BioVector® pMBP-LbCas12a Recombinant Protein Expression Vector / pMBP-LbCas12a 重组蛋白表达与纯化质粒载体

一 产品基本信息与分子生物学背景

• 载体名称：pMBP-LbCas12a（通常由原核高表达骨架与 LbCas12a 融合基因拼接而成）。

• 载体分类：原核表达质粒（大肠杆菌高丰度重组蛋白纯化专用）。

• 质粒大小：约 8.5 - 9.5 kb（具体因融合标签和多克隆位点的优化微调而异）。

• 核心骨架与设计背景：pMBP-LbCas12a 是一株专门为了在大肠杆菌（E. coli）表达系统中高产、高纯度提取 LbCas12a 酶蛋白而优化的工业与科研级原核表达质粒。LbCas12a（旧称 LbCpf1）是源自路氏纤维单胞菌（Lachnospiraceae bacterium ND2006）的 V 型 CRISPR-Cas 核心效应核酸酶。与经典的 Cas9 相比，它仅需要一条较短的 crRNA 引导，识别富含 T 的 PAM 序列（5'-TTTV-3'），并在剪切目标 DNA 时产生具有粘性末端的双链断裂。由于 Cas12a 在靶向结合特异性序列后会触发强烈的顺式（cis）和反式（trans）单链 DNA 非特异性附带剪切活性（Collateral cleavage activity），它已成为现代体外核酸快检技术（如 DETECTR 平台）的绝对核心底层工具酶。为了克服 Cas12a 蛋白本身在大肠杆菌中极易形成不溶性包涵体（Inclusion bodies）的瓶颈，该载体在 LbCas12a 的 N 端融合了麦芽糖结合蛋白（Maltose-Binding Protein, MBP）标签，大幅度飙升了重组蛋白的体外可溶性（Solubility）与产量。

• 核心顺式作用元件与图谱特征：

  • T7 强启动子（T7 Promoter）：强力驱动下游融合蛋白的转录。该启动子受噬菌体 T7 RNA 聚合酶的严密调控，日常处于锁死状态，一旦加入异丙基-$\beta$-D-硫代半乳糖苷（IPTG）诱导，即可启动海量的目的蛋白表达。

  • MBP 融合标签（Maltose-Binding Protein Tag）：位于 LbCas12a 基因的上游。MBP 不仅是强效的可溶性分子伴侣，还能让粗提蛋白通过大麦芽糖亲和层析柱（Amylose Resin Column）进行一步法高纯度亲和纯化。

  • 蛋白酶切位点（TEV / 3C Protease Site）：位于 MBP 标签与 LbCas12a 编码区之间。允许纯化完成后，利用 TEV 蛋白酶或 PreScission Protease 顺式切除 MBP 标签，释放出完全天然、无标签干扰的纯净 LbCas12a 酶。

  • 核定位信号（NLS）与 Poly-His 标签（可选配置）：部分亚型会在 C 端或 N 端额外串联 6×His-tag 用于辅助纯化，或带有 SV40 NLS 信号以便于纯化出的蛋白后续直接用于真核细胞内源基因编辑。

  • 抗性选择标记与复制子：配置有卡那霉素抗性基因（$Kan^R$）或氨苄青霉素抗性基因（$Amp^R$），并基于常规高拷贝原核复制子运行。

二 核心科研价值与体外快检（IVD）转化应用

pMBP-LbCas12a 质粒在大肠杆菌中成功表达纯化出的 LbCas12a 酶蛋白，是现代生物技术中的明星分子：

1. 构建基于 CRISPR 的新型核酸诊断平台（如 DETECTR）：纯化出的 LbCas12a 酶被广泛用于体外超灵敏分子诊断。当环境体系中存在目标病原体 DNA（如新冠病毒、非洲猪瘟病毒等）并被 crRNA 靶向识别结合后，LbCas12a 会疯狂激活其反式剪切（trans-cleavage）活性。通过在体系中加入两端修饰了荧光基团与淬灭基团的单链 DNA 探针（ssDNA F-Q Reporter），LbCas12a 会将探针成百上千次切断，释放出肉眼或仪器可见的荧光，可在 30 分钟内实现单分子级别的基因快检。

