This work was supported by Wellcome Trust grant 088419 to MDB.

GST-태그는 GTPase 1. 정제 <ol><li> 문화 E. 예컨대 220 rpm으로 흔들면서 37 ℃에서 유전자 pGEX-RAC1 O / N로 형질 전환 된 BL21로서 대장균, autoinduction 배지 500 ㎖ (25 mM의 나트륨 2 HPO 4, 25 mM의 KH 2 PO 4, 50 mM의 NH 4 CL, 5 mM의 SO 4 나 2, 2 mM의 황산, 2 mM의 CaCl2를, 0.5 % 글리세롤, 0.05 % 글루코스, 0.2 % 락토오스, 5g 트립 톤, 2.5 g 효모 추출물, 100 μg의 / ㎖ 암피실린). </li><li> 10,000 XG, 4 ° C에서 10 분 동안 원심 분리하여 수확 박테리아. </li><li> 20 ㎖의 단백질 추출 시약, 1X 프로테아제 억제제 세균 펠렛을 재현 탁하고 반전하여 실온에서 20 분 동안 배양한다. </li><li> 30 분 동안 40,000 XG에서 원심 분리하여 해물을 명확히. </li><li> 인산염 완충 식염수로 세척 2 ㎖의 글루타티온 자석 구슬, 추가 (PBS : 10 밀리미터 나 2 HPO 4, 1.8 mM의 KH 2 PO 4, 137 mM의 NaCl을, 2.7 밀리미터의 KCl). </li><li> 4 ° C에서 반전하여 혼합, 2 시간 동안 품어. </li><li> 각 단계에서 비즈를 침전 자분 분류기를 사용하여, 10 ml의 PBS로 단백질 로딩 비드 네 번 씻는다. </li><li> 필요할 때까지 재현 탁의 100 μL 씩에 -80 ° C에서 2 ml의 PBS에서 구슬과 상점을 단백질로드. </li></ol> 는 GTPase 결합 단백질의 2 식 <ol><li> 다음 실험 하루 전에, 녹색 형광 단백질 (GFP)을 코딩하는 플라스미드를 형질 HEK293T 별도 75 cm-2는 GTPase 플라스크에 각각 결합 단백질의 버전 -tagged. 염기로드의 검증을 위해, 트랜의 HEK293T의 세 번째 75 cm 2 플라스크에 TrioD1을 GFP - 태그. <ol><li> 100 μL에 1 ㎎ / ㎖ 멸균 150 mM의 염화나트륨에 polyethylamine을 희석. </li><li> 223 μL 감소 혈청 미디어에 27 μl를 희석 polyethylamine를 추가합니다. </li><li> 250 μL 감소 혈청 배지에 12 μg의 플라스미드 DNA를 추가합니다. </li><li> 실온에서 2 분 동안 각 튜브를 품어. </리&gt; <li> polyethylamine을 결합하고 DNA는 2 분에 대해 하나의 튜브와 소용돌이에 혼합. </li><li> 실온에서 15 ~ 20 분 동안 품어. </li><li> 5 ml의 신선한 성장 배지에 90 % 합류에 HEK293T 성장 배지 (둘 베코 변형 이글 미디어, 10 % 소 태아 혈청, 2 mM L- 글루타민, 항생제)를 대체. </li><li> 플라스크에 결합 polyethylamine / DNA 믹스를 추가하고, 37 ℃, 5 % CO 2에서 O / N을 품어. </li></ol></li></ol> 는 GTPase 결합 단백질의 3 정화 <ol><li> PBS에서 형질 세포의 플라스크를 씻어 무료로 액체를 흡입, 5 분 동안 플라스크 드레인. </li><li> 500 ㎕의 용해 완충액에 세포를 긁어 (50mM 트리스 - 염산 (pH7.8), 1 % Nonidet P-40, 1X 프로테아제 억제제) 미세 원심 분리 튜브에. </li><li> 30 분 동안 4 ° C에서 반전하여 혼합하여 세포를 Lyse. </li><li> 용해시, 세척 사이에 2 분 동안 2,700 XG에 40 μL GFP - 트랩 구슬이 많은 신선한 용해 완충액으로 3 회, 침강 구슬을 씻어. </li><li> 10 분 동안 21,000 XG에서 원심 분리 용 해물을 명확히. </li><li> 전사는 4 ° C에서 반전하여 혼합, 세척 GFP 트랩 비드를 분리 및 GFP 융합 단백질은 2 시간 동안 결합​​하게 경쟁 단백질의 각각의 용 해물을 정화. 