Abrus pulchellus type-2 RIP, pulchellin: heterologous expression and refolding of the sugar-binding B chain.
Abrus pulchellus type-2 RIP, or pulchellin, is a heterodimeric glycoprotein found in A. pulchellus seeds. These chimerolectins, like all type-2 RIPs, are characterized as highly toxic proteins with enzymatic and lectin properties performed by two separate polypeptide subunits. Intending to obtain pure and homogeneous protein for structural and biological studies, the A. pulchellus type-2 RIP lectin subunit or pulchellin binding chain encoding gene fragment (PBC) was cloned. Oligonucleotides based on the sequence homologies between other RIPs like abrin and ricin were synthesized and used to amplify the complete PBC from A. pulchellus genomic DNA. The amplification product was inserted into plasmid pET28a to express the recombinant PBC (rPBC) in Escherichia coli BL21(DE3). The rPBC was expressed as inclusion bodies that were recovered and denatured in a buffer containing urea. Repeated dialysis rounds against the oxidation buffer, which presented the redox pair cysteine-cystine, D-galactose, and decreasing urea concentrations, conducted the protein refolding. The refolding process of rPBC was successfully confirmed by biological assays and circular dichroism.
Goto
Leandro Seiji
LS
Programa de Pós-graduação em Genética e Evolução, Universidade Federal de São Carlos, SP, São Carlos, Brazil.
Beltramini
Leila M
LM
de Moraes
Derminda I
DI
Moreira
Renato A
RA
de Araújo
Ana Paula U
AP
D000036:Abrin / Q000235:genetics; D029912:Abrus / Q000235:genetics*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000737:chemistry; D015870:Gene Expression; D006386:Hemagglutination Tests; D008969:Molecular Sequence Data; D037121:Plant Lectins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D017510:Protein Folding; D021122:Protein Subunits / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012276:Ricin / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D037121:Plant Lectins; D021122:Protein Subunits; D011994:Recombinant Proteins; D000036:Abrin; D012276:Ricin; D005690:Galactose