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国际标准期刊号: 2311-3278
国际标准期刊号: 2311-3278
瓦莱丽娅·扎贝琳娜
重组蛋白在不同的宿主生物体中表达。 大肠杆菌 ( E. coli ) 是中等复杂性生物制药最常用的非哺乳动物生产宿主。全长抗体是目前最畅销的生物制药；然而，一类新的、具有开创性的生物制药包括工程化抗体衍生片段和小支架结合蛋白。这些蛋白质结合特定的靶标，激活或阻断体内的关键反应。它们比抗体小，这使得它们具有更高的组织渗透潜力（例如，它们可以穿过血脑屏障）； 因此，可访问目标的数量增加了。抗原结合片段 ( Fab ) 是源自免疫球蛋白 G (IgG) 分子的单一、单价结合臂。Fab 由轻链 (LC) 和重链 (HC) 组成，通过二硫键连接。由于这些片段的分子尺寸小且复杂性降低， 基于大肠杆菌 的表达系统是哺乳动物细胞培养物的有吸引力的替代品。
Fab 质粒的构建-所有酶和试剂盒均购自 New England Biolabs (NEB, Ipswich, USA)。编码与前导信号序列融合的Fab片段的基因(OmpA SS )购自ATUM(纽瓦克，美国) 。 所有 Fab 的前导序列和间隔序列都是恒定的，但编码 Fab 的序列经过密码子优化。所有 Fab 在恒定区具有相同的 aa 序列，但在可变区具有不同的 Fab 特异性序列。对于PB系统，我们通过Nde I和 Eco 将Fab序列克隆到表达载体pET30a中 RI 限制位点。我们进行聚合酶链式反应 (PCR)，以用 DsbA 信号序列 (DsbA SS ) 替换 OmpA 信号序列 (OmpA SS )。Fab LC 和 HC 分别用携带突出 DsbA SS 序列的有义引物进行扩增（LC 带有 Nde I_DsbA SS – Fab_sense，HC 带有 Bsa I_DsbA SS – Fab_sense）：用于 LC 扩增的反义引物结合在 3' – UTR链接器的末端 （Bsa I_UTR链接器_反义）。
Integration of Fabs into the E. coli Genome - Fabs were amplified from pET30a vectors with TN7/1_pET30a_for and TN7/2_pET30a_back primers (Table S1, Supporting Information). Due to the primer overhangs, the integration cassette was flanked by 50 bp sequences that would anneal to the genomic TN7âsite for integration. Genome integration was performed as described by Sharan et al
µâBioreactor Cultivation- The µâbioreactor system, BioLector (m2pâlabs GmbH, Baesweiler, Germany), was used for cultivation. We performed two types of cultivations. Precultures were grown in 48âwell Flowerplates B (m2pâlabs GmbH) without optodes; the main cultures were grown in FlowerplatesâBOH (m2pâlabs GmbH) equipped with optodes for online measurements of the dissolved oxygen (DO) and pH value. Both plate types accommodated a working volume of 800 µL. Precultures were started from cells scratched off of a frozen research cell bank and cultivated in Luria–Bertani broth (Merck, Darmstadt, Germany) at 37 °C. The main culture was started from precultures, with an initial cell density of OD600 = 0.3, and cultivated at 30 °C. The main culture was cultivated according to protocols developed previously in our group.
Cell Lysis:Frozen cell pellets were thawed and resuspended in 200 µL lysis buffer (30 mm Tris, pH 8.2, 10 mm MgCl2, 30 mm ethylenediaminetetraacetic acid). Then, lysozyme (Merck) and benzonase (SigmaâAldrich) were added to final concentrations of 10 000 U and 2.5 U, respectively. After 10 min of vortexing, 1.5% Triton Xâ100 was added. The lysate was incubated on the vortex for another 10 min. Cell debris was removed with a 10 min centrifugation at 15 000 × g at 4 °C. The inclusion body (IB) fraction was washed twice with 100 mm Tris, pH 8.2, and solubilized by vortexing in 8 m urea and 100 mm Tris, pH 8.2, for 30 min. The final volumes of the soluble fraction and resolubilized IB fraction were equal. The debris was pelleted at 15 000 × g for 10 min and both the soluble and IB fractions were stored at −20 °C until analysis.
Analysis of Fab Expression:Expression of soluble Fab was determined with a sandwich ELISA that only detects correctly assembled Fab. The capture antibody binds only the LC, whereas the detection antibody specifically recognizes the hinge region of the HC. To be in the standard curve range of the ELISA, the Fab concentration range was initially estimated by western blot (WB) analysis. Accordingly, the soluble fractions of cell lysates were adjusted with a dilution buffer (1× phosphateâbuffered saline [PBS], containing 0.1% Tween 20 and 1% bovine serum albumin; Merck). Purified human Fab/κ (Bethyl P80â115; Montgomery, USA) was used from 0.78 ng mL−1 to 100 ng mL−1 to calculate a standard curve. We coated 96âwell plates with antiâhuman IgG (Fabâspecific) goat antibody diluted 1:400 in coating buffer (100 mm NaHCO3 and 40 mm Na2CO3, pH 9.6–9.8). Plates were washed with 1× PBS, containing 0.1% Tween 20, in a HydroFlex microplate washer. The samples were incubated at room temperature (RT) for 1 h to allow the capture antibodies to bind. Antiâhuman IgG mouse that specifically recognizes the hinge region and then antiâmouse IgG goat antibody conjugated to peroxidase were applied as detection antibodies, both diluted 1:1000, and each incubated for 1 h at RT.
Discussion:我们清楚地证明了无质粒表达系统可以极大地促进宿主细胞对 Fab 生产反应的直接表征。将单个 GOI 拷贝定点定向整合到宿主细胞基因组中，消除了由质粒拷贝数、质粒编码的抗生素抗性基因表达和质粒复制的变化引发的对宿主代谢的影响。通过消除这些混杂因素，Fab 生产对宿主细胞代谢的影响变得清晰可见。
结论：
我们建议选择 GI 表达宿主系统来直接表征宿主细胞对重组蛋白表达的反应。我们的结果证实，对于重组蛋白易位到周质而言，GI 生产系统优于传统的 PB 系统。我们证明了 GI 系统的适用性，既作为我们设置的核心部分，用于基础研究，又作为蛋白质生产的有吸引力的替代系统。基于所获得的知识和我们开发的平台，我们为在台式补料分批发酵中使用 GI 生产系统进行进一步详细的宿主细胞反应和过程表征提供了良好的基础。
注：这项工作部分在2018年10月11-12日在俄罗斯莫斯科举行的第21届欧洲生物技术大会上展示