얼음에 GFP - TrioD1 세포에서 해물을 유지합니다. </li><li> 50 mM 트리스 -HCl (pH 7.8), 50 mM의 NaCl을 두번로드 GFP 트랩 구슬 씻어 0.7 % (W / V) Nonidet P-40 회 50 mM 트리스 -HCl (pH 7.6)에서, 20 밀리미터의 MgCl 2 , 세척 사이에 2 분 동안 2,700 XG에 구슬을 침강. </li><li> 40 ㎕의 0.2 M 글리신 (PH 2.5)을 추가하고 30 초 동안 아래로 피펫 팅에 의해 GFP - 융합 단백질을 용출. 4 μL 1 M 트리스 - 염산 (PH 10.4)를 포함하는 새로운 미세 원심 분리 튜브에 21,000 60 초 동안 XG 및 전송 액체에서 즉시 침전물 구슬. 정제 된 단백질의 손상을 제한하기 위해 신속하게이 작업을 수행합니다. </li><li> 정량적 blott를 사용 상대적인 수율을 확립 항 GFP 항체와 함께 웨스턴 블랏에 의한 각 프로브와 정제 된 단백질의 1 μL를 분석제조 업체의 프로토콜에 따라 시스템을 보내고. 대안 적으로, bicinchoninic 산 (BCA) 단백질 분석으로 농도를 결정하지만, 단백질과 동일한 방식으로 분석과 반응하지 않거나 오염 단백질이 존재하면 에러를 도입한다. </li><li> 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2를 첨가하여 단백질의 몰 농도를 등화. </li></ol> 는 GTPase의 4 염기로드 <ol><li> 단계 1에서 제조 한 GST-RAC1 자성 비드, 하나의 분취 량을 해동. </li><li> 각 단계에서 비즈를 침전 자분 분류기를 사용하여, GST-RAC1 비드의 90 μl를 취하여 20 mM 트리스 - 염산 (PH 7.6), 25 mM의 염화나트륨, 0.1 mM의 DTT, 4 mM의 EDTA로 3 회 세척 하였다. </li><li> 대기음 구슬에서 버퍼와 100 μL 20 mM 트리스 - 염산 (PH 7.6), 25 mM의 NaCl을 0.1 mM의 DTT, 4 mM의 EDTA를 추가합니다. </li><li> 국내 총생산 (GDP), GTP 또는 전혀 염기 로딩 경쟁 실험에 필요한지 여부에 따라, 12 μl의 100 mM의 국내 총생산 (GDP), 12 ㎕의 10 mM의 번지를 추가anosine 5 &#39;- [γ-티오] 트리 포스페이트 (GTPγS) 또는 60 μL GST-RAC1 비즈없이 염기. </li><li> 염기 로딩 컨트롤, 세 10 μL 분취에 남아있는 구슬을 분할하여 2 μL 100 mM의 국내 총생산 (GDP), 2 μL 10 밀리미터 GTPγS 각 튜브없이 염기를 추가합니다. </li><li> 교반하면서 30 ℃에서 30 분 동안 비드 혼합물을 배양한다. </li><li> 실험 믹스 3 μL (단계 4.4), 제어 믹스 (단계 4.5)의 각각 0.5 μL 1 M의 MgCl 2를 첨가하여 핵산 - 결합 RAC1 안정화. </li></ol> 바인딩 5. 경쟁. <ol><li> 6의 microfuge 튜브를 설정, 각 포함 : 200 ㎕의 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2 (단계 4.7에서) 10 μL 실험 염기로드 RAC1 비즈 5 μL RAC1 결합 단백질 A (단백질 결합 상수) </li><li> 각 튜브에, 0, 1, 2.5, 5, 10 또는 20 μL의 RAC1 - 결합 단백질 B (가변 결합 단백질)를 추가한다. 