亲和色谱法表达和纯化人基质金属蛋白酶-3
Summary
His-tag纯化，透析和活化用于提高细菌中可溶性活性基质金属蛋白酶-3催化结构域蛋白表达的产量。蛋白质级分通过SDS-PAGE凝胶进行分析。
Abstract
基质金属蛋白酶（MMP）属于麦地金星蛋白酶家族，在细胞外基质（ECM）降解和重塑中起核心作用，以及与几种生长因子和细胞因子的相互作用。特异性 MMP 的过表达是多种疾病的原因，如癌症、神经退行性疾病和心血管疾病。MMPs最近一直是人们关注的焦点，作为开发可以治疗与MMP过表达相关的疾病的治疗方法的目标。
为了研究溶液中的MMP机制，需要更简单和更强大的重组蛋白表达和纯化方法来生产活性，可溶性MMP。然而，由于缺乏翻译后机制，大多数MMPs的催化结构域不能在 大肠 杆菌（大肠杆菌）中以可溶性形式表达，而哺乳动物表达系统通常成本高昂且产量较低。MMP包涵体必须经历繁琐而费力的广泛纯化和重新折叠过程，从而显着降低MPP在天然构象中的产量。本文提出了一种使用Rosetta2（DE3）pLysS（以下简称R2DP）细胞产生基质金属蛋白酶-3催化结构域（MMP-3cd）的方案，该结构域包含N端His-tag，后跟pro-domain（Hisx6-pro-MMP-3cd）用于亲和纯化。R2DP细胞通过含有细菌表达系统中通常罕见的密码子的氯霉素耐药质粒增强真核蛋白的表达。与重组蛋白表达的传统细胞系BL21（DE3）相比，使用这种新菌株的纯化提高了纯化的Hixx6-pro-MMP-3cd的产量。在激活和脱盐后，pro结构域与N端His-tag一起被切割，提供活性MMP-3cd，可立即用于无数 体外 应用。该方法不需要昂贵的设备或复杂的融合蛋白，并且描述了细菌中重组人MMP的快速产生。
Introduction
大多数复杂的真核蛋白在表达后会经历复杂的翻译后修饰，需要高度辅助的蛋白质折叠和辅助因子才能发挥作用1。由于成本高昂且缺乏可靠的表达和纯化方法，即使在小规模的实验室实验中，在细菌宿主中产生大量可溶性人蛋白仍然是一项重大挑战2，3。MMPs，具有大分子量的人内肽酶，当在 大肠杆菌 中表达时，通常表现为不溶性包涵体。提取可溶性人MMP通常会导致费力，耗时的溶解和重新折叠过程4。
MMP在生理和致病过程中都起着关键作用。人MMP是23种锌内肽酶的家族，按结构和底物特异性分类，尽管具有高度保守的催化结构域，但其表达方式不同5，6。MMPs作为无活性酶原分泌，通过翻译后激活及其内源性抑制剂，金属蛋白酶的组织抑制剂（TIMPs）7，8，9，10进行调节。虽然MMP最初因其在ECM周转中的作用而得到认可，但它也与发育，形态发生，组织修复和重塑有关8。MMPs的失调与癌症以及神经退行性疾病，心血管疾病和纤维化疾病以及其他疾病显着相关5，7。
开发强大的大规模MMP生产方法对于确保通过生化和基于细胞的测定对MMP机制的未来研究取得成功至关重要。各种MMP以前已经在细菌中表达11，包括Hisx6标记的MMPs，而不会改变MMP活性12，13，14，15。但是，这些方法包括可能难以复制的繁琐、冗长的步骤。
哺乳动物细胞也可用于表达许多不同的人类蛋白质，同时确保正确的翻译后修饰16。虽然哺乳动物表达系统是生产具有适当翻译后修饰的重组人类蛋白的理想选择，但该方法的主要缺点是初始产量低，生长培养基和试剂昂贵，达到稳定表达系的时间长，以及被真菌或细菌等其他物种污染的风险2，11.此外，哺乳动物细胞系中的MMP产生来自相关细胞蛋白（如TIMPs或纤连蛋白）的杂质11。与在哺乳动物细胞中观察到的缓慢细胞生长不同，细菌表达系统在短时间内提供大规模的蛋白质生产以及更简单的培养基和生长要求。然而，由于细菌表达系统中缺乏其他相关细胞蛋白（即TIMPs），较高浓度的活性MMP通过自体蛋白水解而降解，导致MMP产量低17。
本文描述了使用 大肠杆菌 作为表达宿主的重组Hisx6-pro-MMP-3cd的细菌表达，纯化和活化的详细方法，因为它具有可负担性，简单性并且成功产生更高的MMPs产量2，3，18。由于 大肠杆菌 缺乏重组MMP和其他复杂蛋白质所需的蛋白质折叠机制和翻译后处理，许多 大肠杆菌 菌株已被设计以克服这些限制，使 大肠杆菌 成为更适合表达重组人MMP-3cd的宿主，19，20.例如，本研究中使用的R2DP菌株通过提供含有 大肠杆菌中很少使用的密码子的氯霉素耐药质粒来增强真核表达。
如本方案所述，在R2DP细胞中从pET-3a载体中过表达相对纯的包涵体（图1）后，提取Hisx6-pro-MMP-3催化结构域（MMP-3cd）蛋白并变性4。使用亲和标签色谱纯化Hisx6-pro-MMP-3cd3，19。在重新折叠和透析后，Pro-MMP-3cd（酶原）被4-氨基苯基乙酸汞（APMA）激活，SDS-PAGE分析用于评估产量和进一步纯化的必要性5，21。该协议以可溶性MMP-3cd的表达、纯化和活化为例。然而，它也可以用作具有相似表达和激活机制的其他MMPs和人蛋白酶表达的指南（图2）。对于MMP-3cd以外的其他蛋白质，建议读者在尝试此方案之前确定其靶蛋白的最佳缓冲液组成和方法。
图1：pET-3a-Hisx6-pro-MMP-3cd质粒的质粒图谱。 pET-3a载体包括氨苄西林抗性基因。将N端Hisx6标签序列克隆到基于pET-3a的载体中，包括pro-MMP-3cd，以在BamHI和NdeI限制位点之间的T7启动子的控制下产生pET-3a-Hisx6-pro-MMP-3cd结构。 请点击此处查看此图的放大版本。
图2：Pro-MMP-3cd的细菌表达，纯化，重新折叠和活化。 1.1：pET-3a-Hisx6-pro-MMP-3cd质粒转化为BL21（DE3）或R2DP细胞。1.2：使用IPTG诱导Pro-MMP-3cd蛋白表达。1.3：化学裂解和超声处理用于提取Hisx6-pro-MMP-3cd蛋白，这些蛋白主要不溶于包涵体并存在于包涵体中。尿素用于使包涵体中的蛋白质变性和增溶。变性Hisx6-pro-MMP-3cd蛋白通过亲和色谱纯化纯化。3.在透析过程中，通过逐渐从缓冲液中除去尿素，将洗脱的Hisx6-pro-MMP-3cd缓慢重新折叠。4.最后，通过去除N端前肽结构域，使用APMA激活重新折叠的MMP-3cd蛋白。APMA随后通过脱盐从溶液中除去。这些数字对应于描述这些步骤的协议部分。缩写：MMP-3cd =基质金属蛋白酶-3催化结构域;APMA = 4-氨基苯基汞乙酸酯。 请点击此处查看此图的放大版本。
Protocol
Representative Results
Discussion
