BioVector? pMBP-LbCas12a Recombinant Protein Expression Vector / pMBP-LbCas12a 重組蛋白表達(dá)與純化質(zhì)粒載體

一 產(chǎn)品基本信息與分子生物學(xué)背景

• 載體名稱：pMBP-LbCas12a（通常由原核高表達(dá)骨架與 LbCas12a 融合基因拼接而成）。

• 載體分類：原核表達(dá)質(zhì)粒（大腸桿菌高豐度重組蛋白純化專用）。

• 質(zhì)粒大小：約 8.5 - 9.5 kb（具體因融合標(biāo)簽和多克隆位點的優(yōu)化微調(diào)而異）。

• 核心骨架與設(shè)計背景：pMBP-LbCas12a 是一株專門為了在大腸桿菌（E. coli）表達(dá)系統(tǒng)中高產(chǎn)、高純度提取 LbCas12a 酶蛋白而優(yōu)化的工業(yè)與科研級原核表達(dá)質(zhì)粒。LbCas12a（舊稱 LbCpf1）是源自路氏纖維單胞菌（Lachnospiraceae bacterium ND2006）的 V 型 CRISPR-Cas 核心效應(yīng)核酸酶。與經(jīng)典的 Cas9 相比，它僅需要一條較短的 crRNA 引導(dǎo)，識別富含 T 的 PAM 序列（5'-TTTV-3'），并在剪切目標(biāo) DNA 時產(chǎn)生具有粘性末端的雙鏈斷裂。由于 Cas12a 在靶向結(jié)合特異性序列后會觸發(fā)強(qiáng)烈的順式（cis）和反式（trans）單鏈 DNA 非特異性附帶剪切活性（Collateral cleavage activity），它已成為現(xiàn)代體外核酸快檢技術(shù)（如 DETECTR 平臺）的絕對核心底層工具酶。為了克服 Cas12a 蛋白本身在大腸桿菌中極易形成不溶性包涵體（Inclusion bodies）的瓶頸，該載體在 LbCas12a 的 N 端融合了麥芽糖結(jié)合蛋白（Maltose-Binding Protein, MBP）標(biāo)簽，大幅度飆升了重組蛋白的體外可溶性（Solubility）與產(chǎn)量。

• 核心順式作用元件與圖譜特征：

  • T7 強(qiáng)啟動子（T7 Promoter）：強(qiáng)力驅(qū)動下游融合蛋白的轉(zhuǎn)錄。該啟動子受噬菌體 T7 RNA 聚合酶的嚴(yán)密調(diào)控，日常處于鎖死狀態(tài)，一旦加入異丙基-$\beta$-D-硫代半乳糖苷（IPTG）誘導(dǎo)，即可啟動海量的目的蛋白表達(dá)。

  • MBP 融合標(biāo)簽（Maltose-Binding Protein Tag）：位于 LbCas12a 基因的上游。MBP 不僅是強(qiáng)效的可溶性分子伴侶，還能讓粗提蛋白通過大麥芽糖親和層析柱（Amylose Resin Column）進(jìn)行一步法高純度親和純化。

  • 蛋白酶切位點（TEV / 3C Protease Site）：位于 MBP 標(biāo)簽與 LbCas12a 編碼區(qū)之間。允許純化完成后，利用 TEV 蛋白酶或 PreScission Protease 順式切除 MBP 標(biāo)簽，釋放出完全天然、無標(biāo)簽干擾的純凈 LbCas12a 酶。

  • 核定位信號（NLS）與 Poly-His 標(biāo)簽（可選配置）：部分亞型會在 C 端或 N 端額外串聯(lián) 6×His-tag 用于輔助純化，或帶有 SV40 NLS 信號以便于純化出的蛋白后續(xù)直接用于真核細(xì)胞內(nèi)源基因編輯。

  • 抗性選擇標(biāo)記與復(fù)制子：配置有卡那霉素抗性基因（$Kan^R$）或氨芐青霉素抗性基因（$Amp^R$），并基于常規(guī)高拷貝原核復(fù)制子運(yùn)行。

