pET-32M3C (pET-32M-3C) BioVector? 原核高效融合表達(dá)質(zhì)粒載體pET-32M3C (pET-32M-3C) Prokaryotic High-Level Fusion Expression Plasmid Vector
- 價(jià) 格：￥99860
- 貨 號(hào)：BioVector? pET-32M3C
- 產(chǎn) 地：北京
- BioVector NTCC典型培養(yǎng)物保藏中心
- 聯(lián)系人：Dr.Xu, Biovector NTCC Inc.
電話：400-800-2947 工作微信:1843439339 (QQ同號(hào))
手機(jī)：18901268599
地址：北京
- 已注冊(cè)
BioVector? pET-32M3C (pET-32M-3C) 原核高效融合表達(dá)質(zhì)粒載體
BioVector? pET-32M3C (pET-32M-3C) Prokaryotic High-Level Fusion Expression Plasmid Vector
第一部分 中文說明
一 產(chǎn)品基本信息與設(shè)計(jì)背景
載體名稱：pET-32M3C（常寫作 pET32M3C 或 pET-32M-3C）
載體類型：大腸桿菌（E. coli）高豐度、可溶性、原核融合表達(dá)質(zhì)粒。
骨架起源與工程化改良：
傳統(tǒng)骨架基礎(chǔ)：基于經(jīng)典的 pET-32a(+) 系列骨架優(yōu)化縮小而來。
3C 酶切位點(diǎn)改良：傳統(tǒng)的 pET-32a(+) 載體使用的是腸激酶（Enterokinase, Ek）或 凝血酶（Thrombin） 剪切位點(diǎn)。腸激酶價(jià)格昂貴且在非特異性位點(diǎn)極易發(fā)生漏切、錯(cuò)切。pET-32M3C 通過分子克隆技術(shù)，將純化標(biāo)簽下游的剪切位點(diǎn)特異性替換為 HRV 3C 蛋白酶（人鼻病毒 3C 蛋白酶 / PreScission Protease） 識(shí)別序列。HRV 3C 蛋白酶在 4 攝氏度下即可表現(xiàn)出極高的剪切特異性與活性，能確保融合標(biāo)簽被絕對(duì)精準(zhǔn)、無痕地切除。
M 縮減版設(shè)計(jì)（pET-32M）：去除了原骨架中一些不必要的非編碼冗余片段，使質(zhì)粒分子量由原先的約 5.9 kb 優(yōu)化縮減至約 5.8 kb，進(jìn)一步提高了大片段基因克隆的成功率以及轉(zhuǎn)化效率。
復(fù)制子與抗性：pBR322 復(fù)制 origin（中低拷貝數(shù)，以維持強(qiáng) T7 啟動(dòng)子下的質(zhì)粒穩(wěn)定性）；帶有 氨芐青霉素抗性基因（AmpR / Amplicillin）。
生物安全級(jí)別：1級(jí)（BSL-1）。
二 核心功能元件與轉(zhuǎn)錄/翻譯圖譜
pET-32M3C 載體專門用于解決結(jié)構(gòu)復(fù)雜、易形成不溶性包涵體（Inclusion bodies）的頑固目的蛋白（GOI）的原核表達(dá)問題。其核心功能元件的排列和級(jí)聯(lián)圖譜如下：
T7 啟動(dòng)子 ── lac 操縱子 ── 強(qiáng) RBS ── TrxA 融合標(biāo)簽 ── 6×His 標(biāo)簽 ── HRV 3C 酶切位點(diǎn) ── MCS ── 6×His 標(biāo)簽 (C端) ── T7 終止子強(qiáng)力的 T7lac 轉(zhuǎn)錄軸：
T7 啟動(dòng)子（T7 Promoter）：專門識(shí)別 T7 RNA 聚合酶，驅(qū)動(dòng)極高速度的轉(zhuǎn)錄。
lac 操縱子（lac operator）：緊跟啟動(dòng)子下方，通過結(jié)合 LacI 阻斷未誘導(dǎo)狀態(tài)下的基礎(chǔ)漏表達(dá)（Leaky expression），在加入 IPTG 后解除抑制。
高效助溶與純化雙標(biāo)簽系統(tǒng)（Dual-Tag Framework）：
TrxA 標(biāo)簽（Thioredoxin, 大腸桿菌硫氧還蛋白，~11.9 kDa）：這是 pET-32 系列的核心王牌。TrxA 是一種高度可溶的內(nèi)源性蛋白，作為融合伴侶（Fusion partner），它能夠極大地促進(jìn)下游目的蛋白在胞內(nèi)的正確折疊，強(qiáng)行將原本極易形成不溶性包涵體的毒性/硬核蛋白轉(zhuǎn)化為活性、高豐度的可溶性狀態(tài)（Soluble fraction）。
6×His 標(biāo)簽（六組氨酸純化標(biāo)簽）：在 TrxA 下方及多克隆位點(diǎn)（MCS）的 C 端各內(nèi)置一個(gè) 6×His 標(biāo)簽。無論是全長融合表達(dá)還是標(biāo)簽切除前后，都能配合固定化金屬親和層析（IMAC，如 Ni-NTA 磁珠/填料）進(jìn)行高純度的捕獲和精制。
精準(zhǔn)剪切中樞（HRV 3C Protease Cleavage Site）：
識(shí)別氨基酸核心序列為：Leu-Glu-Val-Leu-Phe-Gln ↓ Gly-Pro。