大规模生产可溶性、人性、重组型MMPs仍然是一项具有挑战性的任务。哺乳动物细胞可以以高成本和长等待时间表达功能性MMP，而 大肠杆菌 迅速产生大量MMP包涵体，必须纯化和重新折叠11，16。R2DP细胞显著提高了MMP包涵体的产量，从而实现了更具成本效益和生产力的MMP再折叠过程。然而， 大肠杆菌 缺乏折叠MMP所需的翻译后机制，尽管工程?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢佛罗里达州杰克逊维尔梅奥诊所的Evette Radisky博士和Alexandra Hockla博士提供pET-3a-pro-MMP-3cd质粒作为克隆Hisx6pro-MMP-3cd基因的模板，以及他们的评论，以及内华达大学里诺分校内华达基因组学中心的Paul Hartley博士进行DNA测序。作者还要感谢Cassandra Hergenrader对部分蛋白质表达的帮助。M.R.-S.我要感谢NIH-P20 GM103650-COBRE综合神经科学赠款和UNR研发mICRO SEED资助奖。
Materials
| 0.22 µm sterile filter | Sigma Aldrich | SLGP033RS | Used to remove some contaminants from the protein extract before purification, and prevent the Ni-NTA column from clogging |
| 1 L Erlenmeyer flasks | Thermo Fisher Scientific | S76106F | n/a |
| 1 L glass bottles | Thermo Fisher Scientific | 06-414-1D | n/a |
| 1.5 mL microfuge tubes | Thermo Fisher Scientific | 02-682-002 | n/a |
| 15 mL conical tubes | Thermo Fisher Scientific | 339650 | n/a |
| 18 G, 1-in. beveled needle | Amazon | B07S7VBHM2 | Used in combination with the dialysis casette |
| 2 mL desalting column | Thermo Fisher Scientific | 89890 | Removes APMA following activation |
| 2-(N-Morpholino)ethanesulfonic acid (MES) | Thermo Fisher Scientific | AAA1610422 | n/a |
| 250 mL conical bottle cushions | Thermo Fisher Scientific | 05-538-53A | Stabilize conical bottles during large-volume centrifugation |
| 250 mL conical bottles | Thermo Fisher Scientific | 05-538-53 | n/a |
| 400 mL stirred cell | Sigma Aldrich | UFSC40001 | Re-concentrates a much larger volume than the centrifugal filter unit. Rosetta2(DE3)pLysS cells produce high volumes of protein that may exceed the 15 mL limit of the centrifugal filter unit |
| 4-aminophenylmercuric acetate (APMA) | Sigma Aldrich | A9563-5G | Activates MMP-3 by cleaving the propeptide |
| 5 mL syringe | Thermo Fisher Scientific | NC0829167 | Used in combination with the dialysis casette |
| 50 mL conical tubes | Thermo Fisher Scientific | 339650 | Used for storage in many purification steps |
| 50 mL re-concentration tube | Sigma Aldrich | UFC901024D | Used for re-concentrating protein samples after dialysis or removing contaminants |
| Agar | Thermo Fisher Scientific | BP1423-500 | Buffer ingredient that solidifies autoclaved LB media upon cooling |
| Ampicillin | Thermo Fisher Scientific | BP1760-25 | Antibiotic used with pET3a vector; used at 100 µg/mL in LB media |
| BamHI | NEB | R3136S | Restriction enzyme to be used with the pET3a vector |
| Calcium chloride (CaCl2) | Thermo Fisher Scientific | 600-30-23 | The calcium ion stabilizes MMP structure |
| Cell spreaders | Thermo Fisher Scientific | 50-189-7544 | Can be used to spread cells across a petri dish after transformation |
| Chloramphenicol | Thermo Fisher Scientific | 22-055-125GM | Antibiotic used with pET3a vector; used at 34 µg/mL in LB media |
| Dialysis Buffer 1 | n/a | n/a | 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM CaCl2, 1 µM ZnCl2, 4 M Urea. |
