SHuffle T7 Express BioVector® 大肠杆菌表达表达菌株

BioVector® SHuffle® T7 Express 大肠杆菌表达表达菌株——产品技术说明书

1 菌株基本信息与工程核心背景

产品名称：BioVector® SHuffle® T7 Express 表达菌株

常用别名：SHuffle T7 Express Competent E. coli，SHuffle T7 表达株

生物学分类：细菌界 Bacteria / 变形菌门 Proteobacteria / 肠杆菌科 Enterobacteriaceae / 肠杆菌属 Escherichia

细胞衍生背景：该菌株衍生自 Escherichia coli B 表达系（具有天然缺乏 Lon 和 OmpT 蛋白酶的优势），通过重组工程对细胞内的氧化还原代谢通路进行了根本性的基因改造。

核心科研与工业价值：SHuffle® T7 Express 是专为在大肠杆菌细胞质内高效表达“富含二硫键、易形成包涵体”的复杂重组蛋白而设计的革命性底盘。 它克服了传统大肠杆菌细胞质高度还原性的限制，广泛应用于单克隆抗体片段（scFv、Fab）、含有大量半胱氨酸的细胞因子、毒素蛋白、受体胞外域以及需要精密正确折叠的工业酶类的体外高活性表达。

2 核心基因型特征与三大重组折叠机制

SHuffle® T7 Express 菌株的核心基因型（以特定改良型为准）蕴含着其三大核心蛋白质折叠优化系统：

• $\Delta gor \Delta trxB$ 双基因缺失（打破细胞质还原环境）：细胞内控制谷胱甘肽还原酶（$gor$）和硫氧还蛋白还原酶（$trxB$）的基因被完全敲除。这使得细胞质由原本的强还原性自发转变为氧化性环境，允许进入细胞质的半胱氨酸巯基（-SH）能够自发氧化形成二硫键（-S-S-）。

• 染色体集成表达 DsbC 巯基/二硫键异构酶（修正错误折叠）：该菌株在染色体上稳定集成并高表达原本属于周质空间的二硫键异构酶 DsbC（去除了周质定位信号，使其驻留细胞质）。DsbC 在细胞质中扮演“折叠监视器”，能够高效催化并断开因氧化环境错误连接的二硫键，并协助其重新排列组合成正确的天然构象，从源头上减少包涵体的产生。

• 染色体集成 T7 RNA 聚合酶（高效转录系统）：其基因组中集成了受 lac 启动子控制的 T7 RNA 聚合酶（类似于 BL21(DE3) 的工作原理）。这使得它完美兼容所有基于 T7 启动子的表达载体（如 pET 系列、pGEX 系列等），只需加入 IPTG 即可启动强效的靶蛋白转录与翻译。

• 抗生素抗性谱：该菌株常染色体天然携带壮观霉素抗性（Spectinomycin-resistant, $Spec^R$），在日常菌种维持时需注意其抗性背景，避免与相同抗性的质粒冲突。

3 专用生长培养基与诱导配方规范

SHuffle® T7 Express 的生长速度和质粒稳定性与培养基质量密切相关。

A 标准 LB 培养基/琼脂平板配方（用于日常常规维持与转化克隆）

• 基础成分：胰蛋白胨（Tryptone）10.0 g，酵母提取物（Yeast Extract）5.0 g，氯化钠 NaCl 10.0 g。

• 琼脂补剂（仅限平板固化）：琼脂粉 15.0 g。

• 溶剂：蒸馏水或去离子水 1000 mL。

• 灭菌规范：121 ℃ 高压蒸汽灭菌 15 分钟。

B 高密度表达培养基选择（推荐）

• TB（Terrific Broth）培养基：相比于 LB，TB 培养基具有更强的缓冲能力和更高的营养浓度（富含甘油作为碳源），能够支持 SHuffle® 菌株生长到更高的菌体密度，从而显著提升单位体积的靶蛋白产率。

