背景:扩展遗传密码是用于修饰重组蛋白性质的常用方法。它涉及将一个密码子重新分配给另一个密码子,例如,不自然,氨基酸,并且需要一对正交tRNA和氨酰基tRNA合成酶的作用,这些合成酶被修饰以仅识别所需的氨基酸。该方法用于在工业酵母巴斯德毕赤酵母中生产携带对叠氮基-L-苯丙氨酸(pAzF)的曲妥珠单抗IgG。将蛋白质折叠和分泌的知识与生物反应器培养相结合,这项工作的目的是使具有扩展遗传密码的单克隆抗体的生产在实验室规模上具有成本效益。
结果:通过分泌信号前肽变化和腔分子伴侣Kar2p和Lhs1p的过表达,蛋白质向内质网的共翻译转运改善了掺入pAzF的曲妥珠单抗IgG及其Fab片段的产生。在Fab的情况下,用于蛋白质降解的液泡靶向敲除进一步提高了蛋白质产量。与筛选相比,工程化巴斯德毕赤酵母菌株的补料分批生物反应器培养将IgG和IgGpAzF的生产率提高了约50倍和20倍,产生多达238mgL-1和15mgL-1的完全组装的四聚体蛋白,分别。通过质谱确认pAzF的成功位点特异性掺入。
结论:巴斯德毕赤酵母成功地用于具有成本效益的实验室规模生产具有非天然氨基酸的单克隆抗体。将这项工作的结果应用于糖工程菌株,并在工艺开发中采取进一步的步骤为利用巴斯德毕赤酵母开发抗体以进行后续缀合开辟了巨大的可能性,例如,生物活性有效载荷。
BACKGROUND: Expansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-L-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale.
RESULTS: Co-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L-1 and 15 mg L-1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry.
CONCLUSIONS: Pichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads.