Purification and characterization of recombinant human p50csk protein-tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL.

Amrein, Kurt E.; Takács, B.; Stieger, Martin; Molnos, Juliette; Flint, Nicholas; Burn, Paul

doi:10.1073/pnas.92.4.1048

Cited by 166 publications

(117 citation statements)

References 31 publications

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“…We also examined and eliminated the possibility of Csk being inactivated by incubation with Src and/or ATPMg. Csk undergoes autophosphorylation to a low extent (Amrein et al, 1995) and this does not signi®cantly a ect its activity (Oetken et al, 1994;Sun et al, 1997). Furthermore, Csk is neither phosphorylated nor inactivated by Src even when a near stoichiometric amount of Src was used (Sun et al, 1997).…”

Section: Resultsmentioning

confidence: 99%

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

1998

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Csk phosphorylates Src family protein tyrosine kinases on a tyrosine residue near their C-terminus and downregulates their activity. We previously observed that this regulation requires a stoichiometric ratio of Csk : Src in a time-independent manner. In this report we examined this unusual kinetic behavior and found it to be caused by Src autophosphorylation. First, pre-incubation of Src with ATP-Mg led to time-dependent autophosphorylation of Src, activation of its kinase activity and loss of its ability to be inactivated by Csk. However, the autophosphorylated Src can still be phosphorylated by Csk. The SH2 binding site for phospho-Tyr of this hyperactive and doubly phosphorylated form of Src is not accessible. Second, dephosphorylation of autophosphorylated Src by protein tyrosine phosphatase 1B allowed Src to be inactivated by Csk. Third, protein tyrosine phosphatase 1B preferentially dephosphorylates the Src autophosphorylation site and allows for Src regulation by Csk. Finally, Yes, another member of the Src family, was also only partially inactivated when a sub-stoichiometric amount of Csk was used. Mutation of the tyrosine autophosphorylation site of Yes to a phenylalanine resulted in a mutant Yes enzyme that can be fully inactivated by a sub-stoichiometric amount of Csk in a time-dependent manner. These results demonstrate that Csk phosphorylation inactivates Src and Yes only when they are not previously autophosphorylated and Src autophosphorylation can block the inactivation by Csk phosphorylation. This conclusion suggests a dynamic model for the regulation of the Src family protein tyrosine kinases, which is discussed in the context of previously reported observations on the regulation of Src family protein tyrosine kinases.

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Section: Resultsmentioning

confidence: 99%

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

1998

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“…Strain BL21(pREP4-groESL), in which overproduction of the C-terminal domain of Wzc was obtained, was reported previously (24). Plasmids pSP72-lacZ and pUC19-phoA were obtained from J. T. Beatty and W. H. Bingle, respectively (25,26).…”

Section: Methodsmentioning

confidence: 99%

Structural Organization of the Protein-tyrosine Autokinase Wzc within Escherichia coli Cells

Doublet

Grangeasse

Obadia

et al. 2002

Journal of Biological Chemistry

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Protein Wzc from Escherichia coli is a member of a newly defined family of protein-tyrosine autokinases that are essential for surface polysaccharide production in both Gram-negative and Gram-positive bacteria. Although the catalytic mechanism of the autophosphorylation of Wzc was recently described, the in vivo structural organization of this protein remained unclear. Here, we have determined the membrane topology of Wzc by performing translational fusions of lacZ and phoA reporter genes to the wzc gene. It has been shown that Wzc consists of two main structural domains: an N-terminal domain, bordered by two transmembrane helices, which is located in the periplasm of cells, and a C-terminal domain, harboring all phosphorylation sites of the protein, which is located in the cytoplasm. In addition, it has been demonstrated for the first time that Wzc can oligomerize in vivo to form essentially trimers and hexamers. Cross-linking experiments performed on strains expressing various domains of Wzc have shown that the cytoplasmic C-terminal domain is sufficient to generate oligomerization of Wzc. Mutant proteins, modified in either the ATP-binding site or the different phosphorylation sites, i.e. rendered unable to undergo autophosphorylation, have appeared to oligomerize into high molecular mass species identical to those formed by the wild-type protein. It was concluded that phosphorylation of Wzc is not essential to its oligomerization. These data, connected with the phosphorylation mechanism of Wzc, may be of biological significance in the regulatory role played by this kinase in polysaccharide synthesis.

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“…However, we were not successful in these attempts, since most of the protein was produced in an insoluble form and we were unable to recover it by solubilization and renaturation. In an attempt to solve this problem, we decided to use folding catalysts to help expression of the protein in a soluble form, an approach that has been used by other investigators with success (20)(21)(22)(23)(24). The simultaneous expression of the bacterial chaperonins GroES/GroEL using the pT-GroE plasmid highly increased the expression of soluble IRP1, as shown in Figure 1.…”

Section: Resultsmentioning

confidence: 99%

“…On the other hand, co-expression of GroES and GroEL increased solubility of four of the proteins tested. Amrein et al (21) described the effect of co-expressing GroES and GroEL in improving solubility of p50csk protein-tyrosine kinase. Mitsuda and Iwasaki (24) observed a great improvement in the expression of membrane-bound cytochrome P450 2B6 when co-expressed with GroES and GroEL in E. coli.…”

Section: Resultsmentioning

confidence: 99%

“…Soluble protein recovery from these inclusion bodies is a difficult and often unsuccessful task. The use of molecular chaperonins to facilitate soluble protein expression has been adopted in some systems with success (20)(21)(22)(23)(24). Chaperonins are ubiquitous proteins that have an important role in the folding of newly synthesized proteins in vivo.…”

Section: Introductionmentioning

confidence: 99%

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Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Carvalho

Meneghini

2008

Braz J Med Biol Res

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Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH 2 -terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

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Purification and characterization of recombinant human p50csk protein-tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL.

Cited by 166 publications

References 31 publications

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

Structural Organization of the Protein-tyrosine Autokinase Wzc within Escherichia coli Cells

Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

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