Molecular, biological, and preliminary structural analysis of recombinant bryodin 1, a ribosome-inactivating protein from the plant Bryonia dioica.

Bryonia dioica (Cucurbitaceae family) produces at least two type I ribosome-inactivating proteins, bryodin 1 (BD1) and bryodin 2 (BD2). A cDNA sequence encoding BD1 was isolated from B. dioica leaf mRNA using degenerative oligonucleotides and codes for a 22 amino acid signal peptide followed by a protein of 267 residues. Expression of two recombinant BD1 (rBD1) forms in Escherichia coli yielded proteins of 267 (to the natural stop codon) and 247 amino acids (to the putative cleavage site yielding the mature protein) that had identical protein synthesis inhibition activity as compared to native BD1. The substitution of Lys for Glu at position 189 near the active site reduced the ability of rBD1 to inhibit protein synthesis by 10-fold. Toxicologic analysis showed that rBD1 was well tolerated in rodents with LD50 values of 40 mg/kg in mice and >25 mg/kg in rats. A crystal of mature rBD1 protein was used to collect X-ray diffraction data to 2.1 A resolution. The rBD1 crystal structure was solved and showed ex...