2. 体外靶向 DNA 基因剪切与打靶验证：用于在体外酶促反应体系中，测试针对特定靶标突变（如肿瘤单核苷酸多态性 SNP 突变位点）设计的 crRNA 的剪切效率与脱靶率（Off-target rate），为体内基因敲除实验提供预实验评估。

3. 重组核质蛋白复合体（RNP）直接转染：将纯化出的 LbCas12a 蛋白与人工体外转录或化学合成的 crRNA 在体外混合，组装形成极为稳定的 RNP 复合体。利用电转（Electroporation）或脂质体直接将其递送入真核细胞或原代免疫细胞（如 CAR-T 细胞）中，可实现即时、高效且极低脱靶风险的无痕基因组编辑。

三 实验室大肠杆菌高丰度诱导表达与一步法纯化标准步骤

1. 表达宿主菌转化与高密度液体扩增

• 推荐表达宿主：大肠杆菌 BL21(DE3) 或 Rosetta(DE3) 蛋白表达专用感受态细胞（必须带有 T7 RNA 聚合酶溶源区）。

• 常规操作：

  1. 采用常规热击法将 pMBP-LbCas12a 质粒转化入 BL21(DE3) 感受态细胞中，涂布于含有对应抗生素（如 50 $\mu$g/mL 卡那霉素）的 LB 平板上，37 ℃ 培养过夜。

  2. 挑取阳性单菌落接种至 5 mL LB 液体培养基中（含抗生素），37 ℃ 振荡培养过夜。

  3. 将预培养的菌液按 1:100 的体积比转接至 1 L 富含营养的 TB 培养基（Terrific Broth）或 2×YT 培养基中。37 ℃、220 rpm 剧烈振荡扩增，直至菌液的菌密度 OD600 达到 0.6 - 0.8 的对数生长中期。

2. 低温 IPTG 诱导蛋白表达（严防包涵体核心控制点）

Cas12a 是分子量超过 140 kDa 的大型多结构域蛋白，如果在高密度下高强度快速表达，极易由于折叠不及发生成片聚集，产生不溶性的包涵体。全流程必须采用“低温慢表达”策略：

1. 冷激预冷（Cold-shock）：当菌液 OD600 达标后，立刻将盛有 1 L 菌液的三角瓶捞出，整体浸入冰水混合物或 4 ℃ 冰箱中静置预冷 30 分钟，强行压制细菌原有的高增殖代谢。

2. 加药诱导：向冷激后的菌液中加入终浓度为 0.1 - 0.5 mM 的 IPTG（低浓度诱导，防止表达过猛）。

3. 低温维持：将摇床温度调至 16 ℃ 极其温和地低速震荡诱导表达 16 - 20 小时（过夜）。低温能显著延缓翻译速度，配合 N 端的 MBP 标签，可逼迫绝大多数 LbCas12a 重组蛋白以完美折叠的可溶态（Soluble form）存活于细菌细胞质中。

4. 细胞收获：4 ℃ 下以 5000 g 离心 15 分钟，收集紧密深黄色的细菌沉淀，可用 PBS 洗涤一次，置于 -80 ℃ 冻存，或直接进入裂解程序。

3. 超声裂解与 Amylose 亲和层析一步法层析纯化

• 全流程要求 0 - 4 ℃ 冰上操作，严防内源蛋白酶降解。

1. 细胞裂解（Lysis）：按每克菌体沉淀加入 5 - 10 mL Lysis Buffer（配方：20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM DTT, 5% 甘油，外加适量无氨基酸不含 EDTA 的蛋白酶抑制剂混合物）。使用微型超声波破碎仪进行冰上超声（参数设置：工作 3 秒，间歇 5 秒，总时间 20-30 分钟，严禁菌液发热升温）。

