链球菌中高分子质量蛋白的纯化
Summary
这里描述的是一个简单的方法，用于纯化链球菌中的基因产物。该技术在蛋白质，特别是膜蛋白和高分子质量蛋白的纯化方面可能有利，可用于其他各种细菌物种。
Abstract
解释基因的功能通常涉及野生型菌株和菌株的类名特征的比较，其中感兴趣的基因已被破坏。基因中断后功能丧失随后通过中断基因产物的外源性添加而恢复。这有助于确定基因的功能。前面描述的方法涉及生成gtfC基因破坏的链球菌菌株。在这里，描述了一种不要求的方法，用于在基因中断后从新生成的S.mutans菌株中纯化gtfC基因产物。它涉及在感兴趣的基因的3°端添加多聚苯乙烯编码序列，允许使用固定金属亲和色谱简单纯化基因产物。在此方法的遗传修饰中，不需要 PCR 以外的酶反应。基因破坏后通过外源性补充恢复基因产物是确定基因功能的有效方法，也可以适应不同的物种。
Introduction
对基因功能的分析通常涉及将野生型菌株的类特征与被破坏的菌株进行比较。一旦产生基因中断的菌株，基因产物的外源性添加允许功能恢复。
获得后续恢复测定所需的纯化基因产物的最常见方法是在大肠杆菌1中执行异体表达。然而，膜蛋白或高分子质量蛋白的表达往往很难使用这个系统1。在这些情况下，目标蛋白通常从通过一系列复杂的步骤原生合成蛋白质的细胞中分离出来，这可能导致基因产物的丧失。为了克服这些问题，根据基因破坏方法2、基于PCR的DNA拼接方法3（指定的两步融合PCR）和基因电穿孔，为基因产物纯化开发了一个简单的程序。在链球菌的转化。在基因产品的C-终点区添加多肽标记（His-tag）有助于通过固定金属亲和色谱（IMAC）进行纯化。
为了分离His-tag表达菌株，感兴趣的基因的整个基因组DNA（在这个His-tag表达基因中断的菌株中）被一个抗生素抗药性标记基因所取代。产生His-tag表达应变的过程与产生基因中断的应变的过程几乎相同，如前面描述的4，5。因此，基因破坏和基因产物分离的方法应作为功能分析的序列实验进行。
在本工作中，多聚苯二烯编码序列被附加到gtfC（GenBank locus标记SMU_1005）基因的3°端，在S.mutans6中编码葡聚酰亚胺转移酶-SI（GTF-SI）。然后，对链球菌物种进行表达研究。大肠杆菌实现异源性gtfC表达是困难的，很可能是因为GTF-SI的分子质量很高。这种菌株被命名为S.穆坦斯·克特夫克。图解图中描绘了野生S.mutans（S.mutans WT）中gtfC和光谱素抗基因盒（spcr）7位数的组织及其衍生物。图 1.GTF-SI是一种分泌蛋白，有助于培育造血性牙科生物膜6。在蔗糖的存在下，在WT S.mutans菌株的光滑玻璃表面上观察到粘附生物膜，但在S.mutans gtfC-中断菌株（S.mutans +gtfC）2、5.当重组GTF-SI的外源性添加后，生物膜形成在S.mutans _gtfC中恢复。应变，S.mutans His-gtfC，然后用于生产重组GTF-SI。
Protocol
Representative Results
Discussion
引体的设计是协议中最关键的一步。gtfC-反向和spcr-前引基的序列根据gtfC的3°端区域和spcr的5°端区域的顺序自动确定。每个引基包括 24 个互补基库，这些基础对 GS 链接器进行编码，并在其 5′ 区域对 His-tag 编码序列进行编码。通过在 3° 端添加 His 标记编码序列，可以避免位于上游侧翼区域的本机调控序列中断。DNA停止科顿必须从gtfC-反向引基器中…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了日本科学促进会（JSPS）（赠款号16K15860和19K10471至T.M.、17K12032至M.I.和18K09926到N.H.）和SECOM科学技术基金会（赠款号2018.09.10号）的支持。
Materials
| Agarose | Nippon Genetics | NE-AG02 | For agarose gel electrophoresis |
| Anaeropack | Mitsubishi Gas Chemical | A-03 | Anaerobic culture system |
| Anti-His-Tag monoclonal antibody | MBL | D291-7 | HRP-conjugated |
| BCA protein assay kit | Thermo Fisher Scientific | 23227 | Measurement of protein concentration |
| Brain heart infusion broth | Becton, Dickinson | 237500 | Bacterial culture medium |
| CBB R-250 | Wako | 031-17922 | For biofilm staining |
| Centrifugal ultrafiltration unit | Sartorius | VS2032 | Buffer replacement and protein concentration |
| Centrifuge | Kubota | 7780II | |
| Chromatographic column | Bio-Rad | 7321010 | For IMAC |
