저자들은 루이스 F. 델가딜로(오하이오 대학교 화학 분자 공학부), 매튜 H. 윌리엄스(오하이오 대학교 화학 분자 공학부), 필리베르토 세데노 로랑 박사(브리검 여성 병원 및 하버드 의과 대학)에게 전문적인 기술 지원을 부탁드립니다. 저자는 또한 겨울 감사 를 원한다 2011 CHE 404/BME 504 그리고 가을 2012 CHE 4830/BME 5830 학생 (화학 및 생물 분자 공학 및 생물 의학 공학 프로그램 학과, 오하이오 대학) 기술 조언 및 토론에 대 한. 이 작품은 국립 과학 재단 (NSF) 주요 연구 계측 보조금 CBET-1039869 (MMB), NSF CBET-1106118 (MMB), 피부과 재단 연구 보조금 A050422 (SRB), 국립 보건원 (NIH) NCI 보조금 1R15CA1611에 의해 지원되었다 (MMB), NIH Kirschstein-NRSA 박사 후 펠로우십 F32CA144219-01A1 (SRB), NIH/NCI R01CA173610 (CJD), NIH NCCAM 보조금 R01AT004628 (CJD).

1. 단백질 A의 선호도 확인
<ol><li>Fc-expressing 단백질(재조합 융합 단백질 또는 IgG 항체, 본선 "단백질"이라고 불리는 IgG 항체)이 단백질 A14-17을 사용하기 전에 단백질A에 대한 허용 가능한 친화력을 가지고 있는지 확인하고, 주식 용액에 단백질의 존재를 테스트한다(예를들어, 세포 배양 과민제는 1,00분에서 원심분리에 의해 정제됨). 유동 세포측정, 서양 블로팅, ELISA 등을 사용하십시오. 실험실 선호도에 따라 Fc의 단백질 기능 및/또는 존재를 감지합니다. 이상적으로, 단백질을 초과하는 것을 피하기 위해 세포 배양 상수(step 6)에서 단백질을 정량화하여 단백질A 막 흡착 용량을 초과하지 않도록 한다. 단백질이 검출되면 진행합니다.</li>
</ol>2. 단백질 A 막 흡착및 세포 배양 상수 준비
<ol><li>멤브레인 흡착기를 준비하기위한 제조업체의 지침을 따르십시오. 공기가 멤브레인 흡착제에 들어가지 마십시오. 멤브레인 흡착을 통해 퍼진 모든 솔루션은 실온에서 0.22 μm 필터를 사용하여 사전 필터링되어야 합니다. 0.22 μm 여과 Dulbecco의 인산완충식염(DPBS) 또는 선택 및 방전 기포버퍼로 10ml 주사기를 채웁니다. DPBS를 퍼퓨즈하여 저장 용액을 제거하고 사용 직전에 멤브레인 흡착기를 평형화합니다.</li>
	<li>진공 구동 0.22 μm 멸균 여과 장치를 사용하여 멤브레인 흡착장치를 통해 관류 직전에 세포 배양 상류제를 필터링합니다.</li>
</ol>3. 단백질 A 멤브레인 애드버로드
<ol><li>흡인 세포 배양은 루어 록 주사기 (30ml 이상)로 상류하고 기포를 배출합니다.</li>
	<li>그림 1에표시된 바와 같이 재료와 장비를 조립합니다. 주사기를 멤브레인 흡착기의 입구에 연결합니다. 멤브레인 흡착 콘센트에 유연한 튜브를 부착합니다. 원하는 경우 주사기와 멤브레인 흡착기 사이에 0.22 μm 주사기 필터를 놓습니다. 플라스크 나 병을 사용하여 흡착기에서 (여과라고도 함)을 통해 흐름을 잡습니다.</li>
	<li>주사기 펌프를 원하는 체피흐름으로 설정하지만 제조업체의 권장유량(예: 10ml/min)을 초과하지 는 않습니다. 멤브레인 흡착을 통해 세포 배양 상류체를 퍼퓨징하여 비커 또는 기타 용기에서 흐름을 수집합니다. 필요한 슈퍼나티로 주사기를 다시 로드합니다.</li>
	<li>원하는 경우, 유동 세포측정, 서양 블로팅, ELISA 등을사용하여 단백질의 존재를 통해 흐름을 테스트한다. 랩 기본 설정에 따라. 일반적으로 흐름을 다시 활성화하는 것은 필요하지 않습니다.</li>
</ol>4. 멤브레인 흡착기의 엘루트 단백질
<ol><li>10ml DPBS로 멤브레인 흡착기를 세척하여 비바운드 단백질을 제거합니다.</li>
