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Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens.

Ye F, Wang C, Fu Q, Zhang LH, Gao YG - Acta Crystallogr F Struct Biol Commun (2015)

Bottom Line: Both SghA and SghR form dimers in solution.The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively.Data were collected and processed, and the crystallographic parameters were within acceptable ranges.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.

ABSTRACT
Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen-host interaction.

No MeSH data available.


Related in: MedlinePlus

(a) Standard calibration curve based on the log(molecular weight) of four different standard proteins plotted against Kav. The circle and triangle on the standard curve indicate the positions of SghR and SghA, respectively (depicted based on the Kav value). (b) Gel-filtration chromatography elution profile of SghA (solid line) and SghR (dashed line) used for molecular-weight calibration.
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fig2: (a) Standard calibration curve based on the log(molecular weight) of four different standard proteins plotted against Kav. The circle and triangle on the standard curve indicate the positions of SghR and SghA, respectively (depicted based on the Kav value). (b) Gel-filtration chromatography elution profile of SghA (solid line) and SghR (dashed line) used for molecular-weight calibration.

Mentions: The recombinant plasmids pET-14b-SghA and pET-14b-SghR encode the corresponding target proteins with an N-terminal His6 tag. Purification of SghA included three chromatographic steps (sequentially, Ni2+-affinity, anion-exchange and gel-filtration chromatography). In contrast to SghA, two chromatographic steps were adopted for the purification of SghR: Ni2+-affinity and gel-filtration chromatography. The purified SghA and SghR proteins both displayed high purity (Figs. 1 ▸a and 1 ▸b), with a final yield of approximately 50 mg protein per litre of culture. It was noted that there was only a subtle difference in lane 6 in Fig. 1 ▸(a) and lane 7 in Fig. 1 ▸(b) from the corresponding nickel-affinity purification. This result suggested that the proteins were quite pure after nickel-affinity purification and that the subsequent anion-exchange or gel-filtration chromatography was not essentially able to enhance the protein purity. From the gel-filtration chromatography profiles, the size of both proteins also appeared to be double that of their corresponding monomers. To verify this, the molecular weights of SghA and SghR were calibrated using a HiLoad Superdex 200 10/300 gel-filtration column and the High Molecular Weight calibration kit (GE Healthcare). The calibration results demonstrated that both proteins indeed exist as dimers in solution (Fig. 2 ▸).


Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens.

Ye F, Wang C, Fu Q, Zhang LH, Gao YG - Acta Crystallogr F Struct Biol Commun (2015)

(a) Standard calibration curve based on the log(molecular weight) of four different standard proteins plotted against Kav. The circle and triangle on the standard curve indicate the positions of SghR and SghA, respectively (depicted based on the Kav value). (b) Gel-filtration chromatography elution profile of SghA (solid line) and SghR (dashed line) used for molecular-weight calibration.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555920&req=5

fig2: (a) Standard calibration curve based on the log(molecular weight) of four different standard proteins plotted against Kav. The circle and triangle on the standard curve indicate the positions of SghR and SghA, respectively (depicted based on the Kav value). (b) Gel-filtration chromatography elution profile of SghA (solid line) and SghR (dashed line) used for molecular-weight calibration.
Mentions: The recombinant plasmids pET-14b-SghA and pET-14b-SghR encode the corresponding target proteins with an N-terminal His6 tag. Purification of SghA included three chromatographic steps (sequentially, Ni2+-affinity, anion-exchange and gel-filtration chromatography). In contrast to SghA, two chromatographic steps were adopted for the purification of SghR: Ni2+-affinity and gel-filtration chromatography. The purified SghA and SghR proteins both displayed high purity (Figs. 1 ▸a and 1 ▸b), with a final yield of approximately 50 mg protein per litre of culture. It was noted that there was only a subtle difference in lane 6 in Fig. 1 ▸(a) and lane 7 in Fig. 1 ▸(b) from the corresponding nickel-affinity purification. This result suggested that the proteins were quite pure after nickel-affinity purification and that the subsequent anion-exchange or gel-filtration chromatography was not essentially able to enhance the protein purity. From the gel-filtration chromatography profiles, the size of both proteins also appeared to be double that of their corresponding monomers. To verify this, the molecular weights of SghA and SghR were calibrated using a HiLoad Superdex 200 10/300 gel-filtration column and the High Molecular Weight calibration kit (GE Healthcare). The calibration results demonstrated that both proteins indeed exist as dimers in solution (Fig. 2 ▸).

Bottom Line: Both SghA and SghR form dimers in solution.The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively.Data were collected and processed, and the crystallographic parameters were within acceptable ranges.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.

ABSTRACT
Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen-host interaction.

No MeSH data available.


Related in: MedlinePlus