J. Plant Biochemistry & Biotechnology Vol. 3, 103-106, July 1994 Cloning of ODAP Degrading Gene and Its Expression as Fusion Protein in Escherichia coli A Jayakumaran Nairl, G S Khatri 2 , I M Santha' and S L Mehta 1 * 1Division of Biochemistry, Indian Agricultural Research Institute, New Delhi 110 012, India 2Centre for Biochemical Technology (CSIR), Mall Road, New Delhi, India A gene responsible for the degradation of B-N-Oxalyl diaminopropionic acid (ODAP) was fused to the malE gene, which codes for maltose binding protein, by cloning into an expression vector pMAL c2. The gene has been expressed as fusion protein of mol wt approximately 62 kD. It has been purified by affinity chromatography. The fusion protein has been cleaved by an endoprotease factor Xa and the presence of maltose binding protein and the product of the cloned gene confirmed. SDS-PAGE has shown that the product of the ODAP degrading gene is a single polypeptide of mol wt of about 20.7 kD. Key words: oxalyl diaminopropionic acid, fusion protein, Lathyrus sativus, maltose binding protein. Lathyrus sativus or Khesari dal is a potential source of protein and is cultivated in India, despite the ban imposed by the Government, mainly because of ease of cultivation. This crop has got a number of good agronomic characteristics. It requires no specific soil type, fertilizers, irrigation, pest control methods or other management practices. Even though it has got all these advantages as a crop, there is a great disad- vantage, namely the presence of a neurotoxin, p-Oxalyl diaminopropionic acid (ODAP) which has made the plant unsuitable for human consumption. Prolonged consumption of Khesari dal will affect central nervous system and lead to paralysis of lower limb, a condi- tion termed as neurolathyrism (1,2). The exceptional qualities of this crop can be fully realised only if the crop is made safe for human consumption either by eliminating or reducing ODAP levels. Although several approaches such as application of physical methods (3-5) mutation breeding and ag- ronomic practices (6) have been adopted in an ef- fort to reduce the neurotoxin content to a nutrition- ally safe level in L. sativus, none of these methods have succeeded in eliminating the toxin from the crop or reducing it to a negligible level. Attempts were made in our laboratory to eliminate the toxin in L. sativus through recombinant DNA techniques for the first time. A bacterial strain capable of using ODAP as the sole source of carbon and nitrogen has been isolated (7). A plasmid, pBYA1 responsible for the degrada- tion of ODAP was isolated from the bacterium and 'Corresponding author a library of the same was constructed in pUC18. A recombinant clone capable of growing in M9 medium supplemented with ODAP contained an insert DNA of the size of 1.8 kb, which was isolated and sequenced (8). The 1.8 kb DNA fragment was found to contain a 55 nucleotide long regulatory region and 576 nucle- otide long stretch, coding for 192 amino acids. But nothing was known about the protein which takes part in the metabolism of ODAP. To understand more about the gene product, its role in ODAP degradation, and also to confirm the proposed reading frame in the 1.8 kb DNA fragment, we adopted the technique of gene fusion and its expression as fusion protein. We re- port here, the cloning of the 1.8 kb DNA fragment to an expression vector and its expression as fusion protein in E. coli, and its purification. Materials and Methods Bacterial strains, plasmids and media-The Escheric fa coli strain DH5a carrying the recombinant clone (BM1) of ODAP deqradinq gene cloned into a Bluescript vector SK- was grown in Luria-Bertani (LB) medium (9) with 100 IJg/ml ampicillin (Amp) at 3rC. The expression vector pMAL c2 was isolated from the transformed TB-1 cells grown in LB medium containing 100 IJg/ml Amp. Bacterial strain T8-1 grown in LB me- dium without any antibiotic was used for transformation. Isolation of plasmids and insert DNA fragment- All preparations of plasmid DNA were carried out by the alkaline lysis method of Birnboim and Doly as described by Sambrook et al (9). The recombinant plasmid isolated from the 8M1 strain was digested