	<li>10-15 ml의 용출 완충액(예: 아민 계 용출 완충제(예: 아민 계 용출 완충제(pH 2.8)을 사용하여 원하는유량(예: 1 ml/min)에서 멤브레인 흡착으로부터의 엘루트 단백질. 용출 부피(1.0-1.5 ml)의 10%에서 중성 화완충(예: 1M Tris(pH 9.4)을 함유한 튜브에서 용출을 잡으세요.</li>
	<li>또는, 1ml 단위로 엘루트 단백질은 10-15ml용출 버퍼의 총 부피를 사용하여 중화 버퍼 100 μl을 포함하는 튜브로 증가한다. 선호하는 특성화 방법을 사용하여 단백질의 존재에 대해 각 분획을 테스트합니다.</li>
	<li>제조업체의 지침에 따라 멤브레인 흡착기를 재생합니다. 0.22 μm 여과DPBS 또는 선택의 버퍼의 10ml, 다음 1 N NaCl에서 0.22 μm 필터링 50 mM NaOH의 10 ml, 그리고 마지막으로 0.22 μm 필터링 DPBS의 10 ml. 4ºC에서 장기 저장을 위해 DPBS에 20 %의 에탄올로 멤브레인 흡착을 채웁니다.</li>
</ol>5. 단백질을 농축하고 투석합니다.
<ol><li>10kDa 분자량 컷오프 원심 필터 유닛에 모든 용출(또는 4.3단계에서 결정된 단백질을 함유한 용출 분수)을 증착한다. 원심분리에 대한 제조업체의 지침을 따르십시오.</li>
	<li>제조업체의 지시에 따라 소량 투석 장치(10kDa 분자량 컷오프)로 재텐테이트를 선택한 버퍼에 대해 투석합니다. 단백질 강수량이 우려되는 경우 완충제는 투석 된 물질에 첨가해야 할 수도 있습니다. 원하는 경우, 투석된 물질은 6단계까지 냉장보관할 수 있지만 방부제 없이 장기간 보관하는 것은 권장되지 않는다.</li>
</ol>6. 신속한 서부 블로팅 절차에서 정제 된 제품을 정량화하고 특성화하십시오.
<ol><li>원하는18-20으로감소 또는 비감소 조건하에서 SDS-PAGE에 의해 선택된 젤에 대한 정제 단백질 및 Fc 표준을 해결한다. 미디 젤이 아닌 미니 젤을 사용하여 런타임을 최소화하십시오.<ol>
<li>아직 알려지지 않은 경우로드량(예: 0.1-1 mg)에 대해 역역 신호가 선형으로 달라지는 Fc 표준 범위를 결정합니다. 정제 된 단백질을 정량화하고 특성화하는 데 사용되는 동일한 젤, 실행 조건, 이송 조건 및 시약을 사용합니다.</li>
			<li>정제된 단백질 밴드 신호가 이미지 분석에서 Fc 표준의 선형 신호 범위 내에 있는지 확인하기 위해 DPBS로 희석된 샘플을 포함하여 다량의 정제된 단백질을 적재합니다.</li>
		</ol>
</li>
	<li>블로터 제조업체의 지시에 따라 반건조블로터(13)를이용하여 불연속 완충 시스템에서 젤에서 폴리비닐리덴 불소(PVDF) 멤브레인으로 단백질을 전달한다. 전송 시간은 일반적으로 5-10 분입니다. 원하는 경우, 쿠마시는 전송효율(21)을확인하기 위해 이송 후 젤을 염색한다.</li>
	<li>진공 보조 단백질 검출 시스템을 사용하여 실험실 선호도에 따라 알칼리인스파타제(AP) 또는 말 무 과산화제(HRP)에 공주된 안티-Fc 또는 안티-IgG로 면역블로팅을 수행한다. 면역 블로팅 시간은 전형적으로 1시간 미만이다.</li>
	<li>기판 및 선택방법(예: AP 기질 및 향상된 케미발광)을 사용하여 면역블롯을 개발한다.</li>
	<li>이미지 분석에 의한 정제 단백질을 정량화합니다. Fc 표준에서 밴드 신호에 선형 회귀를 수행합니다. R2>0.90인 경우, 선의 방정식을 사용하여 대역 신호에서 단백질 양을 계산합니다. 필요한 경우 샘플 희석을 고려합니다. 단백질의 정량화는 Fc에 기초하여 수행되기 때문에, 단백질과 Fc 사이의 분자량 차이에 대한 값을 조정합니다<.</li>
	<li>기능적 분석이나 방법을 사용하여 단백질을 검증하여 유동 세포측정, 서양 블로팅, ELISA 등을통해 Fc를 검출한다. 랩 기본 설정에 따라.</li>
	<li>단백질을 원하는 농도로 희석및/또는 장기간 보관, 냉동 또는 기타 용액을 알리쿼팅하기 전에 정제된 단백질 용액에 원하는 방부제 또는 안정제를 추가하십시오.</li>
</ol>

