背景:大肠杆菌是生产生物制药的最常用宿主生物之一,因为它允许成本有效和快速的重组蛋白表达。然而,具有挑战性的蛋白质通常以低滴度或作为包涵体产生,制造过程需要针对每种蛋白质单独开发。最近,我们开发了CASPONTM技术,用于高滴度可溶性表达的通用基于融合标签的平台过程,包括标准化的下游加工和融合标签的高度特异性酶促切割。为了评估进一步改进N端融合CASPONTM标签的潜在策略,我们修改了编码标签的mRNA的5'UTR和5'区域,以优化核糖体-mRNA相互作用。
结果:在目前的工作中,我们发现,通过修改基于pET30acer质粒系统的5'UTR序列,在实验室规模的碳限制补料分批培养中,融合蛋白CASPONTM-肿瘤坏死因子α的表达发生了变化,但没有实现表达滴度的显著增加。对于携带表达增强子元件的构建体,翻译效率最高,并且在核糖体和mRNA之间具有非常有利的相互作用能(ΔGtotal)。然而,转录效率相对较低的构建体,缺少表达增强子序列,并带有最有利的ΔGtotal测试,与参考pET30a构建体一起导致最高的重组蛋白形成。此外,我们发现,通过在CASPONTM标签的T7AC元件的核苷酸序列中引入同义突变,利用稀有和非稀有密码子的组合,编码CASPONTM标签的转录本的5'末端(-4至37)核苷酸的自由折叠能增加了6kcal/mol。令人惊讶的是,这种新的T7ACrare变体导致重组蛋白滴度提高1.3倍至5.3倍,在工业相关条件下,在实验室规模的碳限制补料分批发酵中显示了三种与工业相关的蛋白质。
结论:本研究揭示了控制重组蛋白表达的核糖体和mRNA之间的一些复杂的相互依赖关系。通过修饰5'UTR以获得mRNA与核糖体之间的优化相互作用能(ΔGtotal),转录水平改变了,突出了个别成绩单的不同翻译效率。已显示,最高的重组滴度不是通过具有最有效翻译的构建体获得的,而是通过具有通常高转录量和有利的ΔGtotal的构建体获得的。此外,通过将包括多个稀有密码子的沉默突变引入CAPONTM标签mRNA的5'末端来增强表达的出乎意料的高潜力。尽管融合蛋白的滴度急剧增加,未观察到包涵体的形成或对细胞生长的负面影响。我们假设滴度的急剧增加很可能是由更好的核糖体结合位点可及性引起的。我们的研究,这证明了在工业相关生产条件下核糖体-mRNA相互作用的变化对蛋白质表达的影响,为使用CASPONTM平台工艺在生物制药行业中的新型T7ACrare标签的适用性打开了大门。
BACKGROUND: Escherichia coli is one of the most commonly used host organisms for the production of biopharmaceuticals, as it allows for cost-efficient and fast recombinant protein expression. However, challenging proteins are often produced with low titres or as inclusion bodies, and the manufacturing process needs to be developed individually for each protein. Recently, we developed the CASPONTM technology, a generic fusion tag-based platform process for high-titer soluble expression including a standardized downstream processing and highly specific enzymatic cleavage of the fusion tag. To assess potential strategies for further improvement of the N-terminally fused CASPONTM tag, we modified the 5\'UTR and 5\' region of the tag-coding mRNA to optimize the ribosome-mRNA interactions.
RESULTS: In the present work, we found that by modifying the 5\'UTR sequence of a pET30acer plasmid-based system, expression of the fusion protein CASPONTM-tumour necrosis factor α was altered in laboratory-scale carbon-limited fed-batch cultivations, but no significant increase in expression titre was achieved. Translation efficiency was highest for a construct carrying an expression enhancer element and additionally possessing a very favourable interaction energy between ribosome and mRNA (∆Gtotal). However, a construct with comparatively low transcriptional efficiency, which lacked the expression enhancer sequence and carried the most favourable ∆Gtotal tested, led to the highest recombinant protein formation alongside the reference pET30a construct. Furthermore, we found, that by introducing synonymous mutations within the nucleotide sequence of the T7AC element of the CASPONTM tag, utilizing a combination of rare and non-rare codons, the free folding energy of the nucleotides at the 5\' end (-4 to + 37) of the transcript encoding the CASPONTM tag increased by 6 kcal/mol. Surprisingly, this new T7ACrare variant led to improved recombinant protein titres by 1.3-fold up to 5.3-fold, shown with three industry-relevant proteins in lab-scale carbon limited fed-batch fermentations under industrially relevant conditions.
CONCLUSIONS: This study reveals some of the complex interdependencies between the ribosome and mRNA that govern recombinant protein expression. By modifying the 5\'UTR to obtain an optimized interaction energy between the mRNA and the ribosome (ΔGtotal), transcript levels were changed, highlighting the different translation efficiencies of individual transcripts. It was shown that the highest recombinant titre was not obtained by the construct with the most efficient translation but by a construct with a generally high transcript amount coupled with a favourable ΔGtotal. Furthermore, an unexpectedly high potential to enhance expression by introducing silent mutations including multiple rare codons into the 5\'end of the CAPONTM tag\'s mRNA was identified. Although the titres of the fusion proteins were dramatically increased, no formation of inclusion bodies or negative impact on cell growth was observed. We hypothesize that the drastic increase in titre is most likely caused by better ribosomal binding site accessibility. Our study, which demonstrates the influence of changes in ribosome-mRNA interactions on protein expression under industrially relevant production conditions, opens the door to the applicability of the new T7ACrare tag in biopharmaceutical industry using the CASPONTM platform process.