er2566  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: Chaperone-assisted expression and purification of the AML-associated Src-family kinase Fgr in Escherichia coli

doi: 10.1016/j.jbc.2025.111099

Figure Lengend Snippet: Coexpression of Escherichia coli GroEL–GroES improves Fgr solubility. A , B acterial cultures expressing Fgr in the absence ( top panel ) or presence ( bottom panel ) of the GroEL–GroES complex were grown and induced as described under the Experimental procedures section. Aliquots were lysed, the soluble and insoluble fractions were separated by centrifugation, and the resulting protein fractions were separated by SDS-PAGE and stained with Coomassie blue . Soluble extracts were then subjected to Ni-IMAC chromatography. SDS polyacrylamide gels of the resulting peak fractions are shown for the samples without ( B ) or with ( C ) GroEL–GroES coexpression. Both gels are stained with Coomassie blue . Positions of the molecular weight markers are indicated on the left of all panels in kilodaltons. Ni-IMAC, nickel immobilized metal affinity chromatography.

Article Snippet: To promote proper folding and solubility, cells were also transformed with the pACYC-Skp-GroEL–GroES expression plasmid encoding cytoplasmic copies of the Skp and GroEL–GroES chaperones (AddGene #83922) ( ).

Techniques: Solubility, Expressing, Centrifugation, SDS Page, Staining, Chromatography, Molecular Weight, Affinity Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Chaperone-assisted expression and purification of the AML-associated Src-family kinase Fgr in Escherichia coli

doi: 10.1016/j.jbc.2025.111099

Figure Lengend Snippet: Washing the column-bound GroEL-Fgr complex with ATP results in partial dissociation of GroEL. A , representative SDS-PAGE of the peak fractions from Ni-IMAC of Fgr coexpressed with GroEL–GroES after washing the column-bound Fgr–chaperone complex with ATP–MgCl 2 buffer. Positions of the molecular weight markers are indicated on the left in kilodaltons. Note the increased ratio of GroEL to Fgr compared with the unwashed column fractions shown in . B , fractions from the ATP–MgCl 2 wash prior to elution. Ni-IMAC, nickel immobilized metal affinity chromatography.

Article Snippet: To promote proper folding and solubility, cells were also transformed with the pACYC-Skp-GroEL–GroES expression plasmid encoding cytoplasmic copies of the Skp and GroEL–GroES chaperones (AddGene #83922) ( ).

Techniques: SDS Page, Molecular Weight, Affinity Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Chaperone-assisted expression and purification of the AML-associated Src-family kinase Fgr in Escherichia coli

doi: 10.1016/j.jbc.2025.111099

Figure Lengend Snippet: Size-exclusion chromatography separates free Fgr from GroEL–GroES. A , representative Superdex 200 size-exclusion chromatogram following Ni-IMAC and ATP wash of the GroEL–Fgr complex. B , fractions from peaks identified by letters A–F in A were collected and analyzed by SDS-PAGE. Peak A, contained the remaining GroEL–Fgr complex, whereas peak D, contained the GroEL-free Fgr with some remaining contaminants. Peak E, while giving strong absorbance in the chromatogram, did not show a band on the gel. This peak may correspond to GroES, which has a subunit molecular weight of 10 kDa and therefore was not resolved. The positions of GroEL and Fgr are indicated by the arrows . Molecular weight markers are shown at left . Ni-IMAC, nickel immobilized metal affinity chromatography.

Article Snippet: To promote proper folding and solubility, cells were also transformed with the pACYC-Skp-GroEL–GroES expression plasmid encoding cytoplasmic copies of the Skp and GroEL–GroES chaperones (AddGene #83922) ( ).

Techniques: Size-exclusion Chromatography, SDS Page, Molecular Weight, Affinity Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Chaperone-assisted expression and purification of the AML-associated Src-family kinase Fgr in Escherichia coli

doi: 10.1016/j.jbc.2025.111099

Figure Lengend Snippet: Assessment of recombinant Fgr kinase activity. Representative progress curves of ADP production by Fgr ( A ) and Hck ( B ) as a function of time with constant protein kinase and substrate concentrations and the ATP concentrations indicated. Hck (positive control) was also expressed in Escherichia coli , although the use of GroEL–GroES was not required. Each data point is the average of four technical replicates ± SD. Solid circles represent the linear portion of each curve that was fit by regression analysis to determine the steady-state reaction rate. At high concentrations of both kinases, the curves plateau and then slope downward as the peptide substrate is exhausted. C , representative concentration–response curve of the steady-state ADP production rate as a function of ATP concentration for near-full-length Fgr versus Hck. Average K m values for ATP ± SD were determined from three independent experiments by nonlinear curve fitting using GraphPad Prism. D , time course of Fgr and Hck autophosphorylation. Purified Fgr and Hck autophosphorylation was followed over the time course shown by immunoblotting with phosphospecific antibodies to the activation loop (pY416) and tail (pY527) tyrosine phosphorylation sites. The experiment was run under conditions that closely mirror the kinetic kinase assays with respect to kinase concentration and with ATP set to the respective K m values.

Article Snippet: To promote proper folding and solubility, cells were also transformed with the pACYC-Skp-GroEL–GroES expression plasmid encoding cytoplasmic copies of the Skp and GroEL–GroES chaperones (AddGene #83922) ( ).

Techniques: Recombinant, Activity Assay, Positive Control, Concentration Assay, Purification, Western Blot, Activation Assay, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Chaperone-assisted expression and purification of the AML-associated Src-family kinase Fgr in Escherichia coli

doi: 10.1016/j.jbc.2025.111099

Figure Lengend Snippet: Overview of chaperone-assisted expression and purification of Fgr in Escherichia coli . A , near-full-length Fgr (SH3–SH2–kinase–tail) was coexpressed in E. coli with the C-terminal Src kinase (Csk) and the protein-tyrosine phosphatase 1B (PTP1B) catalytic domain to promote interaction of the regulatory domains (SH3, red ; SH2, blue ) with the kinase domain ( gray ), resulting in a single, closed conformation ( inset ). Coexpression of the chaperone complex GroEL–GroES was used to improve protein solubility. B , purification strategy. Following induction of Fgr expression, the clarified cell lysate was loaded on a Ni-IMAC column, resulting in capture of the GroEL–GroES–Fgr complex. Dissociation of this complex was achieved by washing the Ni-IMAC column with ATP–MgCl 2 buffer, resulting in partial release of GroEL–GroES from Fgr. Following elution, fractions with Fgr were pooled, concentrated, and applied to a Superdex 200 size-exclusion column, which resulted in the separation of Fgr from GroEL-GroES. These fractions were pooled and further purified by anion exchange chromatography, resulting in highly pure Fgr. Ni-IMAC, nickel immobilized metal affinity chromatography; SH, Src homology.

Article Snippet: To promote proper folding and solubility, cells were also transformed with the pACYC-Skp-GroEL–GroES expression plasmid encoding cytoplasmic copies of the Skp and GroEL–GroES chaperones (AddGene #83922) ( ).

Techniques: Expressing, Purification, Solubility, Chromatography, Affinity Chromatography