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H., Nemes, P., Bucholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Coomassie+brilliant+blue+g250+perchloric+acid+solution+for+staining+in+electrophoresis+and+isoelectric+focusing+on+polyacrylamide+gels.">The use of Coomassie brilliant blue g250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrylamide gels.</a> Anal. Biochem. 64, 509-516 (1975).</li><li>Cedeno-Laurent, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+inhibition+of+galectin-1-binding+carbohydrates+accentuates+antitumor+immunity.">Metabolic inhibition of galectin-1-binding carbohydrates accentuates antitumor immunity.</a> J. Invest. Dermatol. 132, 410-420 (2012).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Desjardins, P., Hansen, J. B., Allen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvolume+Protein+Concentration+Determination+using+the+NanoDrop+2000c+Spectrophotometer.">Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer.</a> J. Vis. Exp. (33), (2009).</li><li>Ernst, O., Zor, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linearization+of+the+Bradford+Protein+Assay.">Linearization of the Bradford Protein Assay.</a> J. Vis. Exp. (38), (2010).</li><li>Simonian, M. H., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+and+colorimetric+determination+of+protein+concentration.">Spectrophotometric and colorimetric determination of protein concentration.</a> Curr. Protoc. Mol. Biol. Chapter. 10, 10-1002 (2006).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150, 76-85 (1985).</li><li>Gravel, C., Li, C., Wang, J., Hashem, A. M., Jaentschke, B., Van Domselaar, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analyses+of+all+Influenza+Type+A+Viral+Hemagglutinins+and+Neuraminidases+using+Universal+Antibodies+in+Simple+Slot+Blot+Assays.">Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays.</a> J. Vis. Exp. (50), (2011).</li></ol>

공개할 것은 없습니다.

본 명세서에 기재된 프로토콜은 제품 회수 또는 팽창 비용을 크게 손상시키지 않으면서 인간 Fc(Gal-1hFc)를 발현하는 키메라 융합 단백질을 신속하게 분리하고 특성화하기 위해 개발되었다. 간소화된 워크플로우의 주요 구성 요소는 절연을 위한 단백질 A 멤브레인 흡착제, 서양 블로팅에 의한 특성화를 위한 반건조 전달 및 진공 보조 면역블로팅입니다.
세포 배양 상류체 300ml?...

