Expression and purification of a small heat shock protein from the plant pathogen Xylella fastidiosa.
The small heat shock proteins (smHSPs) belong to a family of proteins that function as molecular chaperones by preventing protein aggregation and are also known to contain a conserved region termed alpha-crystallin domain. Here, we report the expression, purification, and partial characterization of a novel smHSP (HSP17.9) from the phytopathogen Xylella fastidiosa, causal agent of the citrus variegated chlorosis (CVC). The gene was cloned into a pET32-Xa/LIC vector to over-express the protein coupled with fusion tags in Escherichia coli BL21(DE3). The expressed HSP17.9 was purified by immobilized metal affinity chromatography (IMAC) and had its identity determined by mass spectrometry (MALDI-TOF). The correct folding of the purified recombinant protein was verified by circular dichroism spectroscopy. Finally, the HSP17.9 protein also proved to efficiently prevent induced aggregation of insulin, strongly indicating a chaperone-like activity.
Azzoni
Adriano R
AR
Centro de Biologia Molecular e Engenharia, Genética, Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas, C.P. 6010, Campinas, SP, Brazil. azzoni@unicamp.br
Tada
Susely F S
SF
Rosselli
Luciana K
LK
Paula
Débora P
DP
Catani
Cleide F
CF
Sabino
Adão A
AA
Barbosa
João A R G
JA
Guimarães
Beatriz G
BG
Eberlin
Marcos N
MN
Medrano
Francisco J
FJ
Souza
Anete P
AP
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013997:Time Factors; D014169:Transformation, Bacterial; D044167:Xylella / Q000235:genetics*
D006360:Heat-Shock Proteins; D007328:Insulin; D009841:Oligonucleotides; D011994:Recombinant Proteins