| Dialysis Buffer 2 | n/a | n/a | 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM CaCl2, 1 µM ZnCl2, 2 M Urea. |
| Dialysis Buffer 3 | n/a | n/a | 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM CaCl2 , 1 µM ZnCl2. |
| Dialysis clips | Thermo Fisher Scientific | 68011 | Used in combination with snakeskin dialysis tubing |
| Dialysis tubing | Thermo Fisher Scientific | 88243 | Alternative dialysis method that holds much larger sample volumes, but with higher risk of sample loss |
| Digest buffer | NEB | B7204S | Buffer used in digesting the pET3a vector |
| Disposable cuvettes | Thermo Fisher Scientific | 21-200-257 | Used to measure the bacterial culture OD during growth and expression |
| Dithiothreitol (DTT) | Thermo Fisher Scientific | D107125G | Assists with protein denaturation by reducing any disulfide bonds |
| DNA assembly mix | NEB | E2621S | Used to ligate the Hisx6-pro-MMP-3cd PCR product and digested pET3a vector |
| DNase I | NEB | M0303S | Endonuclease for degrading unfavorable DNA contaminants that could later affect protein purification |
| Ethanol | Thermo Fisher Scientific | A995-4 | n/a |
| Ethylenediaminetetraacetic acid (EDTA) | Thermo Fisher Scientific | J15694-AE | Used in denaturation. Prevents oxidation and subsequent formation of disulfide bonds |
| Gel recovery kit | Promega | A9281 | Isolates and purifies DNA from agarose gels |
| Glycerol | Thermo Fisher Scientific | G33-500 | Used for making glycerol stocks, which are frozen at -80 °C |
| Gravity flow column | BioRad | 7321010 | Used for Ni-NTA purification of recombinantly His-tagged proteins |
| Guanidine hydrochloride (GdnHCl) | Thermo Fisher Scientific | AAA135430B | Second chaotropic agent used for disrupting protein secondary structure. |
| High-transformation efficiency cells | NEB | C2987 | High-transformation efficiency cells with greater chance of success for cloning the N-terminal His-tag into the pET3a-pro-MMP-3cd construct |
| HT Elution Buffer | n/a | n/a | 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 6 M urea, 250 mM imidazole. Adjust pH to 7.4 |
| HT Equilibration Buffer | n/a | n/a | 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 6 M urea. Adjust pH to 7.4 |
| HT Regeneration Buffer | n/a | n/a | 20 mM MES, 0.1 M NaCl. Adjust pH to 5.0 |
| HT Wash Buffer | n/a | n/a | 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 6 M urea, 25 mM imidazole. Adjust pH to 7.4 |
| Hydrochloric acid (HCl) | Thermo Fisher Scientific | A144C-212 | Used to pH buffers |
| Imidazole | Thermo Fisher Scientific | AAA1022122 | Mimics the histidine side group. Used to separate non-specifically binding proteins from the his-tagged target protein |
| Inclusion Body Buffer | n/a | n/a | 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl, 5 mM DTT, 2% v/v Triton X 100, 0.5 M Urea. Adjust pH to 8.0 |
