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.

세포질 행위 항체 엔지니어 인해 이황화 결합 -6,7- 안정화의 형성을 방해 세포질의 환원 환경에 어려운 작업이다. 이것은 그들이 결합 친 화성을 위해 설계되는 이외에 세포질에서 안정성 및 용해도를 위해 설계되지 않는 비활성 세포질 대부분 항체를 야기한다. 파지 디스플레이, 박테리아 표면 디스플레이, 효모 표면 디스플레이 방법은 기존의 방법은 모든 설계 항체의 표시 분비 경로 14-16를 사용하지만, 이러한 방법은 세포 내 접힘 엔지니어링 할 수단이 없다. 타트 경로의 폴딩 품질 관리가 잘못 접힌 세포질 불안정 항체의 전위를 방지하기 때문에 내부 멤브레인 디스플레이를 사용하여 조작 된 항체는 세포 내 안정성 및 용해도를 개선했다. 이 방법은 친 화성 A에 대한 엔지니어링 세포 내 항체의 반복적 인 프로세스를 단순화차 용해도, 두 특성이 한 단계로 설계되어있다. 이 방법은 감소 세포 환경에서 용해도와 항체 공학적 설계되었지만, 또한 단백질이 방법은 페리 플라 즘의 산화성 환경에서 폴딩을 유지하여 설계되기 때문에, 비 환원성 조건에서 작동하도록 설계 항체에 적용될 수있다. 이 기술은 높은 친 화성이 높은 세포질 용해도 항체 엔지니어링 프로세스를 간소화 있지만, 몇 가지 제한 사항이 프로토콜을 사용할 때 고려해야 할 중요하다. 보조 화면 ELISA 신호를 분석 할 때의 scFv 변종 약속 확인하려면, 적절한 항원 결합을 전시하지 않을 수 잠재적으로 흥미 변형과 그 사이의 안목에 대한 임계 값은 몇 클론 추가 특징으로 한 때까지 알 수있을 것 같지 않다. 이 모 항체에 걸쳐 개선 된 결합을 찾기 위해 중요하다; 하나,비정상적으로 높은 신호는 결합력 29 또는 응집 효과 (30), 내부 멤브레인 디스플레이 검사 방식에 고유하지 않은 문제를 나타내는 수 있습니다. 이 프로토콜을 사용하여 패닝 후 spheroplasts를 복구 할 수없는 경우가 아닌 실행 가능한 (게시되지 않은 데이터)만큼 키 제한은 기억한다. 이 항체를 코딩하는 플라스미드를 복구하는 DNA 증폭 및 변환 단계를 필요로한다. 프로토콜의 몇 가지 중요한 단계는 접는과 항체의 결합의 동시 공학을 할 수 있습니다. 심사에 성공하려면, 상영되는의 scFv 라이브러리는 ssTorA 신호 펩타이드 융합으로 표현해야합니다. 이 순서없이, 항체는 문신 경로로 이동하지 않으며, 따라서 페리 플라 즘 (19)에 전좌되지 않습니다. 또한,이 C 말단 에피토프 태그는 bin에 기재된 항체의 검출을 허용하는 항체에 융합하는 것이 필수적이다땡 분석. 분명히, E. 대장균은 또한 필요한 경로 문신 기계가 있어야 scFvs를 표현하는 데 사용하지만, 이는 통상적으로 사용 된 E. 마찬가지다 대장균 균주. 이 프로토콜에 대한 수정이 원하는 특성을 가진 항체를 분리 할 가능성을 향상시킬 수 있습니다. 감산 패닝 단계 전에 원하지 않는 구성 요소의의 scFv 라이브러리를 고갈 표적 항원에 대해 패닝을 완료 할 수있다. 라이브러리 spheroplasts 비 목적 단백질 단독 또는 코팅 BCCP 피복 자성 비드와 함께 배양 할 수 있고, 이러한 비드에 결합하는 spheroplasts 원하는 표적에 대한 결합에 대한 나머지 바운드 spheroplasts 선별 전에 폐기 될 수있다. 대표 결과에 언급 된 바와 같이, 방법은 고립 된 scFv의 친 화성이 spheroplasts에 표시 scFvs과 경쟁 패닝 반응 가용성 선수를 포함하는 개선한다. 가용성 빌려 때문에etitor에는 DNA가 spheroplasts에 표시 scFvs 그렇게 만 서열은 PCR 반응에서 회수되며, 그것에서 증폭되지 않는, 정제 된 단백질이다. 또한,이 방법은 항체 또는 비 - 항체 결합 단백질에 대한 다른 유형의 엔지니어링 확장 될 수있다. 대장균 내부 멤브레인 디스플레이는 높은 친 화성 및 세포 내 용해도가 높은 수준의 엔지니어링 항체를위한 강력한 플랫폼입니다. 이 방법은 세포 내 환경에서 작동하도록 설계 항체의 효율적인 엔지니어링에 특히 적합하다. 이러한 세포 내 항체는 이미 신경 퇴행성 질환, 암 및 바이러스 감염 (31)를 포함하는 필드의 수의 잠재적 치료제로 모색되고있다. 이 기술은 연구와 의학 이러한 필드 및 동일계에서 단백질 표적이 요구된다 공부 다른 필드 도구와 같은 세포 내 항체의보다 광범위한 사용을 가능하게 할 것이다.

