Name: production and purification of baculovirus for gene therapy application
Uploaded: 2018-04-09T13:30:00.000+00:00
Duration: 6 min 11 s
Description: Watch this Scientific Journal Video about Production and Purification of Baculovirus for Gene Therapy Application at JoVE.com

这项工作的部分原因是由辛辛那提儿童研究基金会 (CCRF) 为 M.N. 和创新核心赠款提供资金, 该基金由国家卫生研究院促进翻译科学中心资助。奖项编号 UL1 TR001425。 内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。

有关汇总协议的说明, 请参见图 1 。
1. 杆状病毒质粒 DNA 的纯化
<ol><li>用抗生素在200毫升的 LB 汤中生长含有杆状病毒质粒 DNA (bacmid) 14 的细菌培养;卡那霉素 (50 µg/毫升), 四环素 (10 µg/毫升), 和庆大霉素 (7 µg/毫升), 16 h 在37°c 在一个轨道振动筛设置在 300 rpm。</li>
	<li>使用标准碱性裂解协议15从细菌培养中纯化 bacmid DNA, 并将 bacmid 溶解在 TE 缓冲器中。</li>
</ol>2. 杆状病毒的生产
<ol><li>培养 Sf9 细胞的轨道振动筛孵化器在 135 rpm 和28°c 的聚碳酸酯锥形瓶含有无血清昆虫培养介质1,2,3。 注意:如果 Sf9 细胞从冷冻的库存解冻, 允许至少两个星期的细胞恢复和进入指数阶段的增长。</li>
	<li>用台盼蓝染色 hemocytometer, 计数在生长指数阶段 Sf9 细胞。种子 1 x 106可行的 Sf9 细胞每井在6井组织培养处理的板材在2毫升培养基中。允许单元格在孵化器中的28摄氏度处附加1小时。</li>
	<li>用1毫升无血清 unsupplemented 的昆虫培养基取代生长培养基。</li>
	<li>利用昆虫细胞转染试剂将 bacmid DNA 染到 Sf9 细胞中。<ol>
<li>将8µL 昆虫细胞转染试剂与100µL 的宽限期昆虫培养基混合使用。</li>
		<li>将2µg 的 bacmid DNA 稀释成 unsupplemented 昆虫培养基的100µL, 并通过涡流轻轻混合。</li>
		<li>将稀释后的 DNA 与稀释后的昆虫细胞转染试剂结合, 轻轻混合。室温下孵育15至30分钟。</li>
		<li>将 DNA 脂质混合物滴在细胞上。孵化在28°c 在一个孵化器大约5小时。</li>
	</ol>
</li>
	<li>从细胞中取出转染的混合物, 用2毫升的 PBS 冲洗细胞。</li>
	<li>添加2毫升的昆虫培养基, 并继续培养在28摄氏度的孵化器, 而不改变媒体。 注意:如果 bacmid 含有荧光蛋白, 其表达可以在48小时后转染细胞中检测到。杆状病毒由转染的 Sf9 细胞产生, 并分泌到随后感染 untransfected 细胞的培养基中。整个细胞群在5天内就会感染杆状病毒, 并显示病毒感染的迹象, 也称为病变效应。细胞培养中包括细胞直径的增加, 胞浆中的空泡/颗粒, 细胞生长的停止和死或裂解细胞的存在。然而, 如果转染或感染的效率不是最佳, 文化可能不会显示明显的病变效果。病变效应可以用 200-400X 放大的倒置相显微镜进行可视化。用 anti-gp64 抗体11染色可检测杆状病毒感染细胞。</li>