이 볼륨은 가정pproximately 동일한 주식 상수와 변수 결합 단백질의 농도를 조정해야 할 수도 있습니다. <ol><li> 이 두 단백질의 결합 친화도에 큰 차이가 있으며 이것이 반복 실험을 통해 경험적으로 결정되어야하는 경우 결합 단백질의 양 및 B를 조정한다. 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2를 첨가하여 235 μL로 결합 혼합물의 전체 부피를 차지한다. </li></ol></li><li> 포함하는 미세 원심 분리 튜브를 설정합니다 : 200 ㎕의 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2 (단계 4.7에서) 10 μL 실험 염기로드 RAC1 비즈 10 μL RAC1 결합 단백질 A (단백질 결합 상수) </li><li> 국내 총생산 (GDP), GTPγS없이 염기 제어 튜브를 설정합니다 : 200 ㎕의 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2 10 μL 제어 RAC1 비즈는 국내 총생산 (GDP), GTPγS 또는 전혀 염기와 4.5 단계에서로드 단계 4.7에서 안정화. 180 μL H단계 3.6에서와 같이 제조 EK293T GFP - TrioD1 용 해물, 4 μL 1 M의 MgCl 2 </li><li> 4 ° C에서 반전하여 혼합, 2 시간 동안 상기 혼합물을 배양한다. </li><li> 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2 비드 세 번 세척 하였다. </li><li> 용출 시료 완충액을 20 μL 환원 단백질을 결합 (50 mM 트리스 -HCl (pH 7), 5 % SDS, 20 % 글리세롤, 0.02 ㎎ / ㎖ 브로 모 페놀 블루, 5 % β 메르 캅토 에탄올). </li></ol> 경쟁 6. 분석 <ol><li> 소듐 도데 실 설페이트 폴리 아크릴 아미드 겔 전기 영동 (SDS-PAGE) 및 웨스턴 블롯에 의해 결합 된 단백질 (단계 5.6) 10 μL를 해결. </li><li> 39 °에서 CO 막을 인큐베이션 / N 안티 GFP 항체로 블로킹 완충액으로 1/1000 희석하여 PBS로 1 배 희석하고, 0.1 % 트윈 -20은이 태그는 GTPase - 결합 단백질의 양을 검출한다. </li><li> PBS로 10 분, 0.1 % 트윈 20 막 세 번 씻으십시오. </li><li> 800 - 공액 안티 - 토끼 초 DyLight에 RT에서 30 분 동안 인큐베이션 막을ondary 항체, 버퍼를 차단에서 1 / 10,000을 희석하면, 0.1 % 트윈 20 PBS에 1 배 희석. </li><li> PBS로 10 분, 0.1 % 트윈 20 막 세 번 씻으십시오. </li><li> 제조사의 프로토콜에 따라 밴드 강도를 측정하기 위해 소프트웨어를 사용하여, 적외선 이미지 시스템을 사용하여 멤브레인을 스캔. </li><li> 가변 경쟁자 (단백질 B)의 양에 대해 각 단백질 밴드의 강도를 플롯. </li><li> 라인이 평형이 달성되는 경쟁자 비율을 결정하는 상수 경쟁자 (단백질 A, 5 μL)의 체적 교차하는 점에서 가변 경쟁자의 볼륨을 나눈다. </li><li> 단계 6.1-6.6에 설명 된대로 P21 활성화 키나제 1 (PAK1) (이펙터)와 GFP - TrioD1 (GEF)에 대한 염기 로딩 상태, 프로브 막 확인하십시오. </li></ol>