세포 배양 초월제로부터 면역글로불린 G(IgG) Fc 단편을 발현하는 항체 및 재조합 단백질의 분리및 희석 된 세포 용액은 단백질 A 친화 크롬 토그래피를 사용하여 달성 될 수있다. 기초 과학 및 공학 실험실 및 산업에서다공성 단백질 A-코팅 아가로즈, 유리 또는 폴리머 비드로 포장 된 컬럼은 높은 재정적 비용과 긴 처리 시간1-3에도불구하고 친화성 크로마토그래피에 가장 일반적으로 사용됩니다. 친화성 크로마토그래피는 실험실 및 산업 설정2,4모두에서 가장 큰 비용과 처리 병목 현상을 나타낸다는 것이 좋습니다. 그 결과, 공정열차1,2,5-7에서전체적인 제품 회수를 희생하지 않고 비용 및 처리 시간을 줄이기 위해 수많은 개선이 개발되었다. 특히 항체 및 기타 Fc-발현 단백질의 분리에 대한 유망한 단백질A막흡착제 2,5,7-10을이용한 친화성 크로마토그래피이다. 컬럼 크로마토그래피에서 의Fc 포획은확산제한(즉, Fc-표현 단백질은 내부 모공을 통해 확산되어 단백질 A의 대부분에 도달해야 한다)와 고압 강하량, 멤브레인 흡착제의 질량 수송은 대량 대류에 의해 구동되어 확산한계를 8,9,11,12로방지한다. 또한 멤브레인 흡착기를 가로지르는 압력 강하가 낮기 때문에 구슬이 가득한 컬럼8,9,11,12에비해 관류 유량이 더 빨라집니다. 따라서, 실험실 스케일 절연을 위해 막 크로마토그래피는 절연 시간을 몇 시간 단축하고 컬럼 크로마토그래피에 비해 제품 캡처를 증가시킬 것으로 예상된다. 또한 멤브레인 흡착제에서 IgG 흡착에 대한 수학적 모델이제안되어최종 사용자가 실험실에서 성능을 예측할 수 있도록 합니다.
기초 과학 및 공학 실험실의 또 다른 병목 현상은 Fc 표현 단백질의 특성입니다. 그러나 서양 얼룩과 같은 특성화 를 완료하는 적절한 방법을 선택하면 제품 테스트에 소요되는 시간을 크게 줄일 수 있습니다. 예를 들어, 불연속 완충 시스템에서 반건조 전달은 연속 완충계통(13)에서탱크(젖은) 이송에서 1-2시간 반대로 SDS-PAGE 젤에서 폴리비닐 디플루오리딘(PVDF) 멤브레인으로 단백질의 전기전도 전달을 달성할 수 있다.
인간 IgG의 Fc 단편을 발현하는 키메라 융합 단백질을 분리하고 특성화하는 신속하고 저렴하며 효과적인 프로토콜은 본원에 기재된다. 대부분의 장비는 표준 기본 생체 의학, 생물학, 화학 및 (바이오) 화학 공학 실험실에서 쉽게 사용할 수 있으며 모든 시약은 시판됩니다. 대표적인 결과는 뮤린 갈렉틴-1/인간 Fc 키메라 융합 단백질(Gal-1hFc)의 격리 및 특성화로부터 생성되었지만, 유선형 프로토콜은 항체, Fc 단편 자체 또는 다른 융합 Fc 단백질과 같은 Fc 단편을 소유한 다른 분자의 격리에 적용될 수 있다. 우리의 개선은 표준 방법에 비해 유사하거나 개선 된 제품 복구를 달성하는 동안 (4 시간 이상 일반적으로 ~ 9 시간) 처리 시간을 극적으로 감소.

