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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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Size-exclusion chromatographyverifies that one main species ispresent in the sample. EGF-Ni-claMP elutes slightlyearlier than EGF.
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fig4: Size-exclusion chromatographyverifies that one main species ispresent in the sample. EGF-Ni-claMP elutes slightlyearlier than EGF.

Mentions: The EGFvariants accumulated in the soluble fraction of the cell lysate, asdetermined by SDS-PAGE analysis of the supernatant and pellet fromeach sample following centrifugation of the lysate (data not shown).Following individual processing and purification steps, the yieldof each protein variant was determined (Table 1). SDS-PAGE was performed to examine the purity and amount of proteinpresent at each step, and the pure final product is shown in Figure 3b. Cleavage of the fusion tags from EGF-claMP worked as expected, completely cleaving the fusionprotein and yielding the expected amount of each product. During FactorXa cleavage, a 30% lower yield was observed with the N-terminallytagged construct in comparison to EGF-Ni-claMP (Table 1). The claMP Tag abuts the FactorXa recognition sequence, and therefore, it is hypothesized that cleavageefficiency is reduced because the Ni-claMP complexkinks the protein conformation near the site, limiting access by theprotease to the cleavage site. Sample purity of the final EGF proteinsalso was assessed using size exclusion chromatography (Figure 4). As a control, native EGF was examined, and itelutes at 12 min. EGF-Ni-claMP elutes earlier thanEGF at 10 min, which is expected because it is slightly larger thanEGF.


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)

Size-exclusion chromatographyverifies that one main species ispresent in the sample. EGF-Ni-claMP elutes slightlyearlier than EGF.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Size-exclusion chromatographyverifies that one main species ispresent in the sample. EGF-Ni-claMP elutes slightlyearlier than EGF.
Mentions: The EGFvariants accumulated in the soluble fraction of the cell lysate, asdetermined by SDS-PAGE analysis of the supernatant and pellet fromeach sample following centrifugation of the lysate (data not shown).Following individual processing and purification steps, the yieldof each protein variant was determined (Table 1). SDS-PAGE was performed to examine the purity and amount of proteinpresent at each step, and the pure final product is shown in Figure 3b. Cleavage of the fusion tags from EGF-claMP worked as expected, completely cleaving the fusionprotein and yielding the expected amount of each product. During FactorXa cleavage, a 30% lower yield was observed with the N-terminallytagged construct in comparison to EGF-Ni-claMP (Table 1). The claMP Tag abuts the FactorXa recognition sequence, and therefore, it is hypothesized that cleavageefficiency is reduced because the Ni-claMP complexkinks the protein conformation near the site, limiting access by theprotease to the cleavage site. Sample purity of the final EGF proteinsalso was assessed using size exclusion chromatography (Figure 4). As a control, native EGF was examined, and itelutes at 12 min. EGF-Ni-claMP elutes earlier thanEGF at 10 min, which is expected because it is slightly larger thanEGF.

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