Ehgartner Daniela, Sagmeister Patrick, Langemann Timo, Meitz Andrea, Lubitz Werner, Herwig Christoph
Institute for Chemical, Environmental and Biological Engineering, TU Wien, Gumpendorferstrasse 1a/166, 1060, Vienna, Austria.
CD Laboratory on Mechanistic and Physiological Methods for Improved Bioprocesses, TU Wien, Gumpendorferstrasse 1a/166, 1060, Vienna, Austria.
Appl Microbiol Biotechnol. 2017 Jul;101(14):5603-5614. doi: 10.1007/s00253-017-8281-x. Epub 2017 Apr 20.
Production of recombinant proteins as inclusion bodies is an important strategy in the production of technical enzymes and biopharmaceutical products. So far, protein from inclusion bodies has been recovered from the cell factory through mechanical or chemical disruption methods, requiring additional cost-intensive unit operations. We describe a novel method that is using a bacteriophage-derived lysis protein to directly recover inclusion body protein from Escherichia coli from high cell density fermentation process: The recombinant inclusion body product is expressed by using a mixed feed fed-batch process which allows expression tuning via adjusting the specific uptake rate of the inducing substrate. Then, bacteriophage ΦX174-derived lysis protein E is expressed to induce cell lysis. Inclusion bodies in empty cell envelopes are harvested via centrifugation of the fermentation broth. A subsequent solubilization step reveals the recombinant protein. The process was investigated by analyzing the impact of fermentation conditions on protein E-mediated cell lysis as well as cell lysis kinetics. Optimal cell lysis efficiencies of 99% were obtained with inclusion body titers of >2.0 g/l at specific growth rates higher 0.12 h and inducer uptake rates below 0.125 g/(g × h). Protein E-mediated cell disruption showed a first-order kinetics with a kinetic constant of -0.8 ± 0.3 h. This alternative inclusion body protein isolation technique was compared to the one via high-pressure homogenization. SDS gel analysis showed 10% less protein impurities when cells had been disrupted via high-pressure homogenization, than when empty cell envelopes including inclusion bodies were investigated. Within this contribution, an innovative technology, tuning recombinant protein production and substituting cost-intensive mechanical cell disruption, is presented. We anticipate that the presented method will simplify and reduce the production costs of inclusion body processes to produce technical enzymes and biopharmaceutical products.
将重组蛋白生产为包涵体是生产工业酶和生物制药产品的一项重要策略。到目前为止，包涵体中的蛋白质已通过机械或化学破碎方法从细胞工厂中回收，这需要额外的高成本单元操作。我们描述了一种新方法，该方法使用噬菌体衍生的裂解蛋白从高密度发酵过程的大肠杆菌中直接回收包涵体蛋白：通过使用混合进料补料分批工艺表达重组包涵体产物，该工艺可通过调节诱导底物的比摄取率来调节表达。然后，表达噬菌体ΦX174衍生的裂解蛋白E以诱导细胞裂解。通过离心发酵液收获空细胞包膜中的包涵体。随后的溶解步骤可得到重组蛋白。通过分析发酵条件对蛋白E介导的细胞裂解以及细胞裂解动力学的影响来研究该过程。在比生长速率高于0.12 h且诱导剂摄取速率低于0.125 g/(g×h)时，获得了99%的最佳细胞裂解效率，包涵体滴度>2.0 g/l。蛋白E介导的细胞破碎显示出一级动力学，动力学常数为-0.8±0.3 h。将这种替代性的包涵体蛋白分离技术与通过高压匀浆的技术进行了比较。SDS凝胶分析表明，与研究包括包涵体的空细胞包膜时相比，通过高压匀浆破碎细胞时的蛋白质杂质少10%。在本论文中，提出了一种创新技术，可调节重组蛋白的生产并替代高成本的机械细胞破碎。我们预计，所提出的方法将简化并降低生产包涵体过程以生产工业酶和生物制药产品的成本。
