Pseudomonas aeruginosa recombinant L-asparaginase: Large scale production, purification, and cytotoxicity on THP-1, MDA-MB-231, A549, Caco2 and HCT-116 cell lines

Abstract

In previous studies Pseudomonas aeruginosal-ASNase complete coding sequence gene, 984 bp (GenBank accession number KU161101.2) was isolated by PCR, cloned into pET28a(+) vector, expressed in E. coli DE3(BL21) pLysS, purified to apparent homogeneity and biochemically characterized. In the present work we highlight large scale production, affinity purification of the recombinant enzyme, effect of osmolytes on the stability of the l-ASNase and cytotoxicity on different cancer cell lines. Successful overexpression was achieved in E. coli as a 6-His-Tag fusion protein after 18 h of induction with lactose at a concentration of 2 g/L in fermentation medium and at 37 °C. The recombinant enzyme was purified to homogeneity using Ni²⁺ chelated Fast Flow Sepharose resin with 19758.8 specific activity and 10.28 purification fold. With respect to the effect of osmolytes on the stability of the purified enzyme, the majority of the tested osmolytes namely 5% maltose, 5% mannitol, 30% glycerol and 5% BSA were found to increase the stability of the recombinant l-ASNase as compared to the free enzyme. Triple negative breast cancer cell line, MDA-MB-231 treated with recombinant l-ASNase showed significant morphological changes and the IC₅₀ of the purified enzyme was found to be 3.1 IU. Human leukemia cell line, THP-1 treated with l-ASNase showed apoptotic bodies and morphological changes with IC₅₀ of the purified enzyme 1.75 IU. Moreover, the purified recombinant l-ASNase was found to induced cytotoxic effects on colorectal adenocarcinoma cell line, Caco-2 with IC₅₀ of 68.28 IU. Results of apoptosis assay on THP-1 cells revealed that the purified l-ASNase induced early and late apoptosis at 14.16% and 7.56 respectively as compared to the control untreated cells.

Keywords: Acute lymphoblastic leukemia; Cloning; Cytotoxicity; Expression; l-asparaginase.