3C 蛋白酶在 Gln（谷氨酰胺）和 Gly（甘氨酸）殘基之間特異性切斷。這種剪切往往在 MCS 克隆位點(diǎn)的最前端釋放出目標(biāo)蛋白，避免在切除 TrxA 標(biāo)簽后目的蛋白 N 端殘留冗余的無用氨基酸疤痕（Scar residues）。
三 實(shí)驗(yàn)室標(biāo)準(zhǔn)轉(zhuǎn)化、表達(dá)誘導(dǎo)與蛋白層析純化步驟
克隆與質(zhì)粒轉(zhuǎn)化（Cloning & Transformation）：
將目的基因（GOI）通過常規(guī)酶切連接或重組克隆技術(shù)插入多克隆位點(diǎn)（MCS）中。注意保持讀碼框（Reading Frame）與前端 TrxA/3C 位點(diǎn)的完全一致。
克隆菌株：連接產(chǎn)物首先轉(zhuǎn)化至 DH5$\alpha$、TOP10 等常規(guī)克隆大腸桿菌菌株中進(jìn)行質(zhì)粒擴(kuò)增、測(cè)序鑒定。注：克隆菌株不含 T7 RNA 聚合酶，質(zhì)粒在其中無法進(jìn)行目標(biāo)蛋白的表達(dá)。
宿主菌誘導(dǎo)表達(dá)（Protein Expression & IPTG Induction）：
將測(cè)序正確的質(zhì)粒轉(zhuǎn)化至含有 T7 表達(dá)系統(tǒng)的表達(dá)宿主菌（如 BL21(DE3)、Origami 2(DE3) 或 Rosetta(DE3)）中。注：由于 TrxA 標(biāo)簽常用于促進(jìn)二硫鍵形成，對(duì)于富含二硫鍵的復(fù)雜蛋白，強(qiáng)烈推薦配合使用 Origami 2(DE3) 突變株，其胞內(nèi)氧還環(huán)境能與 TrxA 產(chǎn)生完美的協(xié)同助溶效應(yīng)。
挑取單菌落接種于標(biāo)準(zhǔn) LB 液體培養(yǎng)基（含 100 $\mu$g/ml 氨芐青霉素），37°C 振蕩培養(yǎng)至對(duì)數(shù)中期（OD600 = 0.6–0.8）。
立即向體系中加入終濃度為 0.1 mM 至 1.0 mM 的 IPTG 啟動(dòng)誘導(dǎo)。為了防止過快的翻譯導(dǎo)致折疊速度失衡，建議將溫度下調(diào)至 16°C – 25°C 低溫低速振蕩誘導(dǎo)過夜（12–16小時(shí)），這能使 TrxA 的助溶效果發(fā)揮到極致。
蛋白收獲、親和純化與標(biāo)簽切除（Purification & Tag Removal）：
一純（First IMAC）：超聲波破碎菌體，離心收集上清（可溶性組分）。將上清液上樣至 Ni-NTA 親和層析柱，利用咪唑（Imidazole）梯度洗脫，收集 TrxA-6xHis-3C-GOI 完整融合蛋白。
透析與 3C 酶切（Cleavage）：將洗脫的融合蛋白透析至 3C 酶切緩沖液中以去除高濃度咪唑。按 1:50 或 1:100 的質(zhì)量比加入重組 HRV 3C 蛋白酶，置于 4°C 冰箱中反應(yīng)過夜。
二純（Reverse IMAC / 反向?qū)游觯?/strong>：酶切反應(yīng)混合物中包含了已被切下的 TrxA-6xHis 片段、未切盡的融合蛋白、帶有 His 標(biāo)簽的 HRV 3C 蛋白酶，以及去除了標(biāo)簽的純凈目的蛋白（GOI）。將該混合物重新通過一次全新的 Ni-NTA 柱，此時(shí)標(biāo)簽、殘留融合物和酶將被全部死死吸附在柱子上，而不帶標(biāo)簽的目標(biāo)純凈蛋白將直接從流穿液（Flow-through）中流出，純度可直接達(dá)到 95% 以上。
四 核心科研應(yīng)用方向
結(jié)構(gòu)生物學(xué)與 X 射線晶體學(xué)/冷凍電鏡研究（Structural Biology）：利用 HRV 3C 酶切產(chǎn)生的無痕、無多余殘基的天然結(jié)構(gòu)蛋白，是進(jìn)行蛋白質(zhì)晶體生長、高分辨率核磁共振（NMR）及冷凍電鏡（Cryo-EM）三維構(gòu)象解析的標(biāo)準(zhǔn)首選骨架。
頑固/難折疊哺乳動(dòng)物蛋白的原核廉價(jià)替代表達(dá)：許多來源于哺乳動(dòng)物的激酶、細(xì)胞因子、單鏈抗體片段（scFv），直接在宿主中表達(dá)會(huì)發(fā)生大面積沉淀。pET-32M3C 通過其強(qiáng)力的 TrxA 折疊增溶屏障，實(shí)現(xiàn)了在低成本大腸桿菌體系中對(duì)這些硬核蛋白的大規(guī)模、高活性可溶性工業(yè)化制備。
PART 2 ENGLISH SECTION
I General Information and Design Architecture
Vector Name: pET-32M3C (Commonly cataloged as pET32M3C or pET-32M-3C)
Vector Type: Recombinant E. coli high-yield, high-solubility prokaryotic fusion expression plasmid.
Backbone Matrix and Molecular Streamlining:
Foundational Framework: Optimized and condensed directly from the classical pET-32a(+) parental matrix.