| Dialysis membrane clamp | Fisher brand | 21-153-100 | |
| Dialysis tubing | As One | 2-316-06 | |
| DNA polymerase | Takara | R045A | High-fidelity DNA polymerase |
| DNA sequencing | Eurofins Genomics | ||
| ECL substrate | Bio-Rad | 170-5060 | For western blotting |
| EDTA (0.5 M pH 8.0) | Wako | 311-90075 | Tris-EDTA buffer preparation |
| Electroporation cuvette | Bio-Rad | 1652086 | 0.2 cm gap |
| Electroporator | Bio-Rad | 1652100 | |
| EtBr solution | Nippon Gene | 315-90051 | For agarose gel electrophoresis |
| Gel band cutter | Nippon Genetics | FG-830 | |
| Gel extraction kit | Nippon Genetics | FG-91202 | DNA extraction from agarose gel |
| Imager | GE Healthcare | 29083461 | For SDS-PAGE and western blotting |
| Imidazole | Wako | 095-00015 | Binding buffer and elution buffer preparation |
| Incubator | Nippon Medical & Chemical Instruments | EZ-022 | Temperature setting: 4 °C |
| Incubator | Nippon Medical & Chemical Instruments | LH-100-RDS | Temperature setting: 37 °C |
| Membrane filter | Merck Millipore | JGWP04700 | 0.2 µm diameter |
| Microcentrifuge | Kubota | 3740 | |
| NaCl | Wako | 191-01665 | Preparation of binding buffer and elution buffer |
| NaH2PO4·2H2O | Wako | 192-02815 | Preparation of binding buffer and elution buffer |
| NaOH | Wako | 198-13765 | Preparation of binding buffer and elution buffer |
| (NH4)2SO4 | Wako | 015-06737 | Ammonium sulfate precipitation |
| Ni-charged resin | Bio-Rad | 1560133 | For IMAC |
| PCR primers | Eurofins Genomics | Custom-ordered | |
| Protein standard | Bio-Rad | 161-0381 | For SDS-PAGE and western blotting |
| Solvent filtration apparatus | As One | FH-1G | |
| Spectinomycin | Wako | 195-11531 | Antibiotics; use at 100 μg/mL |
| Sterile syringe filter | Merckmillipore | SLGV004SL | 0.22 µm diameter |
| Streptococus mutans ΔgtfC | Stock strain in the lab. | gtfC replaced with spcr | |
| Streptococus mutans UA159 | Stock strain in the lab. | S. mutans ATCC 700610, Wild-type strain | |
| Sucrose | Wako | 196-00015 | For biofilm development |
| TAE (50 × ) | Nippon Gene | 313-90035 | For agarose gel electrophoresis |
| Thermal cycler | Bio-Rad | PTC-200 | |
| Tris-HCl (1 M, pH 8.0) | Wako | 314-90065 | Tris-EDTA buffer preparation |
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