背景:大肠杆菌中的重组肽生产为对环境有害且大小受限的化学合成提供了可持续的替代方案。然而,高产量的二硫键肽的体内生产仍然具有挑战性,由于宿主蛋白酶/肽酶的降解以及转位到周质空间以形成二硫键的必要性。
结果:在这项研究中,我们建立了在大肠杆菌周质中高效、可溶性生产二硫键肽的表达系统。我们选择了具有不同复杂性的模型肽(大小,结构,二硫键的数量),即甲状旁腺激素1-84,生长抑素1-28,plectasin,和牛胰胰蛋白酶抑制剂(抑肽酶)。所有的肽都在没有和有N末端的情况下表达,低分子量CASPON™标签(4.1kDa),表达盒被整合到宿主基因组中。在微升规模的BioLector™培养过程中,我们发现大多数模型肽只能与CASPON™标签组合充分表达,否则,在SDS-PAGE上的表达仅弱或检测不到。即使使用CASPON™标签,宿主蛋白酶/肽酶的不希望的降解也是明显的。因此,我们通过与共翻译或翻译后信号序列组合表达肽,研究了在易位之前或之后是否发生降解。我们的结果表明,降解主要发生在易位之后,因为降解片段似乎是相同的,独立于信号序列,并且表达与共翻译信号序列没有增强。最后,我们在生物反应器中的C限制补料分批培养过程中,在两种工业相关宿主菌株中表达了所有CASPON™标记的肽。我们发现工艺性能高度依赖于肽-宿主-组合。所达到的滴度在0.6-2.6gL-1之间变化,并且超过先前在大肠杆菌中公开的数据。此外,质谱显示所有的肽都被表达到完成,包括二硫键的完全形成。
结论:在这项工作中,我们证明了CASPON™技术作为在大肠杆菌周质中生产可溶性肽的高效平台的潜力。我们在这里显示的滴度是前所未有的,每当甲状旁腺激素,生长抑素,plectasin或牛胰胰蛋白酶抑制剂在大肠杆菌中产生,从而使我们提出的上游平台优于以前发表的方法和化学合成。
BACKGROUND: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation.
RESULTS: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the
periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L-1, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds.
CONCLUSIONS: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the
periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis.