We thank Tomer Zohar for work on scFv screening and characterization assays. A portion of this work was supported by award number F32CA150622 from the National Cancer Institute (to AJK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

<ol>
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The authors declare that they have no competing financial interests.

工程抗体的细胞质活动是一个艰巨的任务，由于细胞质，阻碍稳定的二硫键6,7-形成的还原环境。这会导致最抗体，除非它们被设计为在细胞质中的稳定性和溶解度，除了被设计为结合亲和力是细胞质无效。噬菌体展示，细菌表面展示，酵母表面展示方法的现有方法都使用分泌途径14-16工程抗体的显示，但这些方法都没有办法工程师胞内折叠。因为Tat途径的折叠质量控制阻止了不良折叠并在细胞质中不稳定的抗体的转用内膜显示工程化抗体具有改善的细胞质的稳定性和溶解性。此方法简化了工程细胞内抗体的迭代过程对亲和性的第二溶解度，因为这两个性质在一个步骤工程改造。虽然这种方法被设计用于工程抗体与降低细胞内环境的溶解度，它也可以适用于工程抗体在非还原条件下发挥作用，因为蛋白质使用这种方法维持其在周质的氧化环境的折叠设计。 尽管该技术简化了工程以高亲和力和高细胞质溶解度抗体的过程中，一些限制是重要的使用该协议时要考虑的。在分析了二次筛选的ELISA信号，以确定有前途的单链抗体变体，潜在的有趣的变体和那些可能不会出现足够的抗原结合之间挑剔的门槛并不容易，直到经过多次克隆得到了进一步的特点是显而易见的。重要的是要寻找改良过的亲本抗体结合是很重要的;然而，异常高的信号可以是指示亲和力29或聚集效应30，其不是唯一的内膜显示筛选方法的挑战。要记住一个关键限制使用此协议时，是无法平移后的恢复原生质球，因为他们都是非可行的（未公布数据）。这就需要DNA扩增和改造步骤来恢复编码抗体的质粒。 该协议的几个关键步骤，使折叠和抗体结合的同步工程。用于筛选是成功的，所述scFv文库被筛选必须表达为融合到ssTorA信号肽。没有该序列中，抗体将不被引导到Tat途径，因而将不被转运到周质19。另外，至关重要的是，C-末端表位标签融合至抗体以允许在bin显示抗体的检测鼎检测。显然， 大肠杆菌大肠杆菌菌株用于表达的scFv还必须有必要的Tat途径机械，但是这是在通常使用的大肠杆菌的真大肠杆菌菌株。 修改这个协议是可能的，以改善其电势以分离具有所需特性的抗体。减色层叠步骤可以淘洗针对靶抗原耗尽非所需成分的抗体库之前完成。库球状体，可以单独用或涂覆涂覆有BCCP用非所需的蛋白的磁珠孵育，并结合于那些珠的球状体可筛选其余未结合的球状体结合于期望的目标之前被丢弃。如在代表结果所提到的，一个方法来改善的分离的scFv的亲和力是包括在平移反应的可溶性竞争者与对球状体显示的scFv竞争。由于可溶性补偿etitor是纯化的蛋白质，没有DNA从它放大，所以只对球状体显示的scFv的序列将在PCR反应中回收。此外，这种方法可以扩展到工程其他类型的抗体或非抗体结合蛋白。 大肠杆菌内的膜显示为工程抗体以高亲和力和高含量的胞内溶解度的有力的平台。这种方法特别适用于设计成在细胞内的环境中发挥作用的抗体的有效的工程。这些细胞内的抗体已经被探索作为在许多领域的潜在治疗剂，包括神经变性疾病，癌症和病毒感染31。这种技术可以使更广泛地使用胞内抗体作为在这些领域的研究和医学并且其中正在学原位蛋白靶标是所期望的任何其他字段的工具。

