이 작품은 미국 국립 보건원 (GM097569)에 의해 지원되었다.

<ol>
	<li>Miles, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tryptophan+synthase:+a+multienzyme+complex+with+an+intramolecular+tunnel.">Tryptophan synthase: a multienzyme complex with an intramolecular tunnel.</a> Chemical Record. 1 (2), 140-151 (2001).</li><li>Raboni, S., Bettati, S., Mozzarelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tryptophan+synthase:+a+mine+for+enzymologists.">Tryptophan synthase: a mine for enzymologists.</a> Cellular and Molecular Life. 66 (14), 2391-2403 (2009).</li><li>Dunn, M. 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저자는 공개하고 경쟁 금융 이익을 선언 할 아무것도 없다.

우리는 성공적으로 구조 기능 상관 관계 연구를위한2β2 βQ114A, α2β2 βK167T,α2 β2 βS377A 세인트TS 복합체를 α 돌연변이 형태를 성공적으로 설계했다. 처음에, 우리는 이전 정화 프로토콜을 사용하여 돌연변이를 정화하기 위해 노력했다22,이는 정제 하는 동안 PEG 8000 및 spermine와 α2β2세인트TS 복합 결정?...

트립토판 신타제(TS) 바이엔자임 복합체(α2 β 2)는알로스터 효소로, 박테리아, 식물, 곰팡이1,2,3에서아미노산 L-트립토판의 생합성에서 마지막 두 단계를 촉매한다. 박테리아 살모넬라 장내 세로바 티피무리움 (St)은인간과 다른 동물에서 심각한 위장 감염을 일으킨다. 인간과 더 높은 동물은 TS(EC 4.2.1.20)가 없기 때문에, S. typhimurium α2β2 TS 복합체 (α2β2StTS)의 억제는 크립토스포리디오스및 결핵4,생식기 및 안구 감염5,그리고잠재적인 그녀의 발동을 위한 잠재적 인 약물 대상으로 탐구되었습니다. α 서브유닛은 인돌-3-글리세롤-인산염(IGP)의 알돌리틱 분열을 글리세랄데히드-3-인산염(GAP)과 인돌로 촉매하여, 인돌닌 타우토머 중간체및 그 후 카본-카본 결합 분열의 형성을 통해 GAP및 인돌3,6을생성한다. β 촉매 부위에는 쉬프 베이스를 통해 β-Lys87에 결합된 피리독살 5′-인산염(PLP) 보조 분자가 포함되어 있으며, 이는 효소 β 서브유닛3,7에서반응하는 과정에서 전자 싱크로 기능한다. β 사이트는 L-트립토판과 PLP 의존 반응에서 물 분자를 제공하기 위해 인돌에 의해 L-세린 사이드 체인 하이드록실의 교체를 촉매한다. 세인트 (주) TS는 다효소 복합체2,3내의 기질 채널링 및 알로스터통신을 조사하는 오랜 모델역할을 한다. TS의 α 및 β 서브유닛 간의 양방향 알로스터 통신은 L-트립토판 합성3동안 촉매 단계를 동기화하고 인돌 방출을 방지할 필요가 있다. 이러한 노력을 확장하기 위해, 우리는 여러 돌연변이 (β-Gln114Ala, β-Lys167Thr 및 β-Ser377Ala)를 단일 지점 돌연변이로 제조하여 효소 구조, 메커니즘 및 세인트TS β 서브 유닛의 촉매 부위에서의 관계의 추가 탐구에 사용할 수 있습니다.
α2β2StTS의 촉매 메커니즘에 대한 자세한 연구는 에디스 W. 마일즈의 연구 그룹에 의해 시작되었습니다. 네이티브 대장균 α2 β2 TS 복합체를 통한 초기 연구는 격리된 α 서브유닛8,9,격리된 β 서브유닛10,11 및 α2 β 2TS 단지의 재구성에 초점을 맞추고 있다12. 정제는 황산염 강수량 암모늄, 샘플 투석, DEAE-세바덱스 크로마토그래피, 투석 및 DEAE-Sephadex 컬럼12에대한 제2 크로마토그래피 라운드에 의해 수행되었다. 또 다른 프로토콜에서, 동일한 복합체의 정제는 DEAE-Sephadex 컬럼에 정제된 세포 용재를 적재하고 세파로즈 4B 컬럼, 황산암모늄 강수량 및투석(13)에크로마토그래피 단계를 거쳐 개선되었다. 두 정화 프로토콜모두 4-5일 동안 지속됩니다. 대장균 α2 β2 TS 복합체는 결정화되었지만 그 당시 의결은 X선 회절에 적합하지 않았다.