C 菌株长期冻存保护液

• 标准配方：无菌 LB 肉汤 80% + 细胞/细菌级纯甘油 20%。与对数生长期的菌液充分混合后，分装保存于深度低温环境中。

4 物理环境控制参数

• 培养温度（常规扩增）：37.0 ℃（最适分裂温度）。

• 诱导折叠温度（红线红字）：20.0 ℃ 至 30.0 ℃（低温诱导）。虽然该菌株支持 37 ℃ 生长，但在加入 IPTG 诱导靶蛋白表达时，强烈建议将温度降低至 16 ℃ - 25 ℃ 展开暗诱导。低温能够显著减缓核糖体的翻译速度，给予 DsbC 异构酶充分的时间去纠正和穿凿二硫键，最大化提升可溶性活性蛋白的比例。

• 气相环境：常规常压无菌空气，需氧震荡培养（建议摇床转速 220-250 rpm，保证充足溶氧）。

5 感受态细胞转化与诱导表达操作规范

标准热激转化步骤：

1. 从 -80 ℃ 超低温冰箱中取出一管 SHuffle® T7 Express 感受态细胞（通常 50 uL/管），快速置于冰上融化（约 2-5 分钟）。

2. 向融化的感受态细胞中加入 1 到 3 uL 构建好的高纯度重组质粒（如 pET-28a-Target），用手指轻弹管底混匀，切勿使用移液枪吹打。

3. 置于冰上静置冰浴 30 分钟。

4. 将离心管快速投入 42.0 ℃ 恒温水浴箱中，准确热击 45 秒，随后立刻秒速移回冰上，静置冰浴 2 分钟（切勿摇动）。

5. 向管中加入 400-500 uL 不含抗生素的预热 SOC 或 LB 液体培养基，置于 37 ℃ 摇床中，以 200 rpm 复苏培养 60 分钟。

6. 取 100 uL 复苏菌液涂布于含有靶向质粒抗性（如卡那霉素 Kan 或氨苄青霉素 Amp）的 LB 琼脂平板上。倒置放入 37 ℃ 孵箱培养 16-20 小时直至长出清晰的小单菌落。

标准重组蛋白诱导流程：

1. 挑取平板上的单菌落，接种至 5 mL 含相应抗生素的 LB 或 TB 肉汤中，37 ℃ 220 rpm 培养过夜作为种子液。

2. 按 1:100 的比例将种子液转接至大体积表达培养基中，37 ℃ 震荡培养直至光吸收值 $OD_{600}$ 达到 0.4 到 0.6 之间。

3. 关键降温阶段：将培养瓶从 37 ℃ 摇床中取出，置于室温或预冷摇床中，使其物理降温至 20 ℃ 保持 15-20 分钟。

4. 加入终浓度为 0.1 到 1.0 mM 的 IPTG（异丙基硫代半乳糖苷）启动诱导。

5. 将摇床参数调整为 20 ℃（或 16 ℃ 保持更长时间），200 rpm 持续诱导表达 12 到 24 小时。

6. 离心收集菌体，通过超声破碎法或温和溶菌酶裂解法收获细胞质总蛋白，进行 SDS-PAGE 以及可溶性/活性分析。

6 长期封存与质控规范

• 生物安全级别：BSL-1（常规实验室安全防护）。属于安全、无致病性的实验室工程安全株。所有废弃培养物按常规高压灭菌程序处理即可。

• 深度超低温保存：甘油冻存管必须永久存放于 -80 ℃ 超低温冰箱或液氮中。由于缺失了 $gor$ 和 $trxB$，该菌株在非保护环境下对环境压力的耐受度低于普通 BL21 菌株，严禁在 -20 ℃ 或 4 ℃ 下长期搁置菌种。

• 蛋白质可溶性抽检质控：在批量使用该表达系统前，建议采用带有特定二硫键标记的对照质粒（如重组绿色荧光蛋白 tGFP，折叠错误时不发光）进行转化测试。通过测定发光效率或可溶性上清中的蛋白收率，确保该批次细胞的 DsbC 辅助折叠活性未发生基因退化或自发突变丢失。

BioVector® SHuffle® T7 Express Competent E. coli Expression Strain Product Datasheet

1 Strain and Engineering Identification General Information

• Product Name: BioVector® SHuffle® T7 Express Expression Strain

• Synonyms: SHuffle T7 Express Competent E. coli, SHuffle T7 Expression Host

• Biological Taxonomy: Kingdom Bacteria / Phylum Proteobacteria / Family Enterobacteriaceae / Genus Escherichia

• Lineage Context: This customized host is derived from the Escherichia coli B strain background (inherently deficient in Lon and OmpT protease systems) and has undergone core genomic rewiring of its intracellular redox metabolic pathways.