能够折叠，并在细胞内环境中起作用的抗体可用于研究和治疗应用中有希望的工具。它们具有通过在细胞内结合到靶蛋白，以防止蛋白质-蛋白质相互作用，破坏蛋白质-核酸相互作用，或阻止对酶1-5基底入口调节蛋白活性的能力。 尽管抗体具有细胞内的应用程序多的潜力，工程他们在细胞内环境正确折叠和溶解度，同时维持对结合靶抗原的能力是具有挑战性的。的还原性的细胞质环境防止通常所需的全长抗体和抗体片段，包括单链可变片段（scFv）抗体6,7的稳定折叠的二硫键的形成。许多定向进化方法已被用来工程用H抗体对于目标IGH亲和力抗原8-10。这些方法通常使用噬菌体展示，酵母表面展示或细菌表面展示筛选抗体11-13的大型图书馆。这些方法是有力及有效用于鉴定结合靶的抗体，但它们依赖于分泌途径运输将要显示14-16蛋白质。分泌途径易位从还原细胞质折叠蛋白成在酵母或进入细菌的周质中的内质网腔。然后将蛋白质在氧化条件下折叠并显示在细胞表面上或包装入噬菌体颗粒来筛选结合亲和力17,18。其结果是，使用这些技术中分离的抗体不一定会在细胞质折好，和细胞内的溶解度通常必须单独设计，如果抗体将在细胞内的应用中使用。 为了改善那些在细胞质以及折叠工程抗体的效率，我们先前报道的MAD-TRAP（对于基于康达识别相关联蛋白的膜锚定显示器）的成功，用于筛选使用大肠杆菌党内的scFv抗体文库的方法膜显示19。细菌内膜显示依赖于双精氨酸易位（TAT）途径用于传输显示抗体，而相比之下，使用分泌途径其他常见的显示方法。 Tat途径包含质量控制机制，只允许可溶的，正确折叠的蛋白从大肠杆菌运大肠杆菌胞质中，穿过内膜并进入周质20,21。 （定位到具有N末端融合到Tat信号肽ssTorA Tat途径即 ，蛋白质），其在细胞质以及折叠过表达的Tat基板形成长寿命易位与N-末端中间体In中的细胞质和周质19中的C末端。这允许正确折叠的Tat基材，包括抗体片段的显示，在大肠杆菌的周质面大肠杆菌内膜。通过酶消化除去外膜以产生原生质球后，抗体被暴露于细胞外空间（ 图1）。这允许对内部隔膜显示达衬底被筛选用于结合到一个特定的目标。重要的是，利用对细胞表面显示Tat途径确保只有在该库被在细胞质以及折叠抗体将被询问的结合，允许结合亲和力和细胞内折叠同步工程。在这个协议中，我们描述了如何显示在E.一个单链抗体库大肠杆菌内膜，平移针对靶抗原的文库，并进行二次筛选，以确定该文库的最有希望的成分。虽然我们关注<html>关于scFv的协议，该方法可以应用于工程的应用要求绑定和细胞内折叠任何蛋白质。 <img alt="图1" src="/files/ftp_upload/54583/54583fig1.jpg" /> 图1.达内膜显示，在E.大肠杆菌，被表达为融合到ssTorA信号序列和正确地在细胞质折叠的scFv抗体穿过内膜运输。 A易位中间形式，其中所述的scFv在与在细胞质N端和在周质的C-末端的内膜锚。 大肠大肠杆菌外膜被酶消化，以形成球状体，从而暴露锚定的抗体与细胞外的空间，使它们可用于检测通过使用结合于所显示的抗体的C-末端融合表位标签的抗体。负载/ 54583 / 54583fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