	<li>在转染和标签为 P1病毒储存后5天收获杆状病毒上清。</li>
	<li>储存的杆状病毒上清, 这是轻敏感, 在黑暗中与0.5% 牛血清白蛋白 (BSA) 在 PBS。贮存在4°c 为短期 (最多90天) 或在-80 摄氏度长期。</li>
	<li>种子 6 x 106 Sf9 细胞在 10 cm 组织培养处理的板材在10毫升昆虫培养基。将1毫升的最初杆状病毒上清 (P1) 添加到细胞中。</li>
	<li>收获杆状病毒上清 (P2) 在 5th天后感染。</li>
	<li>种子50毫升的 Sf9 细胞 (3 x 106细胞/毫升) 的悬浮培养在一个250毫升聚碳酸酯锥形瓶, 并把瓶子在一个轨道振动筛孵化器。将2毫升的 P2库存添加到单元格中, 并在 135 rpm 处晃动, 同时保持28°c 的恒定温度。 注意:感染后三或四天, 整个 Sf9 细胞将显示病变效应, 这是高效杆状病毒产生的征兆。</li>
	<li>依次放大杆状病毒上清液直到获得所需的体积 (p3,p4,...) 通过增加10到50倍的细胞培养量在随后的每一次感染。 注意:p3是 3rd一轮感染, 其中500毫升至 1 L 的 Sf9 细胞培养可感染10至20毫升 P2杆状病毒上清;p4是 4th的一轮感染, 其中5至 10 L 的 Sf9 细胞培养可感染100至200毫升 P3杆状病毒上清, 等等。通常, 在无血清昆虫培养基中, Sf9 细胞的种子为 3 x 106细胞/毫升。</li>
	<li>离心的杆状病毒上清 1000 x g 30 分钟在4°c 去除细胞碎片和治疗杆状病毒上清与50单位/毫升核酸酶 (250 单位/µL 股票) 2 摄氏度, 以消化基因组 DNA 和 RNA。通过0.45 微米的过滤单元过滤上清液。</li>
</ol>3. 色谱系统的制备
<ol><li>用无菌水、1氮氢氧化钠、水和洗涤缓冲器 (20 毫米磷酸钠缓冲液, 含150毫米氯化钠, pH 7.0), 以50毫升/分钟的线性流速, 依次冲洗样品和缓冲线, 制备色谱系统。</li>
	<li>准备一个肝素50µm 柱 (10 x 10 厘米, 7.9 毫升的柱容积 (CV)) 按顺序运行的柱清洁缓冲 (5 cv 无菌20毫米磷酸钠缓冲液含2米氯化钠, pH 7.0) 和 5 CV 洗涤缓冲器在线性流量的7毫升/分钟。</li>
</ol>4. 杆状病毒载体的纯化
<ol><li>建立色谱系统如下:<ol>
<li>使用样品装货入口口岸装载杆状病毒上清。</li>
		<li>使用缓冲入口端口 A1 加载洗涤缓冲器。准备一瓶至少500毫升的洗涤缓冲器, 并将洗涤缓冲线放进瓶子里。</li>
		<li>使用缓冲入口端口 A2, 用于装载洗脱缓冲器 (20 毫米磷酸钠, 1.5 米氯化钠, pH 值 8.0)。准备一瓶至少500毫升洗脱缓冲器, 并将洗脱缓冲线放进瓶子里。</li>
		<li>将50毫升锥形管插入到分数收集器中, 收集柱通过杆状病毒上清, 洗涤缓冲器和洗脱杆状病毒。</li>
	</ol>
</li>
	<li>平衡五7.9 毫升的洗涤缓冲器 (40 毫升) 的肝素柱, 其线性流速为7.0 毫升/分。</li>
	<li>用色谱系统的采样泵 (进样口) 将250毫升的杆状病毒上清液载入肝素柱上, 其线性流速为2.0 毫升/分。</li>
	<li>运行 10 CVs (80 毫升) 的洗涤缓冲通过肝素柱在线性流量2.0 毫升/分钟, 直到紫外线 (紫外线) 吸收曲线 (280 nm) 已经返回到基线, 并变得稳定。</li>
