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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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Cartoon of pET-32 expression construct of recombinant claMP-Tagged EGF.
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fig2: Cartoon of pET-32 expression construct of recombinant claMP-Tagged EGF.

Mentions: Because nativeEGF contains three disulfide bonds, it was importantto determine the extent to which addition of the two cysteine residuesin the claMP Tag might affect protein expressionor lead to non-native disulfide bond formation and misfolding in an E. coli system. Native EGF, like many disulfide-containingproteins, accumulates in inclusion bodies when expressed into thereducing cytosolic environment of E. coli,24,26 and addition of the claMP Tag did not affect this outcome (unpublished data). By expressingEGF in an engineered strain that contains a more oxidizing cytoplasmicenvironment, proper folding is achieved (Figure 2).27,28 Using this approach, EGF and claMP-Tagged EGF variants were produced in the soluble fraction of thecell lysate.


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)

Cartoon of pET-32 expression construct of recombinant claMP-Tagged EGF.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Cartoon of pET-32 expression construct of recombinant claMP-Tagged EGF.
Mentions: Because nativeEGF contains three disulfide bonds, it was importantto determine the extent to which addition of the two cysteine residuesin the claMP Tag might affect protein expressionor lead to non-native disulfide bond formation and misfolding in an E. coli system. Native EGF, like many disulfide-containingproteins, accumulates in inclusion bodies when expressed into thereducing cytosolic environment of E. coli,24,26 and addition of the claMP Tag did not affect this outcome (unpublished data). By expressingEGF in an engineered strain that contains a more oxidizing cytoplasmicenvironment, proper folding is achieved (Figure 2).27,28 Using this approach, EGF and claMP-Tagged EGF variants were produced in the soluble fraction of thecell lysate.

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