<ol>
	<li>Burridge, K., Rho Wennerberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=and+Rac+take+center+stage.">and Rac take center stage.</a> Cell. 116 (2), 167-179 (2004).</li><li>Raftopoulou, M., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+Rho+GTPases+lead+the+way.">Cell migration: Rho GTPases lead the way.</a> Dev Biol. 265 (1), 23-32 (2004).</li><li>Rossman, K. L., Der, C. J., Sondek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GEF+means+go:+turning+on+RHO+GTPases+with+guanine+nucleotide-exchange+factors.">GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors.</a> Nat Rev Mol Cell Biol. 6 (2), 167-180 (2005).</li><li>Worthylake, D. K., Rossman, K. L., Crystal Sondek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=structure+of+Rac1+in+complex+with+the+guanine+nucleotide+exchange+region+of+Tiam1.">structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1.</a> Nature. 408 (6813), 682-688 (2000).</li><li>Scheffzek, K., Ahmadian, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPase+activating+proteins:+structural+and+functional+insights+18+years+after+discovery.">GTPase activating proteins: structural and functional insights 18 years after discovery.</a> Cell Mol Life Sci. 62 (24), 3014-3038 (2005).</li><li>Del Pozo, ., A, M., 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=Integrins+regulate+GTP-Rac+localized+effector+interactions+through+dissociation+of+Rho-GDI.">Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI.</a> Nat Cell Biol. 4 (3), 232-239 (2002).</li><li>Williamson, R. C., 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=Coronin-1C+and+RCC2+guide+mesenchymal+migration+by+trafficking+Rac1+and+controlling+GEF+exposure.">Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure.</a> J Cell Sci. 127 (Pt 19), 4292-4307 (2014).</li><li>Del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+to+the+extracellular+matrix+regulates+the+coupling+of+the+small+GTPase+Rac+to+its+effector+PAK.">Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK.</a> Embo J. 19 (9), 2008-2014 (2000).</li><li>Van Rijssel, J., Hoogenboezem, M., Wester, L., Hordijk, P. L., Van Buul, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-terminal+DH-PH+domain+of+Trio+induces+cell+spreading+and+migration+by+regulating+lamellipodia+dynamics+in+a+Rac1-dependent+fashion.">The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion.</a> PLoS. 7 (1), e29912 (2012).</li><li>Self, A. J., Hall, A. <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+intrinsic+nucleotide+exchange+and+GTP+hydrolysis+rates.">Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates.</a> Methods Enzymol. 256, 67-76 (1995).</li></ol>