| Isopropyl-ß-D-thiogalactopyranoside (IPTG) | Thermo Fisher Scientific | FERR0392 | A reagent that induces target gene expression in pET3a. Make 0.5 mL 1 M aliquots, filter sterilize and store in -20 °C |
| LB Amp CamR media | n/a | n/a | To be poured into a sterible 1 L bottle or 1 L flask. For 1 L, add 25 g LB Broth. Sterilize by autoclaving. Once cooled to below 50 °C, add ampicillin to 100 µg/mL and chloramphenicol to 34 µg/mL |
| LB Amp CamR plates | n/a | n/a | To be poured into sterile petri dishes. Pour until the petri dish lid is completely covered. 1 L of media yields 40-60 plates. For 1 L: 25 g LB Broth, 16 g Agar. Sterilize by autoclaving. Once cooled to below 50 °C, add ampicillin to 100 µg/mL and chloramphenicol to 34 µg/mL |
| LB Broth | Thermo Fisher Scientific | BP1426-2 | Pre-mixed with tryptone, yeast extract, and sodium chloride |
| Lysis Buffer | n/a | n/a | 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl, 0.133 g/mL lysozyme, 0.49% v/v Triton X-100. Adjust pH to 8.0 |
| Lysozyme | MP Biomedicals | 195303 | Used in protein extraction. Enzyme that lyses bacterial cell walls |
| Miniprep kit | Promega | A1330 | If successful, extracts the pET3a-pro-MMP-3cd construct from transformants |
| NdeI | NEB | R0111S | Restriction enzyme to be used with the pET3a vector |
| Ni-NTA resin | Thermo Fisher Scientific | PI88221 | Used to bind recombinant his-tagged proteins. This strong interaction can be displaced with higher concentrations of imidazole |
| PCR mix | NEB | M0492S | A PCR reagent for inserting an N-terminal his-tag into the pET3a-pro-MMP-3cd vector |
| pET plasmid | Addgene | n/a | The pET3a vector offers ampicillin resistance, inducible expression of a target gene, and sequencing with T7 primers |
| Petri dishes | VWR | 25384-342 | Used for plating transformants on LB agar media |
| R2DP cells | Novagen | 714033 | BL21 derivatives with enhanced expression of eukaryotic proteins. Contain tRNAs of codons found to be rare in e. coli |
| SOC growth media | NEB | B9020S | Non-selective growth media for rapid growth during transformation |
| Sodium chloride (NaCl) | Thermo Fisher Scientific | BP358-1 | Used in buffers and helps with protein stability |
| Sodium deoxycholate | Thermo Fisher Scientific | PI89905 | Detergent used in protein extraction. Lyses cell walls |
| Solubilization Buffer | n/a | n/a | 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM DTT, 6 M Urea. Adjust pH to 8.0 |
| Tris base | Thermo Fisher Scientific | BP152-1 | Common buffer used in the physiological pH range. Temperature-sensitive |
| Triton X-100 | Thermo Fisher Scientific | M1122980101 | Detergent used for cell lysis |
| Urea | Thermo Fisher Scientific | AAJ75826A7 | First chaotropic agent for disrupting protein secondary structure |
| Zinc chloride (ZnCl2) | Thermo Fisher Scientific | AAA162810E | Stabilizes MMP structure. The zinc ion is found in the catalytic site of MMP-3 |
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