Extraction, Sequence Alignment and Cloning of Recombinant Papain from Carica papaya

Abstract

Papain is a proteolytic enzyme obtained from the fruits of carica papaya with abundant therapeutic, food, industrial and analytical applications. The challenges related to the development of papain technology on industrial scale include cost of production and downstream processing of the enzyme. In the present research, the responsible for gene encoding papain enzyme was obtained from the carica papaya plant, sequenced and amplified using polymerase chain reaction. This papain gene was initially converted to cDNA and then the papain gene was sequence to identify the region of similarity that may be a classify base on functional, structure, and  phylogenic relationships between the known papain DNA sequence and the sample Papain gene. The papain gene was then clone into the pBR322 cloning vector to create recombinant protein molecules. This showed that the DNA sequencing of the extracted genes exhibited an acceptable level of similarity to the corresponding gene (MEROO647) with identity of 98% and E-value 0.246 X10-9 from the public database NCBI (National Centre for Biotechnology information) In contrast, the similar papain genes sequence (P00784, MEROO647, and AT3G5470) in NCBI extracted from other sources in this study, the papaya gene sequences was obtained from carica papaya (fruit) and in the sequence similarity analysis because the product was amplified from mRNA that was extracted from carica papaya fruit.