Cloning In E Coli, Overexpression And Molecular Characterization Of Novel Glucarpidase: Enzyme Involved In ADEPT For Cancer Treatment

Abstract

Back ground Antibody Directed Enzyme Prodrug Therapy (ADEPT) is a technique which has been used in cancer treatment. This therapy consists of two steps the aim of which is to convert a prodrug to a powerful cytotoxic drug only in the vicinity of the tumor. The technique requires a bacterial enzyme, glucarpidase (former name: carboxypeptidase G2, CPG2). The use of glucarpidase in ADEPT and in detoxification of the cytotoxic methotrexate, MTX is needed to be done in several cycles. This however, hampered by the induced human antibody response to the glucarpidase. The technique therefore will benefit from novel glucarpidase which might avoid the human immune system. Objectives 1.Collections and screening soil samples for novel glucarpidase producing bacteria 2.Characterisation of the isolated bacteria 3.Cloning, overexpression, purification and the molecular characterisation of the novel glucarpidase Methods Isolation of novel glucarpidase producer(s) Over one hundred soil samples were collected from different fields in Qatar where folate rich fruit and vegetables are routinely cultivated. Screening of these samples was performed using folate as the sole carbon and/or nitrogen source. Several molecular techniques were used to characterize the isolated strains. Cloning of the novel glucarpidase gene The gene was cloned by production of genomic library of the novel strain chromosomal DNA and PCR techniques. The gene was subcloned in the overexpression vector pET28a and transformed into E. coli (DE3). The novel gene was expressed by induction using IPTG. The recombinant protein was purified using Ni2+ column. The activity assay was carried out using methotrexate as substrate. Results We successfully isolated two new glucarpidase producing strains, Stenotrophomonas Sp. AA1, and Pseudomonas Sp. AA2. The CPG2 gene from Stenotrophomonas Sp. was cloned and expressed in E. coli using pET28a vector. The SDS analysis showed that the gene was expressed in the soluble form to about 30% of the total protein. The glucarpidase gene was cloned so as to place a polyhistidine tag on the N-terminus of the enzyme. A one-step purification protocol was usually sufficient to obtain essentially pure protein for detailed enzyme assay and characterization. Previous studies indicate that the native form of glucarpidase is Zn2+-dependent. Our data indicate that the recombinant enzyme showed no activity towards the substrate methotraxate in absence of Zn2+ ions. Full activity was restored to the zinc-free enzyme by the addition of Zn2+ to the assay mixture. Therefore, the formation of active enzyme was shown to be dependent on Zn2+. Conclusion We successfully isolated two new glucarpidase producers. We managed to clone express and purify the new glucarpidase gene from one strain. Molecular studies on the novel glucarpidase have been carried out. The novel glucarpidase will pave the way for further application in the cancer drug detoxification and Antibody Directed Enzyme Prodrug Therapy techniques.