Cloning, expression and purification of papain enzyme in E. coli host

BACKGROUND AND OBJECTIVESPapain is a plant proteolytic enzyme that belongs to the cysteine protease family. It is found naturally in papaya and is manufactured from the latex of raw papaya fruits. Papain is able to break down organic molecules made of amino acids, known as polypeptides, and thus plays a crucial role in diverse biological processes in physiological and pathological states, drug designs, industrial uses such as meat tenderizers, and pharmaceutical preparations. Extraction and purification of papain from natural sources is costly and inefficient. Therefore, this study focuses on the cloning, expression, and purification of the papain enzyme in a bacterial host.MATERIALS AND METHODSAn optimized sequence of papain gene was created according to the submitted genes in the NCBI along with a His-tag, in pET۲۱a expression vector by the use of GeneRunner software and Genescript online application. Then the sequence was placed in the ordered vector by a Chinese company.The cloned papain gene was introduced into Escherichia coli BL۲۱(DE۳) as the expression host via CaCl۲ method of competent cell preparation, and the target protein was expressed by IPTG as the inducer.Expression optimization was done in different conditions (IPTG concentration (۰-۱ mM final concentration) and growth temperature (۲۰, ۲۸ and ۳۷ °C)). The produced protein was purified using Ni-NTA column chromatography. SDS-PAGE analysis was performed to evaluate the expression of cloned gene and to follow the purification process. Enzyme activity was monitored via treatment of Skim milk agar (۱% W/V) by different chromatography samples.RESULTS AND DISCUSSIONThe transformed bacteria could easily grow on LB Agar containing Ampicillin (beta lactamase as resistant gene of vector). The most suitable concentration of IPTG at ۲۰°C was ۰.۱ mM, it may be related to the antibacterial activity of papain. During purification with Ni-NTA chromatography and SDS-Page analysis the desired molecules of enzyme emerged in SDS-PAGE and could show protease activity on Skim milk agar.CONCLUSIONOverall, this study demonstrates the successful cloning, expression, and purification of papain enzyme in a bacterial host. Further analysis for optimization of papain purification is now in process in our research group.

کلیدواژه ها:

papain ، His-tag ، chromatography ، SDS-PAGE