저자가 공개하는 게 없다.

This protocol describes a method for comparing the relative affinities of pairs of small GTPase-binding proteins. The key steps are the preparation of purified GTPase-binding proteins and the nucleotide loading of the GTPase. The use of GTPase-binding proteins with the same GFP tag, allows the concentrations at which similar amounts of each competitor binds to be accurately determined. The use of recombinant nucleotide-loaded GTPase allows interrogation of the binding properties of the GTPase under specific activity conditions. This step is also the most sensitive as nucleotides will both hydrolyze and detach from the GTPase if the magnesium conditions are not maintained precisely.
In the cell, the large number of GTPase-binding proteins combined with the rapid nucleotide turnover makes such pathways difficult to interpret. The simplicity of this method in comparing only pairs of binding proteins and using carefully controlled nucleotide-loading conditions allows signaling pathways to be elucidated. However, the greatest strength of the protocol is also the greatest weakness as it is a simplification of the in vivo situation. Competition assays can be used to build a robust hypothesis, but this should then be tested in cells by knockdown experiments.
There are three features that must be considered when selecting the GFP-tagged GTPase-binding proteins to be used in the experiment. First, the fusion proteins must express well in mammalian cells, such as HEK293T, as competition assays require a reasonable amount of protein. Second, it must be possible to purify the recombinant protein without significant degradation, and where this is not possible, cloning of a GTPase-binding fragment should be considered. Third, the two GTPase-binding proteins must resolve from one another on SDS-PAGE to allow analysis in section 6.
There are a number of potential caveats to the experiment that need to be considered, and possibly addressed:
Possible denaturation of purified GTPase-binding proteins during the acid elution step or steric hindrance by the GFP tag. In our hands, these have not been a problem, but must be tested. The purified proteins can be tested in functional assays 10. Commercial kits now exist for testing the activity of GEFs or GAPs without the need for isotope-labeled nucleotides. Sequestering proteins, by their nature protect GTPases from GEF or GAP activity, so can be used as competitive inhibitors in the commercial GEF or GAP assays, as we did in our recent publication 7. The relevant feature of proteins that traffic GTPase are the capacity to bind the GTPase, and this can be tested easily in a pull down assay. An alternative approach to testing protein integrity that is applicable to all binding proteins is to titrate protein eluted from GFP-trap beads with glycine with the same protein removed from GFP-trap beads by enzymatic cleavage. The experiment would be analyzed by probing both the GFP-tagged and cleaved protein with an antibody against the protein itself. If the protein is undamaged by elution, equilibrium should be achieved at a 1:1 ratio. This approach would also indicate whether the presence of the GFP tag itself compromises the binding properties of the candidate protein, though this does require the production of a construct with an enzymatic cleavage site between the tag and the binding protein. Whether the protein is compromised by the tag or the elution step, the problem could be addressed by modifying the protocol to use an alternative purification method. Rather than GFP, binding proteins could be His-tagged, purified using Ni-NTA and analyzed using an antibody against the His-tag. The important feature is that both binding proteins must share a common tag although, if necessary, two tags could be added to a protein, one for purification and the other for detection.
The protocol is designed to investigate competition between interactions with the switch I/II domains. Although the majority of GTPase interactions are mediated by this motif, there are some exceptions, most notably the interactions of GDIs that bind to the prenyl tail, as well as obscuring the switch domains. In principle, the protocol could be adapted to use GTPase purified from mammalian cells, so that the GTPase is prenylated, however, the presence of multiple binding sites or allosteric effects complicate the interpretation of competition-binding data. Further problems associated with such a modification are that GDIs co-purify with GTPase from mammalian cells, compromising the purity of the isolated proteins and the hydrophobic nature of the prenyl groups means that prenylated GTPases are associated with either GDI or lipid membrane and such factors would need to be considered in the experiment.
The amount of GST-Rac1 being used in the assay. The constant GTPase binding protein must be at a greater concentration than the Rac1, or when the competitor is added, it will simply bind to free Rac1. It will be immediately obvious if this has happened as binding of the competitor, without a loss of the constant protein, will be detected in much the same way as when the two competing proteins bind to one another as shown in Figure 3B. As an additional control (Step 5.3), a binding reaction containing double the amount of constant binding protein and no variable binding protein should be included (Step 5.3). If the Rac1 in the titration experiment is saturated, doubling the amount of constant binding protein will have no effect on the output. The volumes suggested in the protocol should be appropriate, but the amount of Rac1 can be easily reduced. If binding of the competitor without loss of the constant binding partner is observed, reducing the amount of Rac1 should be attempted before trying to map binding sites to avoid ternary complex formation.
Non-specific interaction of GTPase-binding proteins with the GST or bead, as well as specifically with Rac1. This problem would be manifested by residual binding of the constant GTPase-binding protein, even when the variable GTPase-binding protein has reached a plateau at high concentration. Identification of this issue will be aided by conducting reciprocal experiments where the constant and variable GTPase-binding proteins are swapped. Reciprocal experiments will also greatly improve the accuracy of the estimate of equilibrium point, so should always be included. In cases of non-specific binding, the relative concentrations at which equilibrium is achieved can still be calculated by comparing band intensity between the maxima and minima for each protein, or by measuring the extent of non-specific binding by using GST beads as bait, rather than GST-Rac1.
Pull down assays using different nucleotide-loading conditions should be used to complement the competition assay described in this protocol. Determining the nucleotide preference of partners is important for both understanding the competition events and understanding the signaling pathway that the GTPase-binding protein is involved in. In Figure 2B we analyze binding of proteins with established preference for GTP-loaded or nucleotide-free GTPase as a means to validate nucleotide loading. However, it is sensible to investigate the effect of nucleotide loading on each of the competitors as well. If the hypothetical competitors show different preferences, competition will make less of a contribution to the signaling pathway, and indeed nucleotide turnover is likely to be the mechanism that directs exchange of the binding proteins.

