Molecular, Biological, and Preliminary Structural Analysis of Recombinant Bryodin 1, a Ribosome-Inactivating Protein from the Plant Bryonia dioica

详细信息    查看全文

• 作者：Susan L. Gawlak ; Michael Neubauer ; Herbert E. Klei ; ChiehYing Y. Chang ; Howard M. Einspahr ; and Clay B. Siegall
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：March 18, 1997
• 年：1997
• 卷：36
• 期：11
• 页码：3095 - 3103
• 全文大小：340K
• 年卷期：v.36,no.11(March 18, 1997)
• ISSN：1520-4995

文摘

Bryonia dioica (Cucurbitaceaefamily) produces at least two type I ribosome-inactivatingproteins, bryodin 1 (BD1) and bryodin 2 (BD2). A cDNA sequenceencoding BD1 was isolated from B.dioica leaf mRNA using degenerative oligonucleotides and codes fora 22 amino acid signal peptidefollowed by a protein of 267 residues. Expression of tworecombinant BD1 (rBD1) forms in Escherichiacoli yielded proteins of 267 (to the natural stop codon) and 247amino acids (to the putative cleavage siteyielding the mature protein) that had identical protein synthesisinhibition activity as compared to nativeBD1. The substitution of Lys for Glu at position 189 near theactive site reduced the ability of rBD1 toinhibit protein synthesis by 10-fold. Toxicologic analysis showedthat rBD1 was well tolerated in rodentswith LD₅₀ values of 40 mg/kg in mice and >25 mg/kg inrats. A crystal of mature rBD1 protein wasused to collect X-ray diffraction data to 2.1 Å resolution. TherBD1 crystal structure was solved andshowed extensive homology with other type I RIPs and A chains of typeII RIPs. The studies describedhere demonstrate that rBD1 retains full biologic activity and serve asa guide for using this potent, yetnontoxic, RIP in the construction of single-chain immunotoxin fusionproteins.