2. 离心澄清：将超声后的浓稠裂解液在 4 ℃ 下以 15,000 g - 18,000 g 超速离心 30 - 45 分钟，极其小心地吸取完全澄清的上清液（弃去不溶性包涵体沉淀）。

3. Amylose 亲和层析柱装载与纯化：

   • 提前用 5 - 10 倍柱体积的 Lysis Buffer 平衡 Amylose Resin 亲和层析介质。

   • 将澄清后的菌体上清重力流速缓慢通过层析柱，使重组蛋白上的 MBP 标签与大麦芽糖基质充分螯合。

   • 使用 20 倍柱体积的 Wash Buffer（高盐洗脱：20 mM Tris-HCl pH 7.5, 1 M NaCl, 5% 甘油）剧烈冲洗层析柱，彻底洗掉非特异性吸附的大肠杆菌宿主杂带和游离核酸。

   • 目的蛋白洗脱：向柱内倒入 Elution Buffer（配方：20 mM Tris-HCl pH 7.5, 500 mM NaCl,10 mM 麦芽糖 Maltose, 5% 甘油）。由于麦芽糖的竞争性结合，富含重组 MBP-LbCas12a 蛋白的组分将呈尖峰状洗脱流出，分管收集各个洗脱峰。

4. TEV 酶切除标签与透析保存（可选）：将收集到的重组蛋白加入适量重组 TEV 蛋白酶，混合置于透析袋中，放入透析缓冲液（20 mM HEPES pH 7.5, 300 mM KCl, 0.5 mM EDTA, 1 mM DTT, 20% 甘油）中 4 ℃ 透析过夜。酶切完成后，再次通过 Amylose 柱（收集未结合的流穿液）或结合 His-tag 负向纯化，即可斩断并彻底清除 MBP 标签，获得超高纯度的天然 LbCas12a 活性酶蛋白。分装后置于 -80 ℃ 锁死长期保存。

Part 2 English Section

I General Information and Molecular Biological Background

• Vector Name: pMBP-LbCas12a (Typically assembled by splicing a prokaryotic high-expression backbone with the LbCas12a fusion construct).

• Vector Classification: Prokaryotic expression plasmid engineered for high-yield recombinant protein purification in E. coli.

• Plasmid Size Scale: Approximately 8.5 - 9.5 kb (subject to custom engineering variations based on chosen fusion tags or multi-cloning site [MCS] fine-tuning).

• Core Backbone Architecture and Development Background:The pMBP-LbCas12a expression vector represents a high-efficiency research and industrial-grade tool meticulously optimized for the production of ultra-pure LbCas12a endonuclease enzyme within Escherichia coli translation systems.LbCas12a (previously designated as LbCpf1) is a Class 2, Type V CRISPR-Cas RNA-guided endonuclease derived from Lachnospiraceae bacterium ND2006. Compared to classical SpCas9, LbCas12a requires a shorter single CRISPR RNA (crRNA) guide guide loop, recognizes a T-rich Protospacer Adjacent Motif (5'-TTTV-3'), and cleaves double-stranded target DNA molecules to yield staggered staggered cuts with distinct cohesive "sticky" cohesive overhangs. Crucially, upon target-specific binding, LbCas12a activates a violent, non-specific single-stranded DNA (ssDNA) trans-cleavage cascade (collateral degradation activity). This distinct diagnostic trait has positioned LbCas12a as the pivotal baseline enzyme for modern in vitro nucleic acid detection technologies, including the standard DETECTR bioassay architecture.To bypass the biological limitation wherein large Cas12a nucleases form insoluble aggregate inclusion bodies during prokaryotic translation, this vector appends a highly soluble Maltose-Binding Protein (MBP) tag to the N-terminus of the LbCas12a open reading frame, vastly augmenting spatial folding, native solubility, and downstream purified protein concentration yields.

• Core Cis-Acting Elements and Map Characterization:

  • Robust T7 Promoter: Drives high-affinity transcription of the downstream fusion construct. Governed strictly by the host’s lysogenic T7 RNA Polymerase, the expression domain remains tightly repressed until the introduction of Isopropyl $\beta$-D-1-thiogalactopyranoside (IPTG) activates high-throughput target accumulation.