<table border="0" cellpadding="0" cellspacing="0" width="1824">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">scFv library</td>
			<td style="width:205px;">Varies</td>
			<td style="width:135px;">N/A</td>
			<td style="width:859px;">A suitable scFv library should be obtained from a commercial or academic source.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MC4100 E. coli cells</td>
			<td>Coli Genetic Stock Center</td>
			<td>6152</td>
			<td>Cells need to be chemically competent or electrocompetent, depending on the selected transformation method.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Difco dehydrated culture media LB Broth, Miller (Luria-Bertani)</td>
			<td style="width:205px;">BD</td>
			<td style="width:135px;">244610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Chloramphenicol (Cm)</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:135px;">BP904-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium phosphate, dibasic (Na2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Potassium phosphate, monobasic (KH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Bovine serum albumin (BSA)</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:135px;">BP9706-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Fisher Scientific</td>
			<td>BP220-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP1521</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminetetraacetic acid (EDTA), 0.5 M</td>
			<td>Fisher Scientific</td>
			<td>BP2482-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Magnesium chloride (MgCl2)</td>
			<td>Fisher Scientific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Lysozyme</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:135px;">L3790-10X1ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex mixer</td>
			<td>VWR</td>
			<td>97043-564</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bicine</td>
			<td>Fisher Scientific</td>
			<td>BP2646100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">D-Biotin</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:135px;">BP232-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Fisher Scientific</td>
			<td>BP1755-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BugBuster Master Mix (cell lysis detergent)</td>
			<td>EMD Millipore</td>
			<td>71456</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vivaspin 2 MWCO, 3000 daltons</td>
			<td style="width:205px;">GE Healthcare Sciences</td>
			<td>28932240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Target antigen</td>
			<td>Varies</td>
			<td>N/A</td>
			<td>Purified target antigen may be purchased or produced/purified.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Dynabeads MyOne Streptavidin T1</td>
			<td style="width:205px;">Invitrogen</td>
			<td style="width:135px;">65601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Dynamag-2 magnet</td>
			<td style="width:205px;">Invitrogen</td>
			<td style="width:135px;">12321D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube rotator</td>
			<td>VWR</td>
			<td>13916-822</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">PCR primers</td>
			<td style="width:205px;">IDT</td>
			<td style="width:135px;">N/A</td>
			<td>Primer sequences are as described in the protocol.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">10X T4 DNA ligase reaction buffer</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">B0202S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">T4 Polynucelotide kinase (PNK)</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">M0201S</td>
			<td>Make sure the T4 ligase buffer used in the primer phosphorylation reaction contains 1 mM ATP.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">5X Phusion HF buffer pack</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">B0518S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Deoxynucleotide (dNTP) solution mix, 10 mM each dNTP</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">N0447L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Phusion DNA polymerase</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">M0530S</td>
			<td>Other high-fidelity polymerases may be used as an alternative, but the annealing temperature in Table 3 must be adjusted.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">C1000 Touch thermal cycler with dual 48/48 fast reaction module</td>
			<td>Bio-Rad</td>
			<td>185-1148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>Promega</td>
			<td>V3121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">SYBR Safe DNA gel stain</td>
			<td style="width:205px;">Invitrogen</td>
			<td style="width:135px;">S33102</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Wizard SV gel and PCR clean-up system</td>
			<td>Promega</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">T4 DNA ligase</td>
			<td style="width:205px;">New England BioLabs</td>
			<td style="width:135px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:627px;">Microdialysis membrane filter</td>
			<td style="width:205px;">EMD Millipore</td>
			<td style="width:135px;">VSWP04700</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>BD</td>
			<td>214030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">96-well polystyrene round-bottom cell culture plates</td>
			<td style="width:205px;">VWR</td>
			<td style="width:135px;">10062-902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Costar general polystyrene assay plate lids</td>
			<td style="width:205px;">Corning</td>
			<td style="width:135px;">3931</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microtitre plate shaker</td>
			<td style="width:205px;">VWR</td>
			<td>12620-926</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Costar 96 well EIA/RIA Easy Wash clear flat bottom polystyrene high bind microplate</td>
			<td style="width:205px;">Corning</td>
			<td style="width:135px;">3369</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Bel-blotter polycarbonate 96-well replicating tool</td>
			<td style="width:205px;">Bel-Art Products</td>
			<td>378760002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Instant nonfat dry milk</td>
			<td>Quality Biological</td>
			<td>A614-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tween 20 (polysorbate 20)</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">PopCulture reagent (concentrated cell lysis detergent)</td>
			<td style="width:205px;">EMD Millipore</td>
			<td>71092-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">Monoclonal ANTI-FLAG M2-Peroxidase(HRP) antibody produced in mouse</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td>A8592</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:627px;">SigmaFast OPD</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:135px;">P9187-50SET</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sulfuric acid (H2SO4), 10N solution</td>
			<td>Fisher Scientific</td>
			<td>SA200-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reynolds Wrap aluminum foil</td>
			<td>VWR</td>
			<td>89079-075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BioTek Epoch microplate spectrophotometer</td>
			<td>Fisher Scientific</td>
			<td>11120570</td>
			<td></td>
		</tr>
	</tbody>
</table>

bacterial inner-membrane display for screening a library of antibody fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