<table border="0" cellpadding="0" cellspacing="0" width="822">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Protein A membrane adsorber (Sartobind Protein A 2 ml)&#160;</td>
			<td style="width:191px;">Sartorius-Stedim</td>
			<td style="width:147px;">93PRAP06HB-12--A</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Amicon Ultra centrifugal filter, 10 kDa, 4 ml</td>
			<td style="width:191px;">Millipore</td>
			<td>UFC801008</td>
			<td>Use 15 ml volume if needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sterile vacuum filter unit, 0.22 &#181;m, 500 ml</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">10 ml Syringes with Luer Lock tips</td>
			<td>BD</td>
			<td>301604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">30 ml Syringes with&#160; Luer Lock tips</td>
			<td>BD</td>
			<td>309650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tygon lab tubing (1/16 in x 1/8 in x 50 ft)</td>
			<td>Cole Parmer</td>
			<td>WU-95903-16</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Slide-A-Lyzer MINI Dialysis Devices (10K MWCO)</td>
			<td>Thermo Scientific</td>
			<td>69576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. antibody collection tray</td>
			<td>EMD Millipore</td>
			<td>WBAVDABTR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. single well blot holder</td>
			<td>EMD Millipore</td>
			<td>WBAVDBH01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Elution buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tris, ultra pure</td>
			<td>Fisher Scientific</td>
			<td>819623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">DPBS</td>
			<td>Thermo Scientific&#160;</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:313px;">DPBS with Ca2+/Mg2+</td>
			<td>Life Technologies</td>
			<td>14080-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">BSA</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Human Fc</td>
			<td>Bethyl Research Laboratories</td>
			<td>&#160;P80-104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-APC</td>
			<td>Jackson Immunoresearch</td>
			<td>109136170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-AP</td>
			<td>Bio-Rad</td>
			<td>170-5018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Alkaline phosphatase substrate</td>
			<td>Promega</td>
			<td style="width:147px;">S3841</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Flow cytometry tubes (5 ml polystyrene or polypropylene)</td>
			<td>BD</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;"></td>
			<td style="width:191px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>55-2226</td>
			<td style="width:173px;">A peristaltic pump is also acceptable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Protein gel electrophoresis system&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>552BR094876</td>
			<td style="width:173px;">Any gel electrophoresis system is acceptable, but mini gel systems run fastest</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Semidry blotter&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td style="width:147px;">690BR006163</td>
			<td style="width:173px;">Any transfer system is acceptable, but discontinuos transfer systems perform electrophoretic transfer fastest</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:313px;">SNAP i.d. vacuum-assisted protein detection system</td>
			<td style="width:191px;">EMD Millipore</td>
			<td>WBAVDBASE</td>
			<td style="width:173px;">An upgraded model has replaced this specific instrument, but either should work just as well</td>
		</tr>
	</tbody>
</table>

isolation and characterization of chimeric human fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow

세포 배양 체와 용세포 용액으로부터 생체 분자의 산업 규모의 정제에 대한 실험실 규모는 친화성 크로마토그래피를 사용하여 달성 될 수있다. 다공성 단백질A아가로즈 구슬을 사용하여 열에 포장된 친화성 크로마토그래피는 틀림없이 IgG의 Fc 단편을 발현하는 항체 및 재조합 단백질의 실험실 스케일 절연의 가장 일반적인 방법이지만, 시간이 많이 걸리고 비용이 많이 드는 과정이 될 수 있다. 희석 솔루션에서 수백 마이크로그램의 단백질을 밀리그램으로 회수해야 하지만 고압 액체 전달 시스템 및/또는 생체 분리 에 대한 전문 지식을 갖춘 인력에 대한 접근이 부족한 소규모 기초 과학 실험실에서는 시간과 재정적 제약이 특히 어렵습니다. 또한, 제품 정량화 및 특성화는 또한 여러 근무일에 걸쳐 처리 시간을 과도하게 연장하고 비용(소모품, 임금 등)을부풀릴 수 있습니다. 따라서, 실험실 규모 격리 및 항체 및 Fc 단편을 소유한 다른 단백질의 특성화를 위해서는 빠르고 저렴하면서도 효과적인 프로토콜이 필요합니다. 이를 위해 20시간 이상의 근무 시간을 요구하는 기존의 방법과는 달리 9시간 미만(약 1일 근무)과 4시간 이내에 제한된 경험의 기술 직원이 완료할 수 있는 프로토콜을 고안했습니다. 대부분의 필수 장비는 표준 생의학 과학, 생화학 및 (바이오) 화학 공학 실험실에서 쉽게 사용할 수 있으며 모든 시약은 시판됩니다. 이러한 프로토콜을 입증하기 위해, 대표적인 결과는 키메라 뮤린 갈렉틴-1이 세포 배양 상수로부터 인간 Fc(Gal-1hFc)에 융합되어 단백질 A막 흡착기를 사용하여 분리되었다는 것을 제시한다. 정제 된 Gal-1hFc는 신속한 서양 블로팅 분석 절차를 사용하여 정량화되었으며 유동 세포 측정을 사용하여 특징지어졌습니다. 간소화된 워크플로우는 항체와 같은 다른 Fc 발현 단백질을 위해 수정하거나 대체 정량화 및 특성화 방법을 통합하도록 변경할 수 있습니다.