	<li>洗脱7.9 毫升肝素柱上的杆状病毒粒子, 其 5 CVs (40 毫升) 的洗脱缓冲器的线性流速为4.0 毫升/分. 观察当杆状病毒粒子与肝素柱分离时, 色谱中蛋白质的尖锐洗脱峰。</li>
	<li>洗脱后, 立即稀释洗脱杆状病毒上清10倍, 使用20毫米磷酸钠缓冲液, 以防止在随后的离心过程中, 杆状病毒颗粒从渗透冲击中失活。</li>
	<li>存储100µL 的每个分数, 包括列流-通过收集在加载的杆状病毒上清, 洗涤缓冲区, 和洗脱液。感染这些杆状病毒分数 HT1080, 以评估纯化过程 (见第 6)。</li>
	<li>用列清理缓冲区和洗涤缓冲区清理列。最后, 用 5 CV 无菌20% 乙醇在水中以7.0 毫升/分钟的线性流速冲洗柱, 并贮存在4摄氏度。</li>
	<li>用水、1氢氧化钠、再用水和20% 乙醇在水中以50.0 毫升/分钟的线性流速清洗色谱系统, 并将系统储存在20% 乙醇中。</li>
</ol>5. 杆状病毒的浓度
<ol><li>使用高压釜预杀菌离心管。添加一个相同数量的杆状病毒上清到每个 ultracentrifuge 管在组织培养罩。通过平衡测量管的重量, 并调整 ultracentrifuge 管的含量与无菌 PBS 的重量。</li>
	<li>将每个 ultracentrifuge 管放置在 SW 32 钛转子的对立桶中。</li>
	<li>在4摄氏度时, 以 8万 x g 运行 ultracentrifuge 90 分钟. </li>
	<li>在组织培养罩中打开 ultracentrifuge 管, 灌装在不去除杆状病毒颗粒的情况下吸入液体。</li>
	<li>并用重悬吹打的200µL PBS, 用 0.5% (w/卷) BSA, 通过有力地将每根管子的颗粒向上和向下。</li>
	<li>将浓缩的杆状病毒转移到 cryovials (每瓶50到100µL), 并存储在-80 摄氏度。</li>
</ol>6. 杆状病毒滴定法
<ol><li>感染前的前一天, 用台盼蓝染色细胞后使用 hemocytometer 计数 HT1080 细胞。种子 1 x 105 HT1080 细胞每井在6井板中的2毫升的 Dulbecco 的改良鹰的培养基 (DMEM) 含有10% 胎牛血清 (血清)。种子几个额外的水井能够确定在感染时存在的细胞的确切数量。在孵化器的37°c 和 5% CO2中培养细胞。 注意:HT1080 细胞用于滴定, 因为它们表达较高水平的硫酸硫酸与其他细胞线16。由于 HT1080 细胞是黏附的, 滴定比使用传统的 Sf9-based 滴定更简单, 更少的时间消耗。</li>
	<li>在感染日, 通过对水井中的细胞计数, 确定每个井的细胞数。通过添加稀释后的原杆状病毒, 在柱纯化之前被搁置, 并通过从柱的流动, 洗涤和洗脱分数的样本来感染细胞。对每个样品, 根据传染性杆状病毒粒子的经验, 确定稀释量和体积, 并对每一个井进行三种感染。</li>
	<li>感染后两天后, 分析细胞对基因表达的兴趣。如果细胞表达荧光蛋白 (例如GFP)17, 则可以使用流式分析仪进行分析。</li>
	<li>根据在感染时存在的细胞数、稀释因子和细胞中荧光蛋白的百分比, 计算出的滴度 (每毫升感染单位): (感染期间细胞数 x 荧光蛋白的百分比阳性细胞 x 稀释因子)/杆状病毒的体积。 注意:例如, 如果感染时每井细胞总数为 1.2 x 105, 荧光蛋白的百分比为 5%, 稀释因子为 100, 稀释杆状病毒的体积增加10µL, 效价为 6 x 107传染性单位 (IU)。毫升.</li>
</ol>