접는 및 세포 내 환경에서 작동 할 수있는 항체는 연구 및 치료 응용 프로그램 모두를위한 유망한 도구입니다. 이들은 단백질 - 단백질 상호 작용을 방지 단백질 - 핵산 상호 작용을 방해하거나 효소 1-5 기판 액세스를 방지하기 위해 셀 내부의 표적 단백질에 결합하여 단백질의 활성을 조절하는 능력을 갖는다. 항체가 세포 내 애플리케이션 많은 잠재력을 가지고 있지만, 표적 항원에 결합하는 능력을 유지하면서 세포 내 환경의 적절한 폴딩 및 용해성을 설계하는 것은 어려운 것이다. 환원 세포질 환경은 일반적으로 전장 항체의 단일 사슬 가변 단편 (scFv를) -6,7- 항체를 포함한 항체 단편의 폴딩 안정에 필요한 이황화 결합의 형성을 방지한다. 지향 진화 접근 방법은 시간과 함께 항체를 엔지니어에게 이용되어왔다대상에 대한 고등학교의 친화력은 8-10 항원. 이러한 접근은 일반적으로 11-13 항체의 대규모 라이브러리를 선별, 파지 디스플레이, 효모 표면 디스플레이, 박테리아 표면 디스플레이를 사용한다. 이러한 방법은 대상에 결합하는 항체를 식별하기위한 강력하고 효과적이다, 그러나 그들은 14 ~ 16을 표시됩니다 단백질을 수송하기 위해 분비 경로에 따라 달라집니다. 분비 경로는 효모 나 박테리아의 페리 플라 즘에 소포체 내강으로 감소 세포질에서 펼쳐진 단백질으로 전위. 단백질은 산화 조건 하에서 접어 세포 표면 상에 표시하거나 친화 17,18 래핑 선별 파지 입자로 패키징된다. 그 결과, 이들 기술을 이용하여 분리 된 항체는 반드시 세포질에서 잘 접하지 않으며, 항체는 세포 내 용도로 사용할 경우, 세포 내 용해도는 종종 개별적으로 설계되어야한다. 개선하기또한 세포질에 접어 공학 항체의 효율성, 우리는 이전 MAD-TRAP (연관 단백질 타트 기반 인식 막 - 고정 디스플레이)의 성공 대장균 inner- 사용 된 scFv 항체 라이브러리를 스크리닝하기위한 방법을보고 막 디스플레이 19. 세균 내 멤브레인 디스플레이 분비 경로를 사용하는 다른 공통의 표시 방법과 대조적으로, 표시된 항체 수송 트윈 아르기닌 전좌 (TAT) 경로에 의존한다. 타트 경로는 수용성 정확하게 접힌 단백질은 E.에서 이송 될 수 있도록하는 품질 제어 메커니즘을 포함 막을 가로 지르는 내측 및 주변 세포질 (20, 21)에 대장균의 세포질. 잘 세포질에 접어합니다 (문신 신호 펩타이드 ssTorA에 N- 말단 융합과 문신 경로를 대상으로 예. 단백질)가 과발현 문신 기판은 N 말단에 중간 긴 수명 전위를 형성 전세포질 및 주변 세포질 (19)의 C 말단 N. 이는 E.의 세포질 표면에 항체 단편을 포함 올바르게 접혀 타트 기판의 표시를 허용 대장균 내부 막. spheroplasts를 생성 효소 절단에 의해 상기 외부 막을 제거한 후, 항체는 세포 외 공간 (도 1)에 노출된다. 이 내막에 표시 문신 기판이 특정 대상에 대한 결합에 대해 스크리닝 할 수 있습니다. 중요한 것은, 세포 표면 디스플레이를 위해 문신 경로를 활용하는 것은 아니라 세포질에 접어 라이브러리 만 항체 친 화성 세포 폴딩 결합 동시 엔지니어링을 가능 바인딩 심문 될 것임을 보장한다. 이 프로토콜에서, 우리는 E.에서의 scFv 라이브러리를 표시하는 방법에 대해 설명합니다 콜라이 내막은 표적 항원에 대한 라이브러리를 패닝 및 라이브러리의 가장 유망한 성분을 식별하기위한 보조 스크린을 수행한다. 우리는 초점 동안<html> scFvs의 프로토콜은,이 방법은 그 응용 결합하고 세포 내 접힘이 필요한 단백질을 설계에 적용 할 수있다. <img alt="그림 1" src="/files/ftp_upload/54583/54583fig1.jpg" /> 그림 1. 문신 내부 멤브레인 표시됩니다. E.에서 대장균은 ssTorA 신호 서열에 융합으로 표현 올바르게 세포질 접어의 scFv 항체는 내막을 가로 질러 전송된다. scFvs가 세포질에서 N 말단 및 주변 세포질의 C- 말단과 내부 막에 고정되는 전위의 중간 형태. E. 대장균 세포 외막 효소는 이에 외 공간에 고정 된 항체를 노광 및 디스플레이 항체의 C 말단 융합 된 에피토프 태그에 결합하는 항체를 사용하여 검출하기에 사용할 수 있도록 spheroplasts을 형성하도록 분해된다.로드 / 54583 / 54583fig1large.jpg &quot;대상 =&quot;_ 빈 &quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.