  • N-Terminal MBP Fusion Domain: Embedded directly upstream of the nuclease frame, the Maltose-Binding Protein operates as a highly potent intramolecular chaperone to stabilize protein solubility while enabling seamless one-step affinity capture using standard amylose resin columns.

  • Proteolytic Cleavage Site (TEV or 3C Boundaries): Positioned strategically between the MBP domain and the LbCas12a coding matrix. This permit investigators to use recombinant TEV protease or PreScission protease to cleanly cleave off the bulky MBP helper molecule post-affinity capture, releasing an un-tagged, completely native LbCas12a enzyme.

  • Nuclear Localization Signals (NLS) & Secondary Tags: Select subconfigurations may embed flanking 6×His-tags to provide secondary purification capability, or incorporate terminal SV40 NLS sequences to facilitate immediate post-purification nuclear trafficking inside mammalian cells via direct Ribonucleoprotein (RNP) transfection pipelines.

  • Prokaryotic Replicon & Selection: Outfitted with a high-copy bacterial origin alongside a functional Kanamycin resistance gene ($Kan^R$) or Ampicillin resistance cassette ($Amp^R$) for solid and liquid cultural selection routines.

II Strategic Research Value and In Vitro Diagnostic (IVD) Applications

The active LbCas12a enzyme harvested from pMBP-LbCas12a platforms serves as a critical macromolecule across several translational fields:

1. Engineering Ultrafast CRISPR Nucleic Acid Biosensors (DETECTR Platforms):Purified LbCas12a is the core enzyme utilized in point-of-care ultra-sensitive diagnostic assays. When specific target viral or bacterial genomic sequences (e.g., SARS-CoV-2, African Swine Fever Virus) are targeted by the complementary crRNA loop, the LbCas12a catalytic core switches on its trans-cleavage activity. By spiking the system with a custom fluorophore-quencher single-stranded DNA reporter probe (ssDNA F-Q Reporter), the activated LbCas12a recursively cleaves the single-stranded linkers, releasing intense fluorescent signals that permit single-molecule viral detection within 30 minutes.

2. In Vitro Target DNA Interception Assays:Deployed inside cell-free setups to validate the targeting kinetics, mismatch tolerances, and real-time off-target cleavage mechanics of novel crRNA configurations before moving into more resource-intensive in vivo gene engineering workflows.

3. Assembling Trace-Free Ribonucleoprotein (RNP) Transfection Complexes:Purified LbCas12a proteins can be complexed with synthetic or in vitro transcribed crRNAs to form stable RNPs. Delivering these fully assembled complexes directly into mammalian lineages or primary human immune cells (such as CAR-T therapeutic platforms) via electroporation allows for high-efficiency locus disruption with minimal off-target risks and zero risk of insertional plasmid integration.

III Laboratory E. coli Induction Expression and One-Step Affinity Purification Protocols

1. Host Strain Transformation and Large-Scale Culture Expansion

• Recommended Expression Hosts: Standard T7-induction strains such as BL21(DE3) or codon-optimized Rosetta(DE3) competent bacteria.

• Expansion Mechanics Sequence:

  1. Deliver the pMBP-LbCas12a construct into competent BL21(DE3) hosts via conventional heat-shock transformation. Plate onto selective LB agar planes containing 50 $\mu$g/mL Kanamycin and incubate at 37 °C overnight.

  2. Harvest a single positive colony into 5 mL of selective LB liquid media and propagate at 37 °C overnight to establish a starter culture.

  3. Inoculate this starter culture at a 1:100 dilution ratio into large culture flasks filled with 1 L of nutrient-dense Terrific Broth (TB) or 2×YT growth medium. Agitate vigorously at 37 °C and 220 rpm until the cell density reaches an OD600 index of 0.6 - 0.8 (mid-logarithmic growth phase).