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本文介绍了在悬浮培养中 Sf9 细胞中杆状病毒的产生和用肝素亲和层析纯化杆状病毒的方法。该协议中使用的参数最大限度地提高了感染杆状病毒的产量, 最大限度地减少了其灭活。此处提供的协议显示, 与其他9实现的恢复相比, 杆状病毒粒子的恢复明显改善。
由于广泛的寄主范围和组织取向, 在中枢神经系统、肝、眼、卵巢、前列腺和睾丸中进行了多项研究...

杆状病毒主要用于生产重组蛋白和疫苗的鳞翅目斜纹 fugiperda (Sf) 9 昆虫细胞使用重组杆状使用杆状 multicapsid 核多角体病毒 (AcMNPV)1,2,3,4. 最近, 它正在成为基因治疗应用5的一个有希望的载体。众所周知, 它具有广泛的宿主和组织取向, 感染静止和增殖细胞, 是非致病性的, 并没有集成到主机染色体4,5,6。此外, 杆状病毒可以在无血清悬浮培养, 可伸缩性, 并允许为未来的临床生产关闭系统处理1。
杆状病毒粒子的纯度对于实现有效的转导非常重要, 同时尽量减少细胞毒性7,8,9。杆状病毒可以通过离心或切向流动过滤 (TFF) 来集中, 对其传染性有有限的影响。然而, 这些程序不仅集中了病毒微粒, 而且还有来自 Sf9 文化的细胞碎片和蛋白质, 这可能是有毒的体外(个人观察), 在使用体内时可能诱发炎症或免疫应答。为了避免这种情况, 特别是在使用高度集中的病毒储存时, 感染性杆状病菌需要纯化, 并与污染微粒分离。
报告了几种用于纯化和浓缩杆状病毒载体的方法10,11,12。在现有的方法中, 肝素亲和色谱允许单步高水平的纯化与低浓度的污染蛋白12。该方法是基于硫酸硫酸根的识别作为杆状病毒13,14的受体。在将 Sf9 细胞上移到杆状病毒的柱上并结合后, 该柱可以用生理 (等渗) 缓冲器冲洗, 以去除未绑定或松散的污染微粒。由于对肝素的结合是可逆的, 杆状病毒颗粒可以洗脱高盐缓冲, 这是立即稀释到生理 (等渗) 盐浓度, 以防止通过渗透冲击 12.此外, 杆状病毒的生产, 以及从色谱柱上捕获和洗脱, 可以使用一个封闭系统的过程, 这是符合当前良好的生产实践 (cGMP)。
在这里, 我们提供了一个详细的协议, 以制造, 纯化和浓缩的传染性杆状病毒使用亲和层析和离心。简要地, 我们生产杆状病毒通过转染 Sf9 细胞与杆状病毒质粒 DNA 在黏附培养和进一步扩大传染性杆状病毒在无血清悬浮培养。我们用肝素亲和层析纯化杆状病毒, 并以离心为最后一步, 高度集中载体进行基因治疗应用。