二 核心科研價值與體外快檢（IVD）轉(zhuǎn)化應(yīng)用

pMBP-LbCas12a 質(zhì)粒在大腸桿菌中成功表達(dá)純化出的 LbCas12a 酶蛋白，是現(xiàn)代生物技術(shù)中的明星分子：

1. 構(gòu)建基于 CRISPR 的新型核酸診斷平臺（如 DETECTR）：純化出的 LbCas12a 酶被廣泛用于體外超靈敏分子診斷。當(dāng)環(huán)境體系中存在目標(biāo)病原體 DNA（如新冠病毒、非洲豬瘟病毒等）并被 crRNA 靶向識別結(jié)合后，LbCas12a 會瘋狂激活其反式剪切（trans-cleavage）活性。通過在體系中加入兩端修飾了熒光基團(tuán)與淬滅基團(tuán)的單鏈 DNA 探針（ssDNA F-Q Reporter），LbCas12a 會將探針成百上千次切斷，釋放出肉眼或儀器可見的熒光，可在 30 分鐘內(nèi)實現(xiàn)單分子級別的基因快檢。

2. 體外靶向 DNA 基因剪切與打靶驗證：用于在體外酶促反應(yīng)體系中，測試針對特定靶標(biāo)突變（如腫瘤單核苷酸多態(tài)性 SNP 突變位點）設(shè)計的 crRNA 的剪切效率與脫靶率（Off-target rate），為體內(nèi)基因敲除實驗提供預(yù)實驗評估。

3. 重組核質(zhì)蛋白復(fù)合體（RNP）直接轉(zhuǎn)染：將純化出的 LbCas12a 蛋白與人工體外轉(zhuǎn)錄或化學(xué)合成的 crRNA 在體外混合，組裝形成極為穩(wěn)定的 RNP 復(fù)合體。利用電轉(zhuǎn)（Electroporation）或脂質(zhì)體直接將其遞送入真核細(xì)胞或原代免疫細(xì)胞（如 CAR-T 細(xì)胞）中，可實現(xiàn)即時、高效且極低脫靶風(fēng)險的無痕基因組編輯。

三 實驗室大腸桿菌高豐度誘導(dǎo)表達(dá)與一步法純化標(biāo)準(zhǔn)步驟

1. 表達(dá)宿主菌轉(zhuǎn)化與高密度液體擴(kuò)增

• 推薦表達(dá)宿主：大腸桿菌 BL21(DE3) 或 Rosetta(DE3) 蛋白表達(dá)專用感受態(tài)細(xì)胞（必須帶有 T7 RNA 聚合酶溶源區(qū)）。

• 常規(guī)操作：

  1. 采用常規(guī)熱擊法將 pMBP-LbCas12a 質(zhì)粒轉(zhuǎn)化入 BL21(DE3) 感受態(tài)細(xì)胞中，涂布于含有對應(yīng)抗生素（如 50 $\mu$g/mL 卡那霉素）的 LB 平板上，37 ℃ 培養(yǎng)過夜。

  2. 挑取陽性單菌落接種至 5 mL LB 液體培養(yǎng)基中（含抗生素），37 ℃ 振蕩培養(yǎng)過夜。

  3. 將預(yù)培養(yǎng)的菌液按 1:100 的體積比轉(zhuǎn)接至 1 L 富含營養(yǎng)的 TB 培養(yǎng)基（Terrific Broth）或 2×YT 培養(yǎng)基中。37 ℃、220 rpm 劇烈振蕩擴(kuò)增，直至菌液的菌密度 OD600 達(dá)到 0.6 - 0.8 的對數(shù)生長中期。

2. 低溫 IPTG 誘導(dǎo)蛋白表達(dá)（嚴(yán)防包涵體核心控制點）

Cas12a 是分子量超過 140 kDa 的大型多結(jié)構(gòu)域蛋白，如果在高密度下高強(qiáng)度快速表達(dá)，極易由于折疊不及發(fā)生成片聚集，產(chǎn)生不溶性的包涵體。全流程必須采用“低溫慢表達(dá)”策略：