새로운 연구에서는, 재조합 및 야생형 형태의 S. typhimurium α2β2 TS 복합체는14,15로정결되었다. 재조합 α2β2stTS 복합체는 pEBA-10 발현 벡터를 운반하는 대장균 CB149로 과발현되었다. 초기 결정화 및 X선 회절 데이터 수집 및 분석α2β2StTS 복합체의 분석은14보고되었다. 그러나, α 같은 길고 얇은 바늘2β2세인트TS 크리스탈 구조 연구를 손상. 더 나은 X선 회절 데이터를 수집하기 위해, 또 다른 정화 프로토콜은 α2β2StTS 복합체15의야생 유형 및 돌연변이 형태를 정화하기 위해 설명되었다. 정화는 스퍼민과 PEG 8,000을 사용하여 초기 침전을 명확히 된 세포 용액으로 수행하였고 큰 부피가 큰 침전물을 원심분리에 의해 제거하였다. α 2 β2세인트TS복합체의 높은 양을 포함하는 상주 분획은 황색 결정이 침전될 때까지 4°C에서 16-48h로 저장하였다. 크리스탈은 다른 버퍼에 대해 세척하고 광범위하게 투석되었습니다. 단백질 복합체는 황산암모늄을 함유하는 완충액으로재결정되었고, 투석15. 단백질 결정화는 용액의 단백질 및 침전물 농도에 따라 달라지지만, 용액에서 다른 돌연변이 형태인 α2β2StTS 콤플렉스에 대한 정화를 모니터링, 예측 및 재현하기는 어렵다. 이 프로토콜은 크로마토그래피 방법을 사용하지 않는다는 장점이 있습니다. 그러나, 단점은 결정화, 투석 및 재결정화에 필요한 긴 정화 시간이며, 전형적으로 5-7일이 필요하다. X선 데이터 수집에 적합한 결정을 얻기 위해 600개 이상의 결정화 조건은 단백질 농도, 온도, 강수제(PEG 4,000, 6,000 및 8,000) 및 첨가제(CaCl2,MnCl2,ZnCl2,카다베린, 퍼드린, 퍼드린, 스프레딘, 스프레시네, 스프레딘, 스프레시네,15. 크리스탈은 더 나은 결정 형태를 가지고 있었고 12 % PEG 8,000 및 2 mM 스페민을 포함하는 조건에서 더 빠르게 성장했습니다. 결정화는 4, 30 또는 42°C보다 25°C에서 더 유리했으며3일 15일이내에 최대 치수로 성장하였다. 몇몇α2β 2StTS 결정 구조물은 그 당시 (1996-1999)16,17,18,19,20,21 및 그밖 많은 구조물이 현재까지 간행되었습니다 보고되었습니다.
여기서 주요 목적은 트립토판 신체화를 정화하고 단백질 결정화를 최적화하기 위한 대체 프로토콜을 제시하는 것입니다. 본 작품은 야생형 절연 α 서브유닛(αStTS), 격리된 β 서브유닛(βStTS), α2 β2st TS 복합체, α2 β2StTS 복합체의 야생형 및 돌연변이 형태 를 정화하는 상당한 개선을보여준다. 정제 시간이 현저히 단축되고 결정화 및 극저온 보호가 최적화되었기 때문에 과거의 프로토콜에 비해 이점이 상당합니다. 이 작품에서 설계된 α2β2세인트TS 복합체의 돌연변이 형태는 야생 형 형태에 사용되는 동일한 조건 근처에서 결정화되었습니다. 그러나, 미세 결정화 최적화는 거의 원자 분해능에서 구조 판정을 위한 충분한 품질의 큰 단결정획득이 필요하였다. 현재까지, 단백질 데이터 뱅크(PDB)에 증착된 134개의 트립토판 신체 결정 구조물이 있으며, 각각 101, 31 및 2개의 결정 구조를 각각 박테리아, 고고학 및 진핵생물에 대해 회계합니다. 멋지게, 73 구조는 S. 엔테리카 세로바 티피무륨에 속하고 5 α 의 5 결정 구조2β2세인트TS 단지는 1.50 Angstroms보다 높은 해상도 한계를 가지고있다. 당연히, 4 아웃 5 우리의 연구 그룹에서 준비 되었다 (PDB IDs:5CGQ 에서 1.18 Å, 4HT3 에서 1.30 Å, 4HPJ 에서 1.45 Å, 6DZ4 에서 1.45 Å 해상도). α2β2StTS 복합체의 돌연변이 형태의 정제 된 결정 구조는 L-트립토판 합성에 관련된 필수 아미노산 잔류물이 연주 되는 메커니즘과 역할에 대한 새로운 통찰력을 제공 할 것으로 예상됩니다.