The actin cytoskeleton that determines the shape, polarity and migratory properties of mammalian cells is regulated by the Rho-family of small GTPases. The Rho-family GTPases include RhoA that stimulates cytoskeletal contraction, Rac1 that stimulates actin branching and membrane protrusion, and Cdc42 that has similar effects on actin polymerization to Rac1 and causes the formation of filopodia 1,2. GTPase signaling activity is determined by binding of a nucleotide, which controls the contraction and relaxation of the switch I and switch II loops that mediate the protein-protein interactions with both regulators and effectors. Guanosine 5&#8217;-triphosphate (GTP)-bound GTPases activate downstream effectors, whereas the Guanosine 5&#8217;-diphosphate (GDP)-bound form is inactive. In the cell, cycles of GTP hydrolysis and nucleotide exchange allow rapid turnover of GTPase signals that are necessary for cytoskeletal dynamics. Nucleotide turnover is regulated by three mechanisms. Guanine nucleotide exchange factors (GEFs) stabilize the nucleotide-free GTPase, catalyzing exchange of GDP for GTP, and thereby stimulating GTPase signaling activity 3,4. GTPase-activating proteins (GAPs) catalyze hydrolysis of GTP to GDP, thereby inhibiting GTPase signaling activity 5. Sequestering molecules such as regulator of chromatin condensation 2 (RCC2) and guanine nucleotide dissociation inhibitors (GDIs) obscure the switch loops and in the case of GDIs remove the GTPase from the membrane by interaction with the prenyl tail 6,7. Each of the three classes of regulatory molecule interact with the switch loops, as do the downstream effectors and some trafficking regulators such as coronin-1C 7. The purpose of this protocol is to measure competition for the switch I/II binding site between putative regulators and downstream signaling molecules. It should be noted that competition assays test binding to a shared binding site, so that this protocol is not suitable for testing interactions with other sites, such as binding of GDIs to the prenyl tail.
The subtlety of the conformation differences between active and inactive forms, combined with the labile nature of the bound nucleotide, has made study of GTPase-binding events difficult. The role of the bound nucleotide means that conventional binding assays such as immunoprecipitation or surface plasmon resonance are not well suited to investigation, as the nucleotide cannot be controlled. This obstacle is compounded by the overlap in the binding sites of GEFs, GAPs, effectors, sequestering molecules and trafficking molecules, which make binding data for a single interaction difficult to interpret in the context of the competition that will occur in the cell. Immunoprecipitation, in particular, is compromised by competition between binding partners, as under certain cellular conditions, one binding partner might be identified at the expense of all others, while under other conditions, another partner might dominate. The dynamic nature of GTPase signaling is essential to GTPase function and must be considered when analyzing the relationships between the binding interactions of different regulators. Indeed, we recently described a pathway that relied heavily on competitive binding. We identified coronin-1C as a trafficking molecule that bound to the switch loops of GDP-Rac1 7. In areas of low GEF activity, trafficking would dominate, removing Rac1 from those regions. However, when Rac1 is delivered to regions of the cell where GEF activity is high, the GEF would outcompete coronin-1C, thereby both activating Rac1 and preventing coronin-1C-mediated removal of Rac1 from that area. The model goes further, because the action of the GEF exchanges bound GDP for GTP, shifting the equilibrium still further from coronin-1C. Consequently, Rac1 activity could be explained entirely in terms of competition and relative affinity.
In this protocol, we describe a method for comparing the relative affinities of different binding partners for small GTPases, using Rac1 as an example. By using a purified protein approach, it is possible to piece together a chain of signaling events by pair wise comparison, in an experiment where the bound nucleotide can be closely controlled.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bugbuster</td>
			<td>Novagen</td>
			<td>70584-3</td>
			<td></td>
		</tr>
		<tr>
			<td>COMPLETE protease inhibitor</td>
			<td>Roche</td>
			<td>05 056 489 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione magnetic beads</td>
			<td>Pierce</td>
			<td align="right">88821</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, branched, average Mw ~25,000</td>
			<td>Sigma Aldrich</td>
			<td>408727-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>OPIMEM</td>
			<td>Life Technologies</td>
			<td>31985-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Media</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10270-1-6</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-Trap_A</td>
			<td>Chromotec</td>
			<td>gta-20</td>
			<td></td>
		</tr>
		<tr>
			<td>GDP</td>
			<td>Sigma Aldrich</td>
			<td>G7127</td>
			<td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td>
		</tr>
		<tr>
			<td>GTP&#947;S</td>
			<td>Sigma Aldrich</td>
			<td>G8634</td>
			<td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td>Sigma Aldrich</td>
			<td>B6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Living Colors</td>
			<td align="right">632592</td>
			<td>Use at 1/1000 dilution</td>
		</tr>
		<tr>
			<td>DyLight 800 conjugated goat anti-rabbit secondary antibody</td>
			<td>Fisher Scientific</td>
			<td align="right">10733944</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PAK1 antibody</td>
			<td>Cell Signaling</td>
			<td>2602S</td>
			<td>Use at 1/1000 dilution</td>
		</tr>
		<tr>
			<td>Odyssey SA Infrared Imaging System</td>
			<td>Li-cor</td>
			<td>9260-11PC</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparing the affinity of gtpase-binding proteins using competition assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