3C Protease Cleavage Site Insertion: Legacy pET-32a(+) matrices utilize Enterokinase (Ek) or Thrombin endopeptidase cleavage sites. Enterokinase is structurally delicate, cost-intensive, and prone to non-specific off-target proteolytic degradation. The pET-32M3C vector replaces these domains with a premium Human Rhinovirus 3C (HRV 3C) Protease recognition linker (equivalent to PreScission Protease). The HRV 3C protease executes highly specific, single-site cleavage even at 4°C, assuring absolute target precision and scarless fusion partner excision.
M-Series Streamlining (pET-32M): Redundant non-coding nucleotide spans have been enzymatically removed, trimming the total vector circumference from ~5.9 kb down to a compact ~5.8 kb, enhancing transformation efficiency and larger gene insert stability.
Replicon & Selection Antibiotic: pBR322 origin of replication (low-to-medium copy configuration calibrated to maintain plasmid structural stability under intense T7 transcription forces); carries the Ampicillin resistance gene (AmpR).
Biosafety Level: BSL-1.
II Core Functional Elements and Expression Map
The pET-32M3C platform is customized to bypass inclusion body blockades of highly complex, hydrophobic, or toxic Genes of Interest (GOIs) inside prokaryotic systems. The linear spatial alignment of its expression cassette reads as follows:
T7 Promoter ── lac Operator ── Strong RBS ── TrxA Tag ── 6×His Tag ── HRV 3C Site ── MCS ── C-terminal 6×His Tag ── T7 TerminatorHigh-Velocity T7lac Transcription Core:
T7 Promoter: Exclusively matched to the bacteriophage T7 RNA Polymerase, driving exceptionally high transcription rates.
lac Operator: Bound downstream of the promoter, it coordinates tightly with the LacI repressor to suppress basal leaky expression before induction. This blockade is instantly lifted upon the introduction of IPTG.
Dual-Tag Solubilization and Affinity Capture Framework:
TrxA Fusion Tag (Thioredoxin, E. coli derived, ~11.9 kDa): The foundational asset of the pET-32 paradigm. TrxA operates as an ultra-soluble native molecular partner. When fused N-terminally to the passenger protein, it acts as a powerful chaperone shield, driving correct intracellular protein folding and pulling heavily aggregated, insoluble proteins into the active, soluble fraction.