1. 冷激預(yù)冷（Cold-shock）：當(dāng)菌液 OD600 達(dá)標(biāo)后，立刻將盛有 1 L 菌液的三角瓶撈出，整體浸入冰水混合物或 4 ℃ 冰箱中靜置預(yù)冷 30 分鐘，強(qiáng)行壓制細(xì)菌原有的高增殖代謝。

2. 加藥誘導(dǎo)：向冷激后的菌液中加入終濃度為 0.1 - 0.5 mM 的 IPTG（低濃度誘導(dǎo)，防止表達(dá)過猛）。

3. 低溫維持：將搖床溫度調(diào)至 16 ℃ 極其溫和地低速震蕩誘導(dǎo)表達(dá) 16 - 20 小時（過夜）。低溫能顯著延緩翻譯速度，配合 N 端的 MBP 標(biāo)簽，可逼迫絕大多數(shù) LbCas12a 重組蛋白以完美折疊的可溶態(tài)（Soluble form）存活于細(xì)菌細(xì)胞質(zhì)中。

4. 細(xì)胞收獲：4 ℃ 下以 5000 g 離心 15 分鐘，收集緊密深黃色的細(xì)菌沉淀，可用 PBS 洗滌一次，置于 -80 ℃ 凍存，或直接進(jìn)入裂解程序。

3. 超聲裂解與 Amylose 親和層析一步法層析純化

• 全流程要求 0 - 4 ℃ 冰上操作，嚴(yán)防內(nèi)源蛋白酶降解。

1. 細(xì)胞裂解（Lysis）：按每克菌體沉淀加入 5 - 10 mL Lysis Buffer（配方：20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM DTT, 5% 甘油，外加適量無氨基酸不含 EDTA 的蛋白酶抑制劑混合物）。使用微型超聲波破碎儀進(jìn)行冰上超聲（參數(shù)設(shè)置：工作 3 秒，間歇 5 秒，總時間 20-30 分鐘，嚴(yán)禁菌液發(fā)熱升溫）。

2. 離心澄清：將超聲后的濃稠裂解液在 4 ℃ 下以 15,000 g - 18,000 g 超速離心 30 - 45 分鐘，極其小心地吸取完全澄清的上清液（棄去不溶性包涵體沉淀）。

3. Amylose 親和層析柱裝載與純化：

   • 提前用 5 - 10 倍柱體積的 Lysis Buffer 平衡 Amylose Resin 親和層析介質(zhì)。

   • 將澄清后的菌體上清重力流速緩慢通過層析柱，使重組蛋白上的 MBP 標(biāo)簽與大麥芽糖基質(zhì)充分螯合。

   • 使用 20 倍柱體積的 Wash Buffer（高鹽洗脫：20 mM Tris-HCl pH 7.5, 1 M NaCl, 5% 甘油）劇烈沖洗層析柱，徹底洗掉非特異性吸附的大腸桿菌宿主雜帶和游離核酸。

   • 目的蛋白洗脫：向柱內(nèi)倒入 Elution Buffer（配方：20 mM Tris-HCl pH 7.5, 500 mM NaCl,10 mM 麥芽糖 Maltose, 5% 甘油）。由于麥芽糖的競爭性結(jié)合，富含重組 MBP-LbCas12a 蛋白的組分將呈尖峰狀洗脫流出，分管收集各個洗脫峰。

4. TEV 酶切除標(biāo)簽與透析保存（可選）：將收集到的重組蛋白加入適量重組 TEV 蛋白酶，混合置于透析袋中，放入透析緩沖液（20 mM HEPES pH 7.5, 300 mM KCl, 0.5 mM EDTA, 1 mM DTT, 20% 甘油）中 4 ℃ 透析過夜。酶切完成后，再次通過 Amylose 柱（收集未結(jié)合的流穿液）或結(jié)合 His-tag 負(fù)向純化，即可斬斷并徹底清除 MBP 標(biāo)簽，獲得超高純度的天然 LbCas12a 活性酶蛋白。分裝后置于 -80 ℃ 鎖死長期保存。

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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