이 프로토콜은 경쟁사의 정확한 농도를 알 필요 (그림 1)하지 않고, RAC1 바인딩 파트너의 상대적 친화도를 계산하도록 설계되었습니다. 단백질 농도의 측정 오류를 소개하고 신호 경로의 분자 사이의 경쟁을 고려할 때 필요하지 않습니다. 그러나, 두 경쟁 분석법으로 상이한 양을 추가 할 때 단순 비율이 계산 될 수 있도록하는 스톡 용액에 동일 몰 농도가 있음을 아는 것이 중요하다. GFP 트랩 비드 40 ㎕를 300 pmol의 그래서 합류 75cm 높은 두 개의 다른 결합 단백질의 제제는 조정 전의 유사 할 것이라는 결과, 비드 포화 것이다 발현 세포의 2 플라스크 ~의 결합능을 가지고 (도 2A). 단백질 중 하나가 잘못 표현하면,이 문제는 셀의 하나 이상의 플라스크에서 그 단백질을 정제에 의해 극복 될 수있다. <p claSS = &quot;jove_content&quot;&gt; 대부분의는 GTPase 이펙터와 규제의 결합은 미끼는 GTPase의 염기 로딩에 따라 달라집니다, 그래서 로딩이 성공 여부를 테스트하는 것이 중요합니다. 로드 중 세포 용 해물로부터 공지 결합 단백질을 침전에 의해 검증 될 수있다. 이러한 GTP-RAC1과에 PAK1 바인드로 이펙터 단백질은 쉽게 해물에서 침전 및 웨스턴 블로 팅 (8) (그림 2B)에 의해 검출 될 수있다. 전이 상태를 안정화시키기는 GTPase없는 뉴클레오티드 우선적 결합 GEFs. GEFs는 일반적으로 비활성 낮은 풍부,의이고 자주 가난시킨다, 그것은 시험 염기 프리는 GTPase에 GEF 또는 GEF 단편을 과발현하는 것이 좋습니다. 우리는 자주 트리오의 첫 번째 DBL 동성을 사용하여, GFP 융합 (GFP - TrioD1 9) (그림 2B)로 표현하지만 어떤 GEF는 작동합니다. 국내 총생산 (GDP)로드는 GTPase에 결합하는 단백질은 희소하다. BINDI로 간단하게 검증 할 수있는 하나의 단백질 (7), 또는 국내 총생산 (GDP) 로딩으로 우리는 최근 RCC2보고도 지구 환경 기금이나 이펙터에 겨. 실험의 출력에 결합는 GTPase 두 GFP 표지 된 결합 파트너를 나타내는 웨스턴 블롯 것이다. 두 단백질을 검출하기 위해 하나의 항체를 사용하여, 두 경쟁 유사한 양 결합되는 농도를 결정할 수 있으므로 상대적 친화 유추. RAC1 인신 매매 단백질의 프로펠러 도메인 사이에이 예 대회에서, coronin-1C (단백질을 RAC1가 결합) 및 RAC1 - 금속 이온 봉쇄 단백질, RCC2은 (단백질 B를 RAC1가 결합), (그림 3A)를 시연한다. coronin-1C 프로펠러 (5 μL)의 일정한 볼륨을 사용하고, RCC2의 증가 볼륨을 추가함으로써, 우리는 RCC2가 강하게 가지고 있음을 보여주는 GFP에서 그 평형이 RCC2 (별표)의 1.25-2.5 μL에 도달시킨다 볼 수 있습니다 coronin-1C보다 RAC1에 대한 친화력. 각 competito 평균치를 정량적 웨스턴 블 롯팅을 사용하여 밴드의 강도를 측정하고, 플롯에 의해R, 평형 점은 볼륨되는 곡선 (도 3b)를 교차를 식별하여 정확하게 계산 될 수있다. 결합 파트너가 서로뿐만 아니라 RAC1 결합에 결합하는 경우 성공적인 경쟁 분석에 사용할 수있는 하나의 장애물이다. 그림 3A의 + B의 우리는 오히려 전체 길이 coronin-1C보다 RCC2과 coronin-1C의 프로펠러 도메인 간의 경쟁을 보여줍니다. 절두 coronin를 사용하는 이유는 coronin-1C는 테일 도메인 통해 RCC2 결합한다는 것이다. 전장 coronin-1C는 RCC2 대해 적정하면, 두 단백질의 결합으로 인해 삼원 복합체 형성보다는 경쟁 (도 3c)로 검출된다. 경쟁이 발생하는 경우, 하나의 단백질의 결합은 다른 감소하면서 증가하고 총 결합 GFP 융합은 일정하게 유지 될 것이다. 경우 삼원 복합체는는 GTPase 결합 단백질 중 하나자를 형성 할 필요가 있기 때문에 여기서 compet적인 억제제는 더 이상 상호 작용하지 않습니다. <img alt="그림 1" src="/files/ftp_upload/53254/53254fig1.jpg" /> 그림 1. 워크 플로우. 경쟁 분석을 사용하여는 GTPase 결합 단백질의 친 화성을 ​​결정하기위한 워크 플로우의 도식 표현. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/53254/53254fig2.jpg" /> 정제 된 단백질의 그림 2. 검증. (A)으로 정제하여 GFP - 태그 두 단백질의 상대 수익률을 결정하는 안티 GFP와 프로빙, 웨스턴 블롯 분석 결합 단백질 RAC1. 실험 기간 동안 보상이 유형들이 결합 실험에 일치하도록 두 단백질의 농도가 조절 될 수있다. (B) GDP, GTPγS없이 염기로드 GST-RAC1이 내생 PAK1 또는 과발현 GFP - TrioD1에 대한 플롯에 의해 감지 단백질 GFP - TrioD1을 표현 HEK293T에서 해물 배양과 결합되었다. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/53254/53254fig3.jpg" /> 상대 단백질의 그림 3. 웨스턴 블롯 분석을 결합. 예 출력을 경쟁 결합 분석에서. (A) RAC1 5 μL GFP - coronin-1C 프로펠러 도메인과 혼합에 적정 된 GFP - RCC2의 볼륨을 증가하고 국내 총생산 (GDP)로드.으로 GFP에 대한 웨스턴 블롯 결합 단백질, 두 단백질의 검출 시차의 문제가 회피되고, GFP 신호는 두 개의 융합 단백질 사이의 몰비를보고한다. 별표는 eithe의 경쟁 비율을 나타냅니다평형 점의 R 측. (B) 3 개의 독립적 인 실험으로부터 바운드 GFP 융합 단백질의 밴드 강도는 평형에 도달하는 데 필요한 형광 접합 된 이차 항체 및 평균 RCC2의 양을 계산하는 플롯하여 정량적 웨스턴 블롯으로 측정 하였다. (C RAC1 결합 단백질 오히려 경쟁보다는 서로에 결합 원 복합체를 형성 실험에서) 예 출력. 국내 총생산 (GDP)이 장착 된 RAC1 5 μL의 GFP - RCC2와 혼합 GFP - coronin-1C 전체 길이의 볼륨을 증가했다가에 적정 하였다. 바운드 GFP - coronin-1C의 증가를 결합 GFP - RCC2의 손실없이이 원 복합체 형성을 나타냅니다. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig3large.jpg" target="_blank">하십시오 이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Comparing the Affinity of GTPase-binding Proteins using Competition Assays at JoVE.com

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

경쟁 분석법을 이용는 GTPase - 결합 단백질의 친화 비교

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...

comparing-affinity-gtpase-binding-proteins-using-competition

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JoVE Journal

Biology

9.0K 조회수.  University of Bristol. This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

비디오: Comparing the Affinity of GTPase-binding Proteins using Competition Assays

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

사용 Schwann - astrocyte 상호 작용 분석 체외에서 Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

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생물학

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Lipidic Mesophases의 막 단백질의 결정화

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Lipidic Bicelle 방법을 사용하여 막 단백질의 높은 처리량 결정

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

탠덤 동질 정제를 사용하여 단백질 나누기 파트너의 식별

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

선호도 태그 재조합 단백질의 높은 처리량 정화

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST - 그의 정화 : 전체 길이 정제 된 단백질을 항복 2 단계 친화력 정화 프로토콜

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

생체 외 분석에서 집계 Hyperphosphorylated 타우 단백질을 사용하여

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

후생 동물 모델 생물을 사용하여 확산 및 독성 프리온 같은 단백질을 조사<em&gt; C. 엘레</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

탠덤 선호도 정화를 사용하여 MyoD 상호 작용 체의 식별 질량 분석에 결합

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...