C1A cysteine peptidases have been shown to play an important role during apicomplexan invasion and egress of host red blood cells (RBCs) and therefore have been exploited as targets for drug development, in which peptidase specificity is deterministic. Babesia bovis genome is currently available and from the 17 putative cysteine peptidases annotated four belong to the C1A subfamily. In this study, we describe the biochemical characterization of a C1A cysteine peptidase, named here BbCp (B. bovis cysteine peptidase) and evaluate its possible participation in the parasite asexual cycle in host RBCs. The recombinant protein was obtained in bacterial inclusion bodies and after a refolding process, presented typical kinetic features of the cysteine peptidase family, enhanced activity in the presence of a reducing agent, optimum pH between 6.5 and 7.0 and was inhibited by cystatins from R. microplus. Moreover, rBbCp substrate specificity evaluation using a peptide phage display library showed a preference for Val > Leu > Phe. Finally, antibodies anti-rBbCp were able to interfere with B. bovis growth in vitro, which highlights the BbCp as a potential target for drug design.

C1A半胱氨酸肽酶已显示在apicomplexan入侵和宿主红细胞（RBCs）的入侵过程中起重要作用，因此已被用作药物开发的靶点，其中肽酶的特异性是确定性的。牛杆状杆菌基因组目前可用，并且从17个假定的半胱氨酸肽酶中注释，其中4个属于C1A亚家族。在这项研究中，我们描述了一种C1A半胱氨酸肽酶的生化特性，此处命名为BbCp（B. bovis半胱氨酸肽酶）并评估其可能参与宿主RBC中的寄生虫无性繁殖周期。重组蛋白是在细菌包涵体中获得的，经过复性过程后，呈现出半胱氨酸肽酶家族的典型动力学特征，在还原剂存在下具有增强的活性，最适pH在6.5至7.0之间，并被来自R. microplus的胱抑素抑制。此外，使用肽噬菌体展示文库的rBbCp底物特异性评估显示了对Val> Leu> Phe的偏爱。最后，抗rBbCp抗体能够在体外干扰牛双歧杆菌的生长，这突出了BbCp作为药物设计的潜在靶标。