<table>
	<tbody>
		<tr>
			<td>15 mL 10 kDa filter</td>
			<td>MilliporeSigma</td>
			<td>UFC901024</td>
			<td>centrifugal filter unit</td>
		</tr>
		<tr>
			<td>15 mL 100 kDa filter</td>
			<td>MilliporeSigma</td>
			<td>UFC910024</td>
			<td>centrifugal filter unit</td>
		</tr>
		<tr>
			<td>2 mL cryogenic vials</td>
			<td>Corning</td>
			<td>CLS430489</td>
			<td>Cryogenic vials</td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-141</td>
			<td>microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>24-well Cryschem Plate</td>
			<td>Hampton Research</td>
			<td>HR3-158</td>
			<td>24-well sitting drop plates</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Fisher Scientific</td>
			<td>O3446I-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>50 mL centrifuge conical tubes</td>
			<td>Thermo Scientific</td>
			<td>12-565-270</td>
			<td>centrifuge conical tubes</td>
		</tr>
		<tr>
			<td>AB15 ACCUMET Basic</td>
			<td>Fisher Scientific</td>
			<td>13-636-AB15</td>
			<td>pH meter</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP1356-100</td>
			<td>Agarose gel</td>
		</tr>
		<tr>
			<td>ammonium sulfate</td>
			<td>Fisher Scientific</td>
			<td>A702-500</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760-5</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Bacterial incubator</td>
			<td>Fisher Scientific</td>
			<td>S35836</td>
			<td>incubator.</td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>New England Biolabs</td>
			<td>R0136S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>bicine</td>
			<td>Fisher Scientific</td>
			<td>BP2646100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Branson 450 Digital Sonifier</td>
			<td>Brason</td>
			<td>B450</td>
			<td>Cell disruptor</td>
		</tr>
		<tr>
			<td>Cesium chloride</td>
			<td>Fisher Scientific</td>
			<td>BP210-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Cesium hydroxide</td>
			<td>Acros Organics</td>
			<td>AC213601000</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Fisher Scientific</td>
			<td>BP904-100</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Fisher Scientific</td>
			<td>D1391</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Fisher Scientific</td>
			<td>BP172-5</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F530S</td>
			<td>HF polymerase</td>
		</tr>
		<tr>
			<td>dNTP Set</td>
			<td>Invitrogen</td>
			<td>10-297-018</td>
			<td>dNTPs set</td>
		</tr>
		<tr>
			<td>EcoRI</td>
			<td>New England Biolabs</td>
			<td>R0101S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Fisher Scientific</td>
			<td>S311-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td height="42">Excella E25R Orbital Shaker</td>
			<td>Eppendorf New Brunswick</td>
			<td>M1353-0004</td>
			<td>Orbital incubator</td>
		</tr>
		<tr>
			<td>GE AKTA Prime Plus</td>
			<td>GE Healthcare</td>
			<td>8149-30-0004</td>
			<td>FPLC</td>
		</tr>
		<tr>
			<td>Gel Extraction Kit</td>
			<td>Invitrogen</td>
			<td>K210012</td>
			<td>DNA purification kit</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>G33-500</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>HindIII</td>
			<td>New England Biolabs</td>
			<td>R0104S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>His-Trap columns</td>
			<td>GE Healthcare</td>
			<td>GE17-5255-01</td>
			<td>5 mL Histrap column</td>
		</tr>
		<tr>
			<td>imidazole</td>
			<td>Fisher Scientific</td>
			<td>O3196-500</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0392</td>
			<td>Inducer</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Kelvinator Series-100</td>
			<td>Kelvinator</td>
			<td>discontinued</td>
			<td>Ultra low freezer</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td>Liquid broth</td>
		</tr>
		<tr>
			<td>Luria Bertani agar</td>
			<td>Fisher Scientific</td>
			<td>BP1425-2</td>
			<td>Solid broth</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>NcoI</td>
			<td>New England Biolabs</td>
			<td>R0193S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Ni-NTA affinity beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>R90115</td>
			<td>Ni-NTA agarose beads</td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>Fisher Scientific</td>
			<td>BP233-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride</td>
			<td>Fisher Scientific</td>
			<td>44-865-0</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>pyridoxal phosphate</td>
			<td>Acros Organics</td>
			<td>AC228170010</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>S-200 HR</td>
			<td>Cytiva</td>
			<td>45-000-196</td>
			<td>Size exclusion column</td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Sorvall RC-5B centrifuge</td>
			<td>Sorvall</td>
			<td>8327-30-1004</td>
			<td>Floor cetrifuge</td>
		</tr>
		<tr>
			<td>Spermine</td>
			<td>Acros Organics</td>
			<td>AC132750010</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Superdex 200 prep grade</td>
			<td>Cytiva</td>
			<td>45-002-491</td>
			<td>Size exclusion column</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td>DNA liagse</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Fisher Scientific</td>
			<td>BP152-500</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Ubl-specific protease 1</td>
			<td>Thermo Scientific</td>
			<td>12588018</td>
			<td>SUMO Protease</td>
		</tr>
	</tbody>
</table>