<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.

E.에 문신 경로의 세포 내 단백질 접힘 품질 제어 메커니즘 대장균 잘 환원 세포질 환경에 접어 단백질 내부 세포 막에 걸쳐 전송을 제한합니다. ssTorA 신호열 (자연스럽게 타트 통로 (20)에 의해 반송되는 토라 단백질로부터의 신호 서열)에 scFv를 융합 과발현으로 전좌가 내막 19 scFvs 표시 결과 스톨된다. 외막 효소의 중단 후, 표시 항체는 항원 - 결합 활성을 스크리닝 가능하게한다. scFv를 표시 용 타트 경로를 이용할 수있는 능력은 칼슨 등으로 도시 하였다. (19) (도 6). scFv가 항체 및 scFv13 scFv13.R4는 인식 아르기닌 - 아르기닌 잔기 쌍을 결여 네이티브 ssTorA 서열 또는 변성 ssTorA 중 하나에 융합 된문신 경로. scFv13.R4는 직접 진화의 네 라운드를 통해 scFv13에서. 마티 등에 의해 설계되고 세포질 9 잘 배 것으로 알려져있다. 이 scFv를는 내막에 표시 되었으나 네이티브 ssTorA 신호 서열에 융합으로 표현 만 (도 6). 반대로, scFv13 잘 세포질 9 절첩 아니므 관계없이 신호 서열의 어떤가 융합하는 내막 잘 표시되지 않는다. 또한, 경우 scFvs은 디스플레이 내부 멤브레인 디스플레이와 문신 경로 사이의 중요한 연결을 보여 관찰되지 않았다는 TatC 단백질의 문신 기계 20,28의 중요한 구성 요소를 결여 세포에서 발현되었다. 이러한 결과는 세포 FOL위한 화면으로서 기능 할 타트 경로를 통해 전송을 허용하는 문신 신호 펩티드를 포함하는 단백질만을 정확하게 내막에 표시되는 세포질 접어 입증땡땡. <img alt="그림 6" src="/files/ftp_upload/54583/54583fig6.jpg" /> 도 내막 표시 scFvs 6. 검출. 유동 세포 계측법 분석은 내막에 잘못 접힌 scFv13 잘 접혀진 scFv13.R4의 표시를 검출하기 위해 수행 하였다. scFvs는 ssTorA 시퀀스에서의 Arg, 인수 쌍리스 -리스로 수정 된 기본 ssTorA 또는 ssTorA (KK)에 융합되었다. scFvs의 C 말단 FLAG 에피토프 태그는 형광 염료 (FITC)로 검출 하였다는 항 FLAG 항체를 π 공역. 플래그 태그없이 TatC 단백질 (ΔtatC) 및 ssTorA-scFv13없이 세포는 대조군으로 시험 하였다. M은 평균 형광 값을 나타냅니다. 허가 기준 19 일부터 재판. <a href="http://www.jove.com/files/ftp_upload/54583/54583fig6large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> (19)을 향상시킨다. 이를 설명하기 위해, β 갈 락토시다 제에 대한 결합 친화도가 낮은 레벨이 scFv13에 기초하여 에러가 발생하기 쉬운 PCR 라이브러리는, 프로토콜에 기재된 방법을 표시하여 패닝 표적 항원 β 갈 락토시다 제에 대해 패닝 하였다. scFv를 1-4 돌연변이 및 패닝 중 하나 라운드 후에 단리하고 scFv13 (도 7a)보다 세포질 용해도보다 높은 레벨 (도 7b) β 갈 락토시다 제를 더 높은 결합 친화도를 나타냈다. scFv를 1-4을 기반으로하는 새로운 라이브러리는 오류가 발생하기 쉬운 PCR을 사용했고, 대해이 2 세대 라이브러리의 패닝했다β - 갈 락토시다 제는 설명 프로토콜의 변형을 사용하여 수행 하였다. 진화의 두 번째 라운드를위한 β 갈 락토시다 제에 대해 패닝의 scFv 1-4보다 높은 친화 클론 분리의 가능성을 개선하기위한 경쟁자로 정제 용해 scFv를 (14)의 존재하에 수행 하였다. 돌연변이 유발 및 패닝의 두 번째 라운드 후, scFv를 2-1 scFv를 2-3은 ELISA 기반 차 심사를 사용하여 분리 하였다. 이러한 scFvs은 scFv13보다 β - 갈 락토시다 아제에 대한 높은 결합 친 화성을 전시뿐만 아니라 1 라운드 클론의 scFv 1-4보다 더 바인딩 전시 없습니다. scFv를 2-1 scFv13.R4 (도 7A)에 필적 결합 β 갈 락토시다 제를 나타냈다. scFv를 2 ~ 3도 용해도 및 항원 결합의 동시 공학을 강조하는 scFv (14)에 비해 세포질 용해도 추가 증가를 보여줍니다. scFvs의 친 화성 및 수용성식이 동시에 스크리닝하기 때문에, 선택된의 scFv는 모드 (mod) 것을 가능생이 용해도하지만 높은 결합 또는 그 반대의 경우도 마찬가지. 예를 들어, 2-1의 scFv의 scFv 2-3보다 낮은 수용성 발현을 가지고 있지만 베타 - 갈 락토시다 제를 더 높은 결합력을 나타낸다. <img alt="그림 7" src="/files/ftp_upload/54583/54583fig7.jpg" /> 그림 7. 대상 바인딩과의 scFv의 세포질 발현이 내부 멤브레인 디스플레이를 사용하여 격리 변형. (A) scFvs는 E.의 세포질에서 발현되었다 대장균 a를 헥사 히스티딘 (6 × -His) 태그합니다 (ssTorA 신호 순서없이 예.) 전지와 니켈 - 니트릴 로트리 아세트산 스핀 컬럼을 사용하여 정제. - 갈 락토시다 아제와 β 정제 scFvs의 결합은 ELISA로 측정 하였다. 정제 scFvs는 β 갈 락토시다 제 - 코팅 ELISA 플레이트 상에 로딩하고, 바인딩은 scFvs 항 6 × -His 항체를 검출 하였다. 데이터는 여섯 복제의 평균이며, 오차 막대는 평균값의 표준 오차를 나타낸다.(B) 세포질 항 6 × -His 항체로 프로빙 웨스턴 블롯에 의해 분석 하였다 scFvs를 발현하는 세포로부터의 세포 용 해물의 가용성 및 불용성 분획. 총 단백질 농도는 시료의 로딩을 표준화 하였다. (A) 재판 및 허가 기준 (19)에서 (B) 적용. <a href="http://www.jove.com/files/ftp_upload/54583/54583fig7large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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.

