Overexpression, purification, and biochemical characterization of GumC, an enzyme involved in the biosynthesis of exopolysaccharide by Xylella fastidiosa.
GumC is one of nine enzymes involved in the biosynthesis of fastidian gum, an exopolysaccharide produced by Xylella fastidiosa that may be linked directly to the pathogenicity of the microorganism. GumC may be responsible for gum polymerization or secretion through the membrane of X. fastidiosa. To perform structure and functions studies, we developed an expression system for the production of GumC as a fusion protein with maltose binding protein (MBP) using pMAL-c2x vector. The GumC-MBP fusion protein was expressed as a 94 kDa protein, which strongly reacts with anti-MBP antibodies. GumC-MBP was isolated by affinity chromatography through an amylose column and used to produce antibodies against the fusion protein. After the enzymatic cleavage of MBP, GumC was purified on a Q Sepharose Fast Flow column. GumC showed a molecular weight corresponding to the expected one (52 kDa) and its N-terminal sequence was identical to that deduced from the DNA. The shape of the circular dichroism spectrum was compatible with a folded protein that contains alpha-helical regions in its structure. Therefore, in this study we describe, for the first time, the production of GumC recombinant protein.
Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
Celina
C
de Pieri
Leila M
LM
Beltramini
Heloisa S
HS
Selistre-de-Araújo
André L
AL
Vettore
Felipe R
FR
da Silva
Paulo
P
Arruda
Glaucius
G
Oliva
Dulce H F
DH
de Souza
D000906:Antibodies / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D058619:Maltose-Binding Proteins; D010957:Plasmids / Q000235:genetics; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D044167:Xylella / Q000737:chemistry*
D000906:Antibodies; D001426:Bacterial Proteins; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011135:Polysaccharides, Bacterial; D011993:Recombinant Fusion Proteins