<table><tbody><tr><td>Akta Avant 150</td><td>GE Healthcare</td><td>28976337</td><td>Chromatography system</td></tr><tr><td>POROS Heparin 50 &amp;micro;m Column</td><td>ThermoFisher Scientific</td><td>4333414</td><td>Heparin column</td></tr><tr><td>Ultracentrifuge</td><td>Beckman-Coulter</td><td>Non-catalog item</td><td>Concentrates virus at high-speed</td></tr><tr><td>Polyallomer Ultracentrifuge tube</td><td>Beckman-Coulter</td><td>326823</td><td>Concentrates virus at high-speed</td></tr><tr><td>MaxQ 8000 orbital shaker incubator&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>Non-catalog item</td><td>Shaker for suspension culture</td></tr><tr><td>250 mL Erlenmeyer flasks</td><td>ThermoFisher Scientific</td><td>238071</td><td>Flask for suspension culture</td></tr><tr><td>1 L Erlenmeyer flasks</td><td>ThermoFisher Scientific</td><td>238072</td><td>Flask for suspension culture</td></tr><tr><td>Microscope (Olympus CKX41)</td><td>Olympus</td><td>Non-catalog item</td><td>Cell monitoring and counting&amp;nbsp;</td></tr><tr><td>Table top centrifuge</td><td>ThermoFisher Scientific</td><td>75253839/433607</td><td>For clarification of Baculovirus supernatant</td></tr><tr><td>50 ml Conical tube</td><td>ThermoFisher Scientific</td><td>14-959-49A</td><td>For collection of Baculovirus supernatant</td></tr><tr><td>6-well plate</td><td>ThermoFisher Scientific</td><td>07-200-80</td><td>Tissue culture treated plate</td></tr><tr><td>10-cm plate</td><td>ThermoFisher Scientific</td><td>08-772E</td><td>Tissue culture treated plate</td></tr><tr><td>Stericup-HV, 0.45&amp;nbsp;&amp;micro;m, PVDF</td><td>EMD-Millipore</td><td>SCHVU05RE</td><td>Filtration unit</td></tr><tr><td>Kanamycin</td><td>ThermoFisher Scientific</td><td>15160-054</td><td></td></tr><tr><td>Tetracycline</td><td>Sigma-Aldrich</td><td>T7660</td><td></td></tr><tr><td>Gentamycin</td><td>ThermoFisher Scientific</td><td>15750-060</td><td></td></tr><tr><td>Bac-to-Bac Vector Kit</td><td>ThermoFisher Scientific</td><td>10360-014</td><td>Baculovirus expression system</td></tr><tr><td>DH10B-T1R&amp;nbsp; Competent cell</td><td>ThermoFisher Scientific</td><td>12331-013</td><td>Competent cell for bacmid</td></tr><tr><td>TE buffer</td><td>In-house</td><td>Non-catalog item</td><td>10 mM Tris-HCl, 1 mM EDTA, pH 8.0.</td></tr><tr><td>Plasmid Maxiprep kit</td><td>ThermoFisher Scientific</td><td>K2100-06</td><td>For bacmid purification</td></tr><tr><td>Sf9 Cells</td><td>ThermoFisher Scientific</td><td>11496-015</td><td>Insect cells</td></tr><tr><td>Grace&amp;rsquo;s Insect Cell Culture Medium</td><td>ThermoFisher Scientific</td><td>11605-094</td><td>Transfection medium</td></tr><tr><td>PBS</td><td>ThermoFisher Scientific</td><td>20012227</td><td>Washing cells, diluting samples</td></tr><tr><td>HyClone SFX-Insect cell media</td><td>GE Healthcare</td><td>SH30278.02</td><td>Serum-free insect cell growth medium</td></tr><tr><td>Benzonase Nuclease</td><td>Sigma-Aldrich</td><td>E1014</td><td>Enzyme to degrade DNA and RNA</td></tr><tr><td>Baculovirus plasmid (bacmid) DNA</td><td>In-house</td><td>Non-catalog item</td><td>Bacmid for Baculovirus Production</td></tr><tr><td>Cellfectin II&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>10362</td><td>Transfection reagent for insect cells</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A4737</td><td>Stabilizes Baculovirus</td></tr><tr><td>Cryovial</td><td>Thomas Scientific</td><td>1222C24</td><td>For storage of Baculovirus</td></tr><tr><td>HT1080 cell line</td><td>ATCC</td><td>CCL-121</td><td>Fibroblast cell line</td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td>Growth media for cell lines</td></tr><tr><td>Wash buffer</td><td>In-house</td><td>Non-catalog item</td><td>20 mmol/l phosphate buffer containing 150 mmol/l sodium chloride</td></tr><tr><td>Elution buffer</td><td>In-house</td><td>Non-catalog item</td><td>20 mmol/l phosphate buffer containing 1.5 mol/l sodium chloride</td></tr><tr><td>Column cleaning buffer</td><td>In-house</td><td>Non-catalog item</td><td>20 mmol/l phosphate buffer containing 2.0 mol/l sodium chloride</td></tr><tr><td>Sterile water</td><td>In-house</td><td>Non-catalog item</td><td>For Akta Avant cleaning</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>1.09137</td><td>For Akta Avant cleaning</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7073</td><td>For Akta Avant cleaning</td></tr></tbody></table>