항체 단편의 라이브러리를 스크리닝에 대한 세균 내부 멤브레인 디스플레이

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

Research

JoVE Journal

Biochemistry

11.4K 조회수.  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.

비디오: 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.

실험실 규모의 생산과 치료 용 항체의 정제

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

연구

생화학

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.

펩티드 Microarrays를 가진 Histone 항체 특이성의 분석

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.

펩 티 드 선호도 시 약 검색 및 분석 결과 분리의 세균성 디스플레이 라이브러리의 반자동된 Biopanning

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.

꼬마 선 충 에서 높은 처리량 화학 심사 하는 간단한 방법

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.

선호도 크로마토그래피 수지의 신속한 개발을 위한 활성화된 상호 연결 아가로스 - 사례 연구로서의 항체 포획

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.

핵산 프로브를 이용한 뉴클레아제 활성의 운동 스크리닝

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.

소분자 억제제 스크리닝을 위한 NADH 결합 ATPase 분석의 반 높은 처리량 적응

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.

결합 단일 도메인 항체 라이브러리의 단계별 파지 선택에 의해 고도로 특이적 화학적으로 유도된 단백질 이분화 시스템 생성

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.

헬름홀츠 젠트럼 베를린에서 결정화 조각 검사를 위한 워크플로우 및 도구

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.

최소 샘플이지만 최대 자동화 모드에서 NMR 기반 단편 스크리닝

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