pcr mutagenesis, cloning, expression, fast protein purification protocols and crystallization of the wild type and mutant forms of tryptophan synthase

살모넬라 타이피무륨의 트립토판 신체(TS) 바이엔자임 복합체(α2β2 TS)를 함유한 구조 연구는 촉매 메커니즘, 알로스터식 거동, 그리고 PLP 의존 효소에서 제품에 기판의 효소 변환에 대한 세부 사항을 더 잘 이해하기 위해 수행되었습니다. 이 작품에서, 고립된 α 및 고립된 β 서브유닛을 생성하는 새로운 표현 시스템은 2일 이내에 고립된 서브유닛으로부터 고량의 순수 서브유닛과 α2 β2stTS 콤플렉스의 정화를 허용하였다. 정제는 친화성 크로마토그래피에 의해 수행되었고, 그 다음으로 친화태그, 황산암모늄 강수량 및 크기 배제 크로마토그래피(SEC)의 분열이 뒤따랐다. 효소 β 부위의 주요 잔류물의 역할을 더 잘 이해하기 위해, 현장 직접 돌연변이 발생은 이전 구조 연구에서 수행되었다. 또 다른 프로토콜은 야생 유형과 돌연변이 α2β2세인트TS 단지를 정화하기 위해 만들어졌습니다. 암모늄 황산염 분획 및 SEC를 사용하는 간단하고 빠르고 효율적인 프로토콜은 하루에2β2StTS 단지의 α 정화할 수 있었습니다. 이 작업에 기재된 두 정제 프로토콜은 이전 프로토콜과 비교하여 PEG 8000및 spermine을 사용하여 동일한 복합체를 정화하여 정화 프로토콜을 따라 α2β2stTS 단지를 결정화할 때 상당한 이점이 있다. 야생 유형 및 일부 돌연변이 형태의 결정화는 PEG 8000 및 spermine을 사용하여 일부 돌연변이체의 정제를 손상시키는 약간 다른 조건에서 발생합니다. 엑스레이 결정학 연구에 적합한 결정을 준비하기 위해 결정화, 결정 품질 및 냉동 보호를 최적화하기 위해 몇 가지 노력을기울였습니다. 여기에 제시 된 방법은 일반적으로 트립토판 synthase 서브 유닛과 야생 유형 및 돌연변이 α2β2세인트TS 단지의 정화에 적용해야합니다.