在大肠杆菌 Tat途径的细胞内蛋白质折叠质量控制机制大肠杆菌限制穿过内细胞膜转运到在还原细胞质环境良好折叠的蛋白质。通过超表达的scFv的ssTorA信号序列（从TORA蛋白，这是自然的Tat途径20传送的信号序列）的融合，易位被停止，从而导致在内侧膜19 scFv的显示。外膜的酶破坏之后，所显示的抗体用于筛选抗原结合活性可用。取Tat途径为单链抗体显示的优点的能力是由Karlsson 等所示的19（ 图6）。所述scFv抗体scFv13和scFv13.R4被融合到缺少由公认的精氨酸 - 精氨酸残基对天然ssTorA序列或修饰的ssTorATat途径。 scFv13.R4通过马丁纽等人通过四轮定向进化工程。从scFv13并且已知在细胞质9折好。这种单链抗体被显示在内膜，但只有当作为融合到天然ssTorA信号序列表达（ 图6）。反之，scFv13没有很好地折叠细胞质9，所以它不是在内部膜显示良好，无论信号序列其所稠合。此外，如果的scFv被在于缺乏TATC蛋白，康达机械20,28的一个重要组成部分，显示没有观察到，显示内膜显示和Tat途径之间的重要链接细胞中表达。这些结果表明，仅包含Tat信号肽和蛋白质正确折叠在细胞质中被显示在内膜，允许传输通过Tat途径充当细胞内FOL一个屏幕鼎。 <img alt="图6" src="/files/ftp_upload/54583/54583fig6.jpg" /> 图6.检测上内膜显示scFv的流式细胞仪分析，以检测在其内的膜折叠很差scFv13和良好的折叠scFv13.R4的显示。的scFv进行融合到天然ssTorA或ssTorA（KK），其中精氨酸 - 精氨酸对在ssTorA序列修饰成Lys赖氨酸。用异硫氰酸荧光素（FITC）检测上的scFv的C-末端FLAG表位标签缀合抗FLAG抗体。没有TATC蛋白（ΔtatC）和ssTorA-scFv13没有FLAG标记细胞进行了测试作为对照。 M表示中间荧光值。从权限参考19转载。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54583/54583fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 19。为了证明这一点，根据scFv13，其具有对β半乳糖苷酶结合亲和力的低电平的易错PCR文库，是针对靶抗原β半乳糖苷酶使用显示器和平移在协议中所述方法平移。 scFv的1-4被一轮诱变和淘选后分离，并表现出较高的结合亲和力β-半乳糖苷比scFv13（ 图7A）和细胞质溶解度更高水平（ 图7B）。 一个新的图书馆，以单链抗体1-4，使用易错PCR制成，以及对第二代文库淘β半乳糖苷酶使用所描述的协议的变形完成。第二轮演进抗β半乳糖苷酶的声像是在纯化的，可溶性scFv 14的存在下完成的作为竞争对手，以改善与比的scFv 1-4更高的亲和性分离的克隆的可能性。第二轮诱变和平移后，单链抗体2-1和2-3单链抗体使用基于ELISA的二次筛选分离。这些scFv的不仅表现出对β半乳糖苷酶比scFv13更高的结合亲和力，但也表现出比第一轮克隆的scFv 1-4更好的结合。的scFv 2-1表现出β半乳糖苷酶结合比得上scFv13.R4（ 图7A）的。的scFv 2-3还示出了在细胞质溶解度进一步增加相比的scFv 14，突出的溶解性和抗原结合的同步工程。由于scFv的亲和力和可溶性表达筛选，用于同时，可能的是一个选定的scFv已国防部中心提供全方位溶解性，但高结合，反之亦然。例如，单链抗体2-1具有比单链抗体2-3可溶表达低，但它具有较高的结合亲和力β-半乳糖苷。 <img alt="图7" src="/files/ftp_upload/54583/54583fig7.jpg" /> 图7.目标结合和scFv细胞质表达变异体内部采用膜分离出的显示（A）的scFv在大肠杆菌的细胞质中表达大肠杆菌细胞（ 例如 ，没有ssTorA信号序列）与六组氨酸（6×-His）标签，并使用镍-氮川三乙酸自旋柱纯化。对β-半乳糖苷酶的结合的纯化的scFv的用的ELISA测定。纯化的scFv被装载到β半乳糖苷酶包被ELISA板，并用抗6×-His抗体检测结合的scFv的。的数据是六个重复的平均，误差棒表示平均值的标准误差。（B）中的细胞裂解物的从表达细胞质通过用抗6×-His抗体探测Western印迹分析的scFv细胞的可溶和不溶组分。总蛋白质浓度用于标准化样品的加载。转载（A）和经许可的参考19改编（B）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54583/54583fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments at JoVE.com

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

细菌内膜显示筛查抗体片段的图书馆

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

bacterial-inner-membrane-display-for-screening-library-antibody

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.4K 次观看.  University of Maryland. We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

视频: Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

实验室规模的生产和治疗性抗体的纯化

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

科研

生物化学

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

用肽微阵列分析组蛋白抗体特异性

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

使用核酸探针进行核酸活性的动力学筛选

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results.
Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY&#160; II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input.
Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.

Crystallographic fragment screening at the Helmholtz-Zentrum Berlin is performed using a workflow with dedicated compound libraries, crystal handling tools, fast data collection facilities&#160;and largely automated data analysis. The presented protocol intends to maximize the output of such experiments to provide promising starting points for downstream structure-based ligand design.

赫尔姆霍尔茨-曾特鲁姆柏林水晶碎片筛选工作流程和工具

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

基于核磁共振的片段筛选，样品最少，自动化程度最高

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...