2. Cold-Shock Pre-cooling and Low-Temperature IPTG Induction

Because Cas12a is a large multi-domain protein (~140 kDa), high-temperature expression causes translational overcrowding, forcing the nascent polypeptide chains to aggregate into insoluble inclusion bodies.A strict low-temperature propagation strategy must be implemented:

1. Cold-Shock Matrix Stabilization: The moment cultures hit the target OD600 parameters, transfer the 1 L vessels out of the incubator and submerge them entirely in an ice-water slurry or a 4 °C cold room for 30 minutes. This cold-shock rapidly arrests high-velocity host growth and slows cellular metabolic rates.

2. Chemical Induction Induction: Spike the chilled culture with 0.1 - 0.5 mM IPTG (low-concentration induction to prevent protein synthesis overcrowding).

3. Low-Velocity Overnight Incubation: Secure the cultures inside a pre-cooled shaking incubator calibrated to 16 °C and agitate gently at 150 - 180 rpm for 16 - 20 hours (overnight). The lowered kinetic energy slows down translation speeds, enabling the N-terminal MBP chaperone to guide the nascent LbCas12a polymers into a fully soluble, active conformation within the host cytoplasm.

4. Biomass Harvesting: Centrifuge the induced cultures at 5,000 g for 15 minutes at 4 °C. Collect the dense bacterial pellets, rinse once with cold PBS, and flash-freeze at -80 ℃ or proceed immediately to mechanical lysis.

3. Cryo-Ultrasonic Lysis and One-Step Amylose Affinity Chromatography

• Maintain strict temperature parameters (0 - 4 °C) throughout to protect against endogenous host protease activity.

1. Mechanical Cell Disruption (Lysis):Resuspend the bacterial pellet at a ratio of 5 - 10 mL of ice-cold Lysis Buffer per gram of wet biomass. (Lysis Buffer Formulation: 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM DTT, 5% Glycerol, supplemented with a standard EDTA-free cocktail of protease inhibitors). Subject the chilled suspension to micro-tip sonication on ice (Parameters: 3 seconds active pulse, 5 seconds delay, 20-30 minutes total time. Keep the container iced to prevent thermal denaturation).

2. Clarification Centrifugation: Spin the raw lysate at 15,000 g - 18,000 g inside a pre-cooled centrifuge at 4 °C for 30 - 45 minutes. Carefully transfer the clear aqueous supernatant to a sterile container, completely isolating it from the pellet of insoluble cell debris.

3. Amylose Affinity Chromatography Operation:

   • Pre-equilibrate an appropriate volume of Amylose Resin matrix with 5 - 10 column volumes (CV) of Lysis Buffer.

   • Pass the clarified supernatant slowly through the matrix using gravity flow, allowing maximum contact time for the MBP tags to complex with the immobilized amylose beads.

   • Flush the matrix with 20 CV of high-salt Wash Buffer (20 mM Tris-HCl pH 7.5, 1 M NaCl, 5% Glycerol) to strip away weakly bound host proteins and residual background nucleic acids.

   • Target Nuclease Elution: Gravity-feed the column with specialized Elution Buffer (Formulation: 20 mM Tris-HCl pH 7.5, 500 mM NaCl,10 mM Maltose, 5% Glycerol). The high-affinity free maltose displaces the bound tags, releasing the consolidated MBP-LbCas12a enzyme in a sharp protein peak. Collect sequential fractions across the elution profile.

4. Tag Cleavage and Dialysis Consolidation (Optional):Pool the protein fractions and introduce an appropriate unit ratio of recombinant TEV protease directly into the mixture. Seal the solution inside a dialysis membrane and submerge in standard Storage Dialysis Buffer (20 mM HEPES pH 7.5, 300 mM KCl, 0.5 mM EDTA, 1 mM DTT, 20% Glycerol) at 4 °C overnight. This simultaneous cleavage and dialysis step separates the MBP tag from the LbCas12a core. Pass the mixture through a secondary negative-selection amylose resin step to trap the liberated MBP, yielding an ultra-pure, un-tagged, fully functional LbCas12a active nuclease bank. Distribute into single-use aliquots and store long-term at -80 °C.

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