重组蛋白的高效表达，通常以下几个方面会涉及：
宿主体系选择
蛋白需求不同，那么表达宿主体系也不尽相同。原核表达体系常用的宿主为大肠杆菌和枯草杆菌，前者表达量高，后期产品需要去除内毒素；后者蛋白产量相对较低，但不产生内毒素。重组蛋白大肠杆菌表达常采用BL21（DE3）和K-12衍生菌株作为表达宿主与枯草杆菌作为表达菌株的主要区别如下所示：
| 项目 | 大肠杆菌表达宿主 | 枯草杆菌表达宿主 |
| 优点 | 应用最为广泛，操作简单，大规模生产成本低廉 | 生产成本低廉，不产内毒素，能够分泌表达蛋白 |
| 缺点 | 分泌能力差 | 产量相对较低 |
| 常用宿主 | Rosetta(DE3), Rosetta(DE3)pLysS, Rosetta 2(DE3)pLysS, Origami 2(DE3), Rosetta-gami 2(DE3)pLysS | WB600,WB800N, Bacillus Subtilis 168 |
表达质粒体系选择
目前最为常用的重组蛋白表达质粒载体融合了复制子(Replicon)、启动子(promoter)、筛选标签(selection marker)、多克隆位点（MCS）和融合蛋白移除策略(fusion tag removal strategy)，如图所示
卡梅德生物的科学家通过改造pET载体，获得双表达质粒（BiPlasmid vector），该质粒含有双MCS位点，双T7启动子，双lac操纵子和双核糖体结合位点，利用该质粒我们可以为客户进行一次转染操作，而获得两种蛋白的独立表达。同时我们构建了含有低温诱导蛋白表达元件的pCold系列载体，该载体能够在16℃条件下经过IPTG诱导表达大分子量蛋白，如上清表达150kDa活性蛋白。
融合标签选择
亲和标签：蛋白重组表达过程中采用亲和标签融合表达有两方面的作用：一方面是为了使蛋白纯化过程变得更加容易；另一方面是为了促进某些不溶性蛋白可溶。蛋白标签可分两类：一类是短肽类标签；一类是长肽以融合蛋白（fused partner）形式共表达。使用不同的蛋白标签需要根据具体案例来分析，卡梅德生物总结了常用的一些蛋白标签性质，如表1所示。
| 表1 蛋白标签主要特征 | ||||
| 肽标签 | 残基/MW (kDa) | 配体/基质 | 纯化条件 | |
| Poly-Arg | ~5/0.80 | 阳离子交换树脂 | NaCl线性洗脱(0–400 mM) | |
| Poly-His | ~6/0.84 | Ni2+琼脂柱 | 20–250 mM咪唑/低pH | |
| FLAG | 8/1.01 | FLAG抗体亲和琼脂柱 | 2–5 mM EDTA | |
| Strep-tag II | 8/1.06 | 链亲和素 | 2–25 mM脱硫生物素 | |
| c-myc | 11/1.20 | myc抗体亲和琼脂柱 | 低pH | |
| S-tag | 15/1.75 | S蛋白琼脂柱 | 3 M异硫氰酸; 0.2 M柠檬酸钾, pH 2或3 M MgCl2 | |
| 融合伴侣蛋白 | ca.（计算分子量） | 可溶性促进 | ||
| Fh8 | 69/8.0 | Ca2+依赖性苯基琼脂糖凝胶 | 10 mM EDTA | ND |
| Trx | 109/11.7 | 4氨基氧化苯胂琼脂凝胶柱 | 5–1000mM 2-巯基乙醇 | +++ |
| SUMO | ca. 100/12.0 | 亲和标签纯化（His） | ++++ | |
| BRT17 (β roll tag) | 153/14.7 | 25–75 mM Ca2+溶液沉淀 | ND | |
| GST | 211/26.0 | 谷胱甘肽琼脂柱 | 10–20 mM还原谷胱甘肽 | + |
| HaloTag7 | ca. 300/34.0 | 氯烷烃配体琼脂柱 | 带标签挂柱蛋白酶裂解 | ND |
| MBP | 396/ca. 42.5 | 交联支链淀粉 | 10 mM麦芽糖 | +++ |
| ELPs | 550/ca.47.0 | 高浓度NaCl (>1.5 M)或温度转变方式 | ND | |
| NusA | 495/54.8 | 亲和标签纯化（His） | ++ | |
蛋白重组表达策略组
重组蛋白的表达往往不像理论那样一帆风顺，在实际的表达过程中有可能产生各种各样的问题。在选择一个最优的蛋白表达策略非常困难的情况下，那么采用看似笨拙的方法，有可能会成为一种意想不到的“成功捷径”，比如项目要求构建表达两种重组蛋白时，构建6种不同的表达质粒，意味着有36种不同的表达条件组合，通过高通量微量蛋白表达实验一次性就可获得最佳蛋白表达条件组合，后续中式飞行试验和成本控制会变得相对容易
蛋白原核重组表达服务具体流
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