Polyhistidine (6×His) Tracks: Flanked both directly downstream of the TrxA element and at the extreme C-terminus of the Multiple Cloning Site (MCS). This enables standardized immobilized metal affinity chromatography (IMAC, utilizing Ni-NTA resins or magnetic beads) at multiple stages of purification.
High-Fidelity Cleavage Junction (HRV 3C Site):
Core Recognition Sequence: Leu-Glu-Val-Leu-Phe-Gln ↓ Gly-Pro.
The protease cuts precisely between the Gln (Q) and Gly (G) residues. Positioned at the N-terminal boundary of the MCS, this site allows the clean removal of the massive TrxA assembly, preventing the retention of unwanted amino acid residues on the N-terminus of the target protein.
III Standard Protocols for Transformation, Expression Tuning, and Reverse-IMAC Purification
Cloning and Target Plasmid Prototyping:
Clone the target Gene of Interest (GOI) into designated restriction configurations within the Multiple Cloning Site (MCS). Ensure the insert's open reading frame (ORF) aligns perfectly with the N-terminal TrxA and HRV 3C translation frame.
Cloning Strain Proliferation: Transform ligation solutions into standard cloning-grade E. coli host strains like DH5$\alpha$ or TOP10 for sequence validation and high-yield plasmid extraction. Note: Cloning strains lack the T7 RNA Polymerase gene, making target protein expression impossible inside these lines.
Expression Host Selection & Metabolic Induction:
Deliver verified pET-32M3C constructs into specialized DE3 expression hosts (such as BL21(DE3), Origami 2(DE3), or Rosetta(DE3)). Note: Because TrxA excels at guiding proper disulfide bond creation, complex target proteins with dense disulfide bridges should ideally be expressed in Origami 2(DE3). This host's mutated, oxidized cytoplasmic environment works synergistically with the TrxA tag.
Inoculate a verified single colony into standard liquid LB medium containing 100 $\mu$g/mL Ampicillin, shaking at 37°C until it enters mid-log phase (OD600 = 0.6–0.8).
Initiate target protein expression by supplementing the culture with 0.1 mM to 1.0 mM IPTG. To maximize soluble protein accumulation and prevent translation crowding, lower the temperature to 16°C–25°C for slow, low-temperature overnight induction (12–16 hours).
Cell Harvest, Primary Capture, and Tag Removal Routine:
Primary IMAC Capture: Disrupt the cells via sonication and clarify the lysate via high-speed centrifugation to collect the soluble fraction. Load the supernatant onto a Ni-NTA affinity column, and run an imidazole gradient to isolate the intact TrxA-6xHis-3C-GOI fusion product.
Dialysis & 3C Protease Cleavage: Dialyze the eluted fraction into standard 3C cleavage buffer to strip away excess imidazole. Supplement the dialyzed protein pool with recombinant His-tagged HRV 3C protease at a mass ratio between 1:50 and 1:100, then incubate at 4°C overnight.
Reverse IMAC Polishing: The digested mixture now contains cleaved TrxA-His tags, the His-tagged HRV 3C protease itself, minor uncleaved fusion remnants, and the untagged, pure target protein (GOI). Pass this entire slurry through a fresh Ni-NTA column. All His-tagged components (tags, enzymes, and remnants) will bind to the matrix, while the highly pure, untagged target protein passes directly through into the flow-through fraction, routinely yielding >95% purity.
IV Strategic Research Fields
Structural Biology & High-Resolution Macromolecular Analytics: The generation of untagged proteins with native N-terminal sequences makes this vector an ideal tool for producing samples for X-ray crystallography, high-resolution Nuclear Magnetic Resonance (NMR), and Cryo-Electron Microscopy (Cryo-EM) structural determinations.
Soluble Prototyping of Challenging Mammalian Targets: Many eukaryotic kinases, signaling cytokines, and single-chain variable fragments (scFvs) form inclusion bodies when expressed in standard bacterial setups. The pET-32M3C vector leverages its built-in TrxA solubility shield to enable low-cost, scalable production of these difficult targets within an E. coli platform.
BioVector NTCC質(zhì)粒載體菌株細(xì)胞蛋白抗體基因保藏中心
電話:400-800-2947
工作QQ/微信同號(hào):1843439339
網(wǎng)址http://www.nedfriskphoto.com
- 公告/新聞