α정화- 및 트립토판 신체의 β서브 유닛 살모넬라 타이피무륨 트립토판 신체의 α-서브유닛(αSt TS)과 β-서브유닛(βStTS)은 수정된 pET SUMO 벡터에서 분리하였다. 도 1A는 His6-SUMO-αStTS(레인 αON) 및 His6-SUMO-βStTS(레인 βON) 융합 단백질에 대응하는 두 개의 강력?...

Watch this Scientific Journal Video about PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase at JoVE

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

이 문서는 살모넬라 타이피무륨 트립토판 synthase의 발현 및 정화를 위한 일련의 연속적인 방법을 제시하며, 이 프로토콜은 하루에 단백질 복합체를 정화하는 신속한 시스템이다. 커버되는 방법은 현장 지향 돌연변이 발생, 대장균의단백질 발현, 친화 성염색로그래피, 젤 여과 크로마토그래피 및 결정화입니다.

PCR 돌연변이 발생, 복제, 발현, 빠른 단백질 정화 프로토콜 및 트립토판 신타제의 야생 유형 및 돌연변이 형태의 결정화

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

pcr-mutagenesis-cloning-expression-fast-protein-purification

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JoVE Journal

Biochemistry

4.2K 조회수.  University of California-Riverside. 이 문서는 살모넬라 타이피무륨 트립토판 synthase의 발현 및 정화를 위한 일련의 연속적인 방법을 제시하며, 이 프로토콜은 하루에 단백질 복합체를 정화하는 신속한 시스템이다. 커버되는 방법은 현장 지향 돌연변이 발생, 대장균의단백질 발현, 친화 성염색로그래피, 젤 여과 크로마토그래피 및 결정화입니다.

비디오: PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

항온, 발현, 정제, 인간 세로토닌 전달체 결정화 바운드<em&gt; S</em&gt; 시탈 로프 람

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

연구

생화학

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

진핵 세포에서 단백질 복합체의 탠덤 선호도 정화

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis is an obligate pathogen that causes fatal inhalational anthrax. Bacillus cereus is considered a useful model for studying B. anthracis due to its close evolutionary relationship. The gene cluster ba1554 - ba1558 of B. anthracis is highly conserved with the bc1531- bc1535 cluster in B. cereus, as well as with the bt1364-bt1368 cluster in Bacillus thuringiensis, indicating the critical role of the associated genes in the Bacillus genus. This manuscript describes methods to prepare and characterize a protein product of the first gene (ba1554) from the gene cluster in B. anthracis using a recombinant protein of its ortholog in B. cereus, bc1531.

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Bacillus anthracis is the obligate pathogen of fatal inhalational anthrax, so studies of its genes and proteins are strictly regulated. An alternative approach is to study orthologous genes. We describe biophysicochemical studies of a B. anthracis ortholog in B. cereus, bc1531, requiring minimal experimental equipment and lacking serious safety concerns.

재조합 단백질 발현, 결정화 및 생물 물리학 연구<em&gt; 바실러스</em&gt; 보존 된 뉴클레오티드 피로 인산화 효소, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize proteins, DNAs, RNAs, and their complexes can however not be predicted. Employing a broad variety of conditions is a way to increase the yield of quality diffraction crystals. Two fully automated systems have been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization screening against 1,920 initial conditions by vapor diffusion in nanoliter droplets. Semi-automated protocols have also been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefly discussed. Taken together, they enable convenient and highly efficient macromolecular crystallization in a multi-user facility, while giving the users control over key parameters of their experiments.

Automated systems and protocols for the routine preparation of a large number of screens and nanoliter crystallization droplets for vapor diffusion experiments are described and discussed.

분자 생물학의 MRC 실험실에서 고분자 결정에 대 한 프로토콜을 자동

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 1: 원스텝 단백질 단백질과 단백질 대사 산물 단지의 병렬 분석에 대 한 친 화력 정화

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

표현과 포유류 Bestrophin 이온 채널의 정화

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

카페인 추출, 효소 활동 및 식물 세포 정지에서 카페인 Synthase의 유전자 발현

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

표현, 가용 화, 그리고 진 핵 Borate 전송기의 정화

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.