production and purification of baculovirus for gene therapy application

杆状病毒传统上被用于生产重组蛋白和疫苗。然而, 最近, 杆状病毒正逐渐成为基因治疗应用的一个有希望的载体。在这里, 杆状病毒是由 Sf9 细胞黏附培养的杆状病毒质粒 DNA (bacmid) 的瞬态转染而产生的。杆状病毒随后在无血清悬浮培养的 Sf9 细胞中展开, 直到获得所需的体积。然后用肝素亲和层析法从培养上清液中纯化。由于肝素对杆状病毒包络糖蛋白的亲和性, 在上清液中将杆状病毒颗粒结合在一起的肝素柱上上清。该列用缓冲区冲洗, 以去除污染物, 而杆状病毒则是用高盐缓冲柱洗脱的。洗脱液被稀释为一个等渗盐浓度, 杆状病毒粒子进一步集中使用离心。使用这种方法, 杆状病毒可以集中到500倍, 25% 的传染性微粒的恢复。虽然这里描述的协议表明了从培养到1升的杆状病毒的生产和纯化, 该方法可以扩大在封闭系统悬浮培养, 以产生一个临床级的载体, 用于基因治疗的应用。

所提供的协议在流程图中 (图 1)。步骤包括对 bacmid DNA 的 Sf9 细胞进行瞬态转染, 在板的黏附培养中产生杆状病毒, 随后在无血清悬浮培养中扩增, 核酸酶处理和离心和过滤澄清,用肝素亲和色谱法纯化后, 离心浓度。
解冻后, Sf9 细胞需要至少两个星期的时间来恢复和进入指数阶段的增长。积极生长的 Sf9 细?...

Watch this Scientific Journal Video about Production and Purification of Baculovirus for Gene Therapy Application at JoVE.com

Production and Purification of Baculovirus for Gene Therapy Application

杆状病毒基因治疗应用的生产与纯化

在该协议中, 杆状病毒是通过瞬态转染杆状病毒质粒进入 Sf9 细胞并在无血清悬浮培养中扩增而产生的。用肝素亲和层析法纯化上清液, 进一步浓缩离心。该协议对用于基因治疗的杆状病毒的制备和纯化具有重要意义。

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

production-purification-baculovirus-for-gene-therapy

Research

JoVE Journal

Genetics

14.7K 次观看.  Cincinnati Children's Hospital Medical Center. 在该协议中, 杆状病毒是通过瞬态转染杆状病毒质粒进入 Sf9 细胞并在无血清悬浮培养中扩增而产生的。用肝素亲和层析法纯化上清液, 进一步浓缩离心。该协议对用于基因治疗的杆状病毒的制备和纯化具有重要意义。

视频: Production and Purification of Baculovirus for Gene Therapy Application

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

科研

遗传学

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.

哺乳动物 Bestrophin 离子通道的表达与纯化

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

生物化学

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

使用巴库洛病毒表达载体系统生产重组PRMT蛋白

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. ...

表达和纯化溶质载体转运蛋白的策略

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

作者聚焦:使用密码子优化基因表达和纯化人溶质载体转运蛋白 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

用于结构和生化研究的人溶质载体的高通量表达和纯化

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

生物学

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-他的净化：一个两步亲和纯化协议屈服全长纯化的蛋白质

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

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

杆状病毒基因治疗应用的生产与纯化

摘要

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此视频中的章节

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

哺乳动物 Bestrophin 离子通道的表达与纯化

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