Fc 발현 단백질의 격리 및 특성화를 위한 간소화된 프로토콜은 희석세포 배양 초나티로부터 인간 Fc(Gal-1hFc)에 융합된 키메라 뮤린 갈렉틴-1을 처리하는 데 일상적으로 사용됩니다. 그림 2의 순서도는 프로토콜의 각 단계에 대한 워크플로우와 시간을 보여 줍니다. 300ml의 전형적인 미퍼나탄의 경우, 용출 분획의 선택적 유동 세포측정 시험이 수행될 때 총 처리 시간은 약 9시간입니다. 모...

Watch this Scientific Journal Video about Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow at JoVE.com

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

단백질 A 아가로즈 비드가 포장된 컬럼을 이용한 전통적인 친화성 크로마토그래피와 비교하여, 단백질 A 멤브레인 흡착제는 항체 및 기타 Fc 단편 발현 단백질의 실험실 규모 절연을 크게 가속화할 수 있습니다. 적절한 분석 및 정량화 방법은 단백질 처리를 더욱 가속화할 수 있으므로 20시간 이상의 근무 시간이 아닌 한 작업 시간에 격리/특성을 완료할 수 있습니다.

단백질 A 멤브레인 흡착제 및 간소화된 워크플로우를 사용하여 키메라 인간 Fc 발현 단백질의 격리 및 특성화

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

isolation-characterization-chimeric-human-fc-expressing-proteins

Research

JoVE Journal

Bioengineering

7.1K 조회수.  Ohio University. 단백질 A 아가로즈 비드가 포장된 컬럼을 이용한 전통적인 친화성 크로마토그래피와 비교하여, 단백질 A 멤브레인 흡착제는 항체 및 기타 Fc 단편 발현 단백질의 실험실 규모 절연을 크게 가속화할 수 있습니다. 적절한 분석 및 정량화 방법은 단백질 처리를 더욱 가속화할 수 있으므로 20시간 이상의 근무 시간이 아닌 한 작업 시간에 격리/특성을 완료할 수 있?...

비디오: Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

단백질과 세포 교도소에 대한 2 차원 문양의 기판 만들기

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

연구

생체공학

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Photoacoustic Flowmetry를 사용하여 순환 흑색증 전지의 탐지 및 격리

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

막 척추 : 막 단백질의 단백질 - 단백질 상호 작용을 식별 할 수있는 생화학 적 도구<em&gt; 생체</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

테인 매개 인공 단백질 히드로 겔의 합성

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

사례 연구로 담배에 과도 단백질 발현 : 실험 접근의 디자인을 사용하여 복잡한 시스템의 특성

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

신경 조직의 재생을위한 가용성 및 불용성 바이오틴 단백질의 발현, 분리 및 정제

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

발광 공명 에너지는 포유 동물 세포에 표현 막 단백질의 구조 변화를 연구에 전송

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

3 차원 조직 공학 인간의 식도 점막 모델의 제조, 특성 및 사용 가능성

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

들여 쓰기 및 인장 시험을 사용하여 인간의 부드러운 조직의 생체 역학적 특성

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

제조 및 Macrostructure를 포함 하는 광학 조직 팬텀의 특성화

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...