Pulchellin, a highly toxic type 2 ribosome-inactivating protein from Abrus pulchellus. Cloning heterologous expression of A-chain and structural studies.
Pulchellin is a type 2 ribosome-inactivating protein isolated from seeds of the Abrus pulchellus tenuiflorus plant. This study aims to obtain active and homogeneous protein for structural and biological studies that will clarify the functional aspects of this toxin. The DNA fragment encoding pulchellin A-chain was cloned and inserted into pGEX-5X to express the recombinant pulchellin A-chain (rPAC) as a fusion protein in Escherichia coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (> 86%) with the A-chain of abrin-c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. In order to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reduced/oxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of approximately 60 kDa, similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection of different dilutions into mice. The rPAB was able to kill 50% of the tested mice with doses of 45 microg x kg(-1). Our results indicated that the heterodimer showed toxic activity and a conformational pattern similar to pulchellin. In addition, rPAC produced in this heterologous system might be useful for the preparation of immunoconjugates with potential as a therapeutic agent.
Silva
André L C
AL
Centro de Biotecnologia Molecular Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo, Brazil.
Goto
Leandro S
LS
Dinarte
Anemari R
AR
Hansen
Daiane
D
Moreira
Renato A
RA
Beltramini
Leila M
LM
Araújo
Ana P U
AP
D029912:Abrus / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D007274:Injections, Intraperitoneal; D051379:Mice; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012639:Seeds / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D021122:Protein Subunits; D012331:RNA, Fungal; D018749:RNA, Plant; D012335:RNA, Ribosomal; D011993:Recombinant Fusion Proteins; D009699:N-Glycosyl Hydrolases