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claMP Tag: a versatile inline metal-binding platform based on the metal abstraction peptide.

Mills BJ, Mu Q, Krause ME, Laurence JS - Bioconjug. Chem. (2014)

Bottom Line: This approach has been much more effective with large lanthanide series metals than smaller transition metals.The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag.The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The University of Kansas , Lawrence, Kansas 66045, United States.

ABSTRACT
Molecularly targeted research and diagnostic tools are essential to advancing understanding and detection of many diseases. Metals often impart the desired functionality to these tools, and conjugation of high-affinity chelators to proteins is carried out to enable targeted delivery of the metal. This approach has been much more effective with large lanthanide series metals than smaller transition metals. Because chemical conjugation requires additional processing and purification steps and yields a heterogeneous mixture of products, inline incorporation of a peptide tag capable of metal binding is a highly preferable alternative. Development of a transition metal binding tag would provide opportunity to greatly expand metal-based analyses. The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag. The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

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Addition of the claMP Tag to either the N- orC-terminus of EGF has little to no effect on the function of the protein.The decrease in cell viability of A431 cells caused by addition ofEGF is comparable to that of both Ni-claMP-EGF andEGF-Ni-claMP.
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fig8: Addition of the claMP Tag to either the N- orC-terminus of EGF has little to no effect on the function of the protein.The decrease in cell viability of A431 cells caused by addition ofEGF is comparable to that of both Ni-claMP-EGF andEGF-Ni-claMP.

Mentions: In order to be used successfully as aninline metalcarrier, the claMP Tag cannot be deleterious to thefunction of the protein. A standard cell viability assay using theA431 cell strain was completed to assess the impact of the claMP Tag on EGF function. This strain is known for itsoverexpression of the epidermal growth factor receptor (EGFR) on thecell surface.31 Overexpression of EGFRleads to the unexpected outcome of growth inhibition upon stimulationwith EGF.31 The N- and C-termini of EGFare not responsible for high-affinity binding to the receptor,32 so placement of the claMP Tagon either terminus would not be expected to diminish EGF activity,and indeed it does not. EGF or either claMP-Taggedvariant of EGF elicits a similar cytostatic response (Figure 8). At nanomolar concentrations, addition of EGFor either Ni-claMP-EGF protein results in a 50% lossin cell viability in the A431 cell line, confirming EGF remains activein the presence of the tag at either position.


claMP Tag: a versatile inline metal-binding platform based on the metal abstraction peptide.

Mills BJ, Mu Q, Krause ME, Laurence JS - Bioconjug. Chem. (2014)

Addition of the claMP Tag to either the N- orC-terminus of EGF has little to no effect on the function of the protein.The decrease in cell viability of A431 cells caused by addition ofEGF is comparable to that of both Ni-claMP-EGF andEGF-Ni-claMP.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215913&req=5

fig8: Addition of the claMP Tag to either the N- orC-terminus of EGF has little to no effect on the function of the protein.The decrease in cell viability of A431 cells caused by addition ofEGF is comparable to that of both Ni-claMP-EGF andEGF-Ni-claMP.
Mentions: In order to be used successfully as aninline metalcarrier, the claMP Tag cannot be deleterious to thefunction of the protein. A standard cell viability assay using theA431 cell strain was completed to assess the impact of the claMP Tag on EGF function. This strain is known for itsoverexpression of the epidermal growth factor receptor (EGFR) on thecell surface.31 Overexpression of EGFRleads to the unexpected outcome of growth inhibition upon stimulationwith EGF.31 The N- and C-termini of EGFare not responsible for high-affinity binding to the receptor,32 so placement of the claMP Tagon either terminus would not be expected to diminish EGF activity,and indeed it does not. EGF or either claMP-Taggedvariant of EGF elicits a similar cytostatic response (Figure 8). At nanomolar concentrations, addition of EGFor either Ni-claMP-EGF protein results in a 50% lossin cell viability in the A431 cell line, confirming EGF remains activein the presence of the tag at either position.

Bottom Line: This approach has been much more effective with large lanthanide series metals than smaller transition metals.The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag.The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The University of Kansas , Lawrence, Kansas 66045, United States.

ABSTRACT
Molecularly targeted research and diagnostic tools are essential to advancing understanding and detection of many diseases. Metals often impart the desired functionality to these tools, and conjugation of high-affinity chelators to proteins is carried out to enable targeted delivery of the metal. This approach has been much more effective with large lanthanide series metals than smaller transition metals. Because chemical conjugation requires additional processing and purification steps and yields a heterogeneous mixture of products, inline incorporation of a peptide tag capable of metal binding is a highly preferable alternative. Development of a transition metal binding tag would provide opportunity to greatly expand metal-based analyses. The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag. The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

Show MeSH