencing a plurality of template nucleotide sequences includes immobilizing the plurality of template nucleotide sequences on a substrate. A first subset of the plurality of template nucleotide sequences is immobilized in a first field of view and a second subset of the plurality of template nucleotide sequences is immobilized in a second field of view. The first and second subsets are hybridized to a caged primer. The caged primer includes a caging group. The method further includes lysing the caging group from the caged primer in the first field of view and observing the first field of view to detect sequencing of the first subset of the plurality of template nucleotide sequences.

Abstract translation: 对多个模板核苷酸序列进行测序的方法包括将多个模板核苷酸序列固定在底物上。 多个模板核苷酸序列的第一子集被固定在第一视场中，并且多个模板核苷酸序列的第二子集被固定在第二视野中。 第一和第二子集与笼式引物杂交。 笼状引物包括笼养组。 该方法还包括在第一视场中从笼式引物裂解笼基组，并观察第一视野以检测多个模板核苷酸序列的第一子集的测序。

公开(公告)号：US20110236983A1

公开(公告)日：2011-09-29

申请号：US12980817

申请日：2010-12-29

Applicant: Joseph Beechem ,  Theofilos Kotseroglou ,  William Michael Lafferty ,  Mark F. Oldham

Inventor： Joseph Beechem ,  Theofilos Kotseroglou ,  William Michael Lafferty ,  Mark F. Oldham

IPC: G01N33/50 ,  G01N21/64 ,  B82Y99/00

CPC classification number: G01N21/6408 ,  C12Q1/6818 ,  G01N21/6428 ,  G01N21/6458 ,  G01N2021/6441 ,  Y10T436/143333 ,  C12Q2563/173 ,  C12Q2563/155 ,  C12Q2561/12

Abstract: A nucleic acid detection system and method are provided, in which excitation energy is transmitted from a pulsed excitation source to a reaction site including a fluorescence resonance energy transfer (FRET)-based dye system to generate a fluorescent signal at the reaction site, the fluorescent signal is detected by a detector from the reaction site, and detection of the fluorescent signal is respectively blocked and permitted at the detector by a detector gate this is timed based on an emission start time of the transmitted excitation energy.

Abstract translation: 提供了一种核酸检测系统和方法，其中激发能量从脉冲激发源传输到包括基于荧光共振能量转移（FRET）的染料系统的反应位点，以在反应位点产生荧光信号，荧光 信号被来自反应部位的检测器检测，并且荧光信号的检测分别被检测器门阻断并在检测器处允许，其基于发射的激发能量的发射开始时间来定时。

公开(公告)号：US20110200989A1

公开(公告)日：2011-08-18

申请号：US13009442

申请日：2011-01-19

Applicant: Gordon A. Janaway ,  Christina E. Inman ,  Joseph Beechem

Inventor： Gordon A. Janaway ,  Christina E. Inman ,  Joseph Beechem

IPC: C12Q1/68 ,  C12M1/34 ,  G01N21/75 ,  G01N21/63 ,  G01N21/64

CPC classification number: G01N21/6452 ,  C12Q1/6818 ,  C12Q1/6869 ,  G01N21/6428 ,  G01N2021/6441 ,  Y10T436/143333 ,  C12Q2563/131 ,  C12Q2523/313

Abstract: A system for detection of nucleic acids can include an excitation source configured to transmit excitation energy to a reaction site including a single molecule of nucleic acid reacted with a two-photon absorption moiety. The system also can include an optical system configured to focus the excitation energy transmitted from the excitation source to a focal region containing the reaction site, wherein said excitation energy within the focal region is sufficient to cause two-photon absorption by the two-photon absorption moiety. The system can further include a detector configured to detect emissions generated at the reaction site resulting from two-photon absorption of the excitation energy by the two-photon absorption moiety.

Abstract translation: 用于检测核酸的系统可以包括被配置为将激发能量传递到包括与双光子吸收部分反应的单分子核酸的反应位点的激发源。 该系统还可以包括配置成将从激发源传输的激发能量聚焦到包含反应位点的聚焦区域的光学系统，其中聚焦区域内的所述激发能足以通过双光子吸收引起双光子吸收 部分。 该系统还可以包括检测器，其被配置为检测由双光子吸收部分的激发能量的双光子吸收引起的在反应位置产生的发射。

公开(公告)号：US20090005267A1

公开(公告)日：2009-01-01

申请号：US12041482

申请日：2008-03-03

Applicant: Bradley Love ,  Brian Dwyer ,  George Klee ,  Joseph Beechem ,  Ravinder Singh ,  Stefan Grebe

Inventor： Bradley Love ,  Brian Dwyer ,  George Klee ,  Joseph Beechem ,  Ravinder Singh ,  Stefan Grebe

IPC: C40B40/10 ,  G01N33/53 ,  G01N33/536 ,  G01N33/543 ,  G06F19/00 ,  G01N33/545 ,  G01N33/544

CPC classification number: G01N33/743 ,  G01N33/6854

Abstract: The present disclosure provides an immunoassay involving a multiplex of antibodies that recognize the same analyte but that have a different cross-reactivity to structurally similar compounds. Data obtained from the immunoassay involving observed analyte concentrations is input into an algorithm to determine the true concentration of the analyte in a sample.

Abstract translation: 本公开提供了涉及识别相同分析物但具有与结构上类似化合物不同的交叉反应性的抗体多重化的免疫测定。 从包括观察到的分析物浓度的免疫测定中获得的数据被输入到算法中以确定样品中分析物的真实浓度。