Abstract

Trypsin is a key enzyme under the serine proteases that is found in the pancreas which plays a key role in protein digestion. It cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. This enzyme has received greater attention mainly due to its increased use in the removal of fusion tags during protein purification and its role in the processing of biosimilars like insulin. The present study was carried out to develop a clone with Novel TrpLE1413(TrpE) Fusion Tag for enhanced expression of trypsin which helps in cost reduction of biosimilar processing. In our experiment we have used a synthetic bovine trypsin gene containing a novel fusion tag TrpE at its N terminus, which was cloned into the pET41b (+) vector and expressed in E. coli BL21 (DE3) in a lab-scale bioreactor. Using the optimized fermentation process with TrpE Fusion Tag, 27.8 g/L inclusion bodies were produced at the end of fermentation, of which 209 mg/L of active trypsin was obtained after purification. In contrast, previous reports have claimed to produce a maximum of 60 mg/L of the enzyme without the fusion tag. Thus based on our findings, the small size (less than 2 kDa) of TrpE tag and its hydrophobicity may reduce the loss incurred during the purification process. Hence, it could be discerned that the use of the TrpE fusion tag along with a robust fermentation process led to 3– 4 fold higher yield making it a commercially viable process facilitating an improved recovery of the enzyme.

抽象的

胰蛋白酶是在胰腺中发现的丝氨酸蛋白酶之下的关键酶，它在蛋白质消化中起关键作用。它主要在氨基酸赖氨酸或精氨酸的羧基侧切割肽链。该酶受到越来越多的关注，主要是因为它在蛋白质纯化过程中越来越多地用于去除融合标签，并且在生物类似物（如胰岛素）的加工中具有重要作用。进行本研究以开发具有新型TrpLE1413（TrpE）融合标签的克隆，以增强胰蛋白酶的表达，从而有助于降低生物仿制药的成本。在我们的实验中，我们使用了一个合成的牛胰蛋白酶基因，该基因在其N端含有一个新的融合标签TrpE，该基因被克隆到pET41b（+）载体中并在大肠杆菌中表达实验室规模的生物反应器中的BL21（DE3）。使用带有TrpE Fusion Tag的优化发酵工艺，在发酵结束时产生了27.8 g / L的包涵体，其中纯化后获得了209 mg / L的活性胰蛋白酶。相比之下，以前的报道声称最多可产生60 mg / L的无融合标签的酶。因此，根据我们的发现，TrpE标签的尺寸小（小于2 kDa）及其疏水性可以减少纯化过程中的损失。因此，可以看出，使用TrpE融合标签以及强大的发酵过程可以使产量提高3-4倍，从而使其在商业上可行，从而可以提高酶的回收率。