• Core Research & Industrial Value: SHuffle® T7 Express is a revolutionary bacterial chassis engineered specifically for the high-yield expression of complex, disulfide-bond-rich recombinant proteins that typically form insoluble inclusion bodies in standard E. coli. By converting the highly reducing cytoplasmic baseline into a functional oxidative workspace, it serves as an international gold standard for producing monoclonal antibody fragments (scFv, Fab), cysteine-dense cytokines, mammalian receptors, and properly folded industrial enzymes within the cytoplasm.

2 Core Genotypic Hallmarks & Triple Folding Optimization Mechanisms

The comprehensive genomic configuration of the SHuffle® T7 Express strain drives three independent, synergistic protein-folding engines:

• $\Delta gor \Delta trxb$ Double Deletion (Eliminating Cytoplasmic Reducing Power): The simultaneous knockout of the glutathione reductase ($gor$) and thioredoxin reductase ($trxB$) genes prevents the reduction of cysteines. This converts the cytoplasm into a stably oxidative environment, allowing cysteine sulfhydryl groups (-SH) to actively mature into disulfide bonds (-S-S-) directly upon translation.

• Chromosomally Integrated DsbC Disulfide Isomerase (Correcting Misfolded Intermediates): The strain features a permanent genomic integration of the disulfide isomerase DsbC, stripped of its periplasmic leader sequence so it remains active within the cytoplasm. DsbC acts as an intracellular chaperone that scans newly formed proteins, cleaving non-native or mismatched disulfide linkages and isomerizing them into their correct biological architecture, significantly lowering inclusion body formation.

• Chromosomal T7 RNA Polymerase (High-Efficiency Transcription): Harbors a lysogenic copy of the T7 RNA polymerase gene controlled by the inducible lac promoter (equivalent to the expression dynamics of a BL21(DE3) setup). This allows seamless compatibility with any T7-driven vector systems (such as pET or pGEX arrays) via standard IPTG induction.

• Antibiotic Resistance Profile: Naturally carries a chromosomally encoded Spectinomycin resistance ($Spec^R$) cassette. Researchers must note this background selection marker to ensure it does not conflict with incoming experimental plasmid resistance.

3 Dedicated Growth Media and Induction Formulations

The doubling speed and plasmid retention profile of SHuffle® T7 Express rely heavily on premium nutrient selection.

A Standard LB Medium / Agar Plates (For Clone Maintenance and Transformation Verification)

• Basal Macro-Nutrients: Tryptone 10.0 g, Yeast Extract 5.0 g, Sodium Chloride (NaCl) 10.0 g.

• Solidifying Matrix (For plates only): Agar powder 15.0 g.

• Solvent Matrix: Distilled or Deionized Water 1000 mL.

• Sterilization Cycle: Autoclave at 121 ℃ for 15 minutes.

B High-Density Expression Media Recommendations

• TB (Terrific Broth) Medium: Strongly recommended over standard LB for large-scale production runs. TB possesses enhanced buffering capacities and increased nutrient density (utilizing glycerol as a primary carbon feed), enabling SHuffle® cultures to achieve significantly higher optical densities ($OD_{600}$) and superior volumetric protein target yields.

C Cryopreservation Archiving Fluid

• Glycerol Stock Formula: 80% Sterile LB Broth Matrix + 20% Analytical/Bacterial Grade Pure Glycerol. Blend uniformly with active log-phase cultures prior to long-term storage under ultra-low freezing configurations.

4 Controlled Environmental Parameters

• Incubation Temperature (Growth): 37.0 ℃ (Optimum operating index for rapid binary fission).

• Induction Folding Temperature (Critical Threshold): 20.0 ℃ to 30.0 ℃ (Low-Temperature Induction Window). While the biomass expands efficiently at 37 ℃, it is imperative to drop the workstation temperature to 16 ℃ - 25 ℃ during active IPTG induction. Lowering thermal parameters slows down ribosomally mediated translation kinetics, granting the cytoplasmic DsbC machinery adequate time to scan, dock, and isomerize complex disulfide configurations, maximizing the soluble target ratio.

• Gas Phase Configuration: Standard atmospheric oxygen environment with active agitating aeration (Orbital shaker set to 220-250 rpm to avoid hypoxia).

5 Transformation and Recombinant Expression Protocols

Standard Heat-Shock Transformation Protocol:

1. Retrieve a 50 uL aliquot of frozen SHuffle® T7 Express competent cells from -80 ℃ storage and immediately place on ice to thaw gently (approx 2-5 minutes).

2. Introduce 1 to 3 uL of high-purity recombinant plasmid DNA (e.g., pET-28a construct) directly into the thawed matrix. Mix gently by tapping the bottom of the tube with a finger; never pipette or vortex the fragile cells.

3. Incubate the cellular mixture on ice for exactly 30 minutes.

4. Transfer the tube instantly into a 42.0 ℃ calibrated water bath for a strict 45-second heat shock. Transfer immediately back onto ice for 2 minutes without agitating.

5. Add 400-500 uL of pre-warmed, antibiotic-free SOC or LB media to the tube, and incubate in a shaking incubator at 37 ℃, 200 rpm for 60 minutes to recover phenotype expression.

6. Spread a 100 uL aliquot of the recovered pool onto an LB agar plate supplemented with the target plasmid antibiotic (e.g., Kanamycin Kan or Ampicillin Amp). Invert the plates and incubate at 37 ℃ for 16-20 hours until single colonies emerge.

Standard Inducible Protein Expression Pipeline:

1. Inoculate a single isolated colony from the transformation plate into 5 mL of LB or TB broth containing appropriate selection antibiotics. Shake over-night at 37 ℃ at 220 rpm to establish a starter seed stock.

2. Inoculate the starter culture at a 1:100 dilution ratio into the final production volume. Shake at 37 ℃ at 220 rpm until the cell density reaches an $OD_{600}$ index of 0.4 to 0.6.

3. Thermal Equilibrium Phase: Remove the production vessels from the 37 ℃ environment and transition them to a pre-cooled workspace calibrated at 20 ℃ for 15-20 minutes to bring the liquid matrix to the correct induction temperature.

4. Introduce IPTG (Isopropyl $\beta$-D-1-thiogalactopyranoside) to a final operational concentration of 0.1 to 1.0 mM.

5. Operate the shaker platform at 20 ℃ (or 16 ℃ for longer runs) at 200 rpm for a continuous induction window of 12 to 24 hours.

6. Pelleting out the biomass via centrifugation, and disrupt the cell walls utilizing ultrasonic sonication or mild lysozyme enzymatic digestion to isolate the cytoplasmic soluble fraction for downstream SDS-PAGE or functional assay validation.

6 Long-Term Preservation and Quality Control Metrics

• Biosafety Classification: BSL-1 (Standard laboratory safety parameters). Represents a non-pathogenic, safe laboratory cloning and expression host. Discard spent matrices following routine institutional autoclaving disinfection runs.

• Ultra-Low Freeze Archiving: Glycerol vials must reside permanently at -80 ℃ or within liquid nitrogen repositories. Due to the deletion of cellular pathways ($gor$/$trxB$), this host exhibits reduced fitness to environmental fluxes compared to wild-type BL21; keeping liquid stocks at 4 ℃ or -20 ℃ for extended intervals will lead to sharp viability drops.

• Folding System Validation QC: Periodically evaluate the cytoplasmic folding integrity by transforming a control plasmid encoding a disulfide-dependent reporter protein (such as a recombinant green fluorescent protein tGFP variant that requires proper disulfide loops to fluoresce). Quantifying the specific fluorescence output or measuring soluble yield against a baseline BL21 control verifies that the genomic DsbC and pathway knockouts remain intact without phenotypic drifting.

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