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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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Schematicthat illustrates the unique method by which metal isinserted into the claMP Tag. The claMP Tag is reacted with a chelated metal ion (i.e., Ni-NTA), whichleads to metal insertion into the tag. A metal ion with a 2+ charge, such as Ni(II), reacted with the net neutral claMP Tag sequence generates a single product with a charge of 2–. EGF has a 4– charge, resultingin a net charge of 6– for the inline claMP Tag conjugate.
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fig1: Schematicthat illustrates the unique method by which metal isinserted into the claMP Tag. The claMP Tag is reacted with a chelated metal ion (i.e., Ni-NTA), whichleads to metal insertion into the tag. A metal ion with a 2+ charge, such as Ni(II), reacted with the net neutral claMP Tag sequence generates a single product with a charge of 2–. EGF has a 4– charge, resultingin a net charge of 6– for the inline claMP Tag conjugate.

Mentions: Our lab discovered the metal abstraction peptide (MAP) and itsnovel chemistry that binds transition metals and develops a uniquelystructured product with compatible properties for use in clinicalapplications.18 The structure is extremelystable under basic conditions, resisting metal release and loss tohigh affinity chelators in competition experiments, but undergoesrearrangement upon acidification, permitting rapid exchange or releaseof the metal (manuscript in preparation). MAP is composed of a threeamino acid sequence (Asn-Cys-Cys; NCC), and when positioned inline,the metal is coordinated in a square planar organization by two thiolateligands and two deprotonated amide nitrogens from the peptide backbone.19 Because the sequence is composed of naturalamino acids, it can be engineered as a tag into a genetic construct.The desired protein product encodes the inline metal carrier, whichwe refer to as the claMP Tag. In order to generatethe specific metal-bound peptide structure having these unique properties,the claMP Tag must be reacted with a metal ion thatis partially coordinated by a chelator (Figure 1), such as those used in immobilized metal affinity chromatography(IMAC), e.g., nitrilotriacetic acid (NTA). The peptide abstracts themetal ion from the high-affinity chelator; this reaction is extremelyefficient and yields a highly stable, single product.19−21


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)

Schematicthat illustrates the unique method by which metal isinserted into the claMP Tag. The claMP Tag is reacted with a chelated metal ion (i.e., Ni-NTA), whichleads to metal insertion into the tag. A metal ion with a 2+ charge, such as Ni(II), reacted with the net neutral claMP Tag sequence generates a single product with a charge of 2–. EGF has a 4– charge, resultingin a net charge of 6– for the inline claMP Tag conjugate.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Schematicthat illustrates the unique method by which metal isinserted into the claMP Tag. The claMP Tag is reacted with a chelated metal ion (i.e., Ni-NTA), whichleads to metal insertion into the tag. A metal ion with a 2+ charge, such as Ni(II), reacted with the net neutral claMP Tag sequence generates a single product with a charge of 2–. EGF has a 4– charge, resultingin a net charge of 6– for the inline claMP Tag conjugate.
Mentions: Our lab discovered the metal abstraction peptide (MAP) and itsnovel chemistry that binds transition metals and develops a uniquelystructured product with compatible properties for use in clinicalapplications.18 The structure is extremelystable under basic conditions, resisting metal release and loss tohigh affinity chelators in competition experiments, but undergoesrearrangement upon acidification, permitting rapid exchange or releaseof the metal (manuscript in preparation). MAP is composed of a threeamino acid sequence (Asn-Cys-Cys; NCC), and when positioned inline,the metal is coordinated in a square planar organization by two thiolateligands and two deprotonated amide nitrogens from the peptide backbone.19 Because the sequence is composed of naturalamino acids, it can be engineered as a tag into a genetic construct.The desired protein product encodes the inline metal carrier, whichwe refer to as the claMP Tag. In order to generatethe specific metal-bound peptide structure having these unique properties,the claMP Tag must be reacted with a metal ion thatis partially coordinated by a chelator (Figure 1), such as those used in immobilized metal affinity chromatography(IMAC), e.g., nitrilotriacetic acid (NTA). The peptide abstracts themetal ion from the high-affinity chelator; this reaction is extremelyefficient and yields a highly stable, single product.19−21

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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