Abstract
Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding.
Highlights
Proteases are a class of enzymes that occupies a crucial position with respect to physiological roles as well as various industrial and therapeutic applications
The ends of the resulting double-stranded complementary DNA (cDNA) molecules were repaired by adding a single ‘A’ base and Illumina adapters were ligated to the repaired ends. cDNA fragments of 250–350 bp were purified from the gel and subjected to further template enrichment by PCR using two primers that anneal to the ends of the adapters to construct a fragmented cDNA library
After trimming the adapter sequences and removing low-quality sequences, 171,253,393 clean reads remained for the normalized cDNA library, with an average GC content of 42.99% (Table 2)
Summary
Proteases are a class of enzymes that occupies a crucial position with respect to physiological roles as well as various industrial and therapeutic applications. Latex from plants has been considered an important source of papain-like cysteine proteases, which are promising candidates with high proteolytic activity and unique characteristics as biocatalysts. (Asclepiadaceae family) is a tropical plant that has been widely used in Indian traditional medicinal system. The latex from this plant has various therapeutic uses, including as hepatoprotective, anti-arthritic, anti-inflammatory, antipyretic, and anticancer treatments [1,2,3,4,5]. Ramos et al described purification of several cysteine proteases (Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3) from the latex of Calotropis procera [6,7,8]. The preparation of purified enzymes from tropical plant sources depends on several factors, such as environmental conditions for plant growth and the techniques involved in enzyme purification processes which determine activity, purity, and yield
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