A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli.
Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa.
Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas, Campinas, SP, Brazil.
Clelton A
CA
Santos
Lilian L
LL
Beloti
Marcelo A S
MA
Toledo
Aline
A
Crucello
Marianna T P
MT
Favaro
Juliano S
JS
Mendes
André S
AS
Santiago
Adriano R
AR
Azzoni
Anete P
AP
Souza
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D002850:Chromatography, Gel; D004926:Escherichia coli; D008074:Lipoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010457:Peptidoglycan / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D058849:Protein Refolding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D044167:Xylella
D001426:Bacterial Proteins; D008074:Lipoproteins; D010457:Peptidoglycan