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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.

Show MeSH
Ni(II) is successfullyincorporated into the claMP Tag in the presence ofEGF. (a) Absorption spectroscopy validatesNi(II) incorporation into the claMP Tag. In comparisonto EGF, EGF-Ni-claMP contains distinct features inthe visible region, which confirm Ni(II) incorporation. (b) Anionexchange chromatography demonstrates the difference in net chargebetween EGF and EGF-Ni-claMP due to the additionof Ni-claMP.
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fig7: Ni(II) is successfullyincorporated into the claMP Tag in the presence ofEGF. (a) Absorption spectroscopy validatesNi(II) incorporation into the claMP Tag. In comparisonto EGF, EGF-Ni-claMP contains distinct features inthe visible region, which confirm Ni(II) incorporation. (b) Anionexchange chromatography demonstrates the difference in net chargebetween EGF and EGF-Ni-claMP due to the additionof Ni-claMP.

Mentions: The Ni-claMP complex of the correct structure has a rusty orange color, allowingincorporation in the context of the protein to be confirmed with absorptionspectroscopy, as established with the peptide system.21 In the EGF-Ni-claMP spectrum, there arebroad features present in the visible region that reflect the structureof the unique metal complex, as seen in Figure 7a. These features are absent from the control spectrum of nativeEGF, which confirms they are due to correct Ni-claMP formation and not nonspecific binding of Ni(II) to EGF.


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)

Ni(II) is successfullyincorporated into the claMP Tag in the presence ofEGF. (a) Absorption spectroscopy validatesNi(II) incorporation into the claMP Tag. In comparisonto EGF, EGF-Ni-claMP contains distinct features inthe visible region, which confirm Ni(II) incorporation. (b) Anionexchange chromatography demonstrates the difference in net chargebetween EGF and EGF-Ni-claMP due to the additionof Ni-claMP.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Ni(II) is successfullyincorporated into the claMP Tag in the presence ofEGF. (a) Absorption spectroscopy validatesNi(II) incorporation into the claMP Tag. In comparisonto EGF, EGF-Ni-claMP contains distinct features inthe visible region, which confirm Ni(II) incorporation. (b) Anionexchange chromatography demonstrates the difference in net chargebetween EGF and EGF-Ni-claMP due to the additionof Ni-claMP.
Mentions: The Ni-claMP complex of the correct structure has a rusty orange color, allowingincorporation in the context of the protein to be confirmed with absorptionspectroscopy, as established with the peptide system.21 In the EGF-Ni-claMP spectrum, there arebroad features present in the visible region that reflect the structureof the unique metal complex, as seen in Figure 7a. These features are absent from the control spectrum of nativeEGF, which confirms they are due to correct Ni-claMP formation and not nonspecific binding of Ni(II) to EGF.

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