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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 claMP Tag to EGF did not significantlyhinder expression or purification of desired product. Coomassie stained18% tris-tricine gels illustrating the expression of pET-32-EGF, pET-32-claMP-EGF, and pET-32-EGF-claMP and finalpurified EGF products. (a) (Lane 1) molecular weight standard, (lanes2, 4, 6) E. coli lysate of pET-32-EGF,pET-32-claMP-EGF, and pET-32-EGF-claMP before IPTG induction, (lanes 3, 5, 7) E. coli lysate of pET-32-EGF, pET-32-claMP-EGF, and pET-32-EGF-claMP 16 h after IPTG induction. (b) (Lane 1) molecularweight standard, (lane 2) purified EGF-Ni-claMP,(lane 3) purified EGF, and (lane 4) purified Ni-claMP-EGF.
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fig3: Addition of claMP Tag to EGF did not significantlyhinder expression or purification of desired product. Coomassie stained18% tris-tricine gels illustrating the expression of pET-32-EGF, pET-32-claMP-EGF, and pET-32-EGF-claMP and finalpurified EGF products. (a) (Lane 1) molecular weight standard, (lanes2, 4, 6) E. coli lysate of pET-32-EGF,pET-32-claMP-EGF, and pET-32-EGF-claMP before IPTG induction, (lanes 3, 5, 7) E. coli lysate of pET-32-EGF, pET-32-claMP-EGF, and pET-32-EGF-claMP 16 h after IPTG induction. (b) (Lane 1) molecularweight standard, (lane 2) purified EGF-Ni-claMP,(lane 3) purified EGF, and (lane 4) purified Ni-claMP-EGF.

Mentions: SDS-PAGE was used to verify the claMP Tagdidnot adversely affect expression of claMP-Tagged EGF;all EGF variants showed excellent expression and reasonably similarexpression levels (Figure 3a). The molecularweight of each fusion protein was expected to be approximately 24kDa. As shown in Figure 3a, no band is presentat this size in the preinduction samples (lanes 2, 4, and 6), buta band appears in the postinduction sample for each of the variousconstructs (lanes 3, 5, and 7), confirming successful expression.Native EGF was used as a standard to establish relative expressionof the claMP-Tagged forms. N-terminal and C-terminalplacement led to a 14% and 29% decrease in EGF expression yield, respectively.Differences in expression among the variants were on the same orderas batch-to-batch variation among replicates of the same protein.Therefore, insertion of the claMP Tag into the proteinsequence has a negligible effect on protein expression, as determinedby densitometric analysis of whole cell lysates.


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 claMP Tag to EGF did not significantlyhinder expression or purification of desired product. Coomassie stained18% tris-tricine gels illustrating the expression of pET-32-EGF, pET-32-claMP-EGF, and pET-32-EGF-claMP and finalpurified EGF products. (a) (Lane 1) molecular weight standard, (lanes2, 4, 6) E. coli lysate of pET-32-EGF,pET-32-claMP-EGF, and pET-32-EGF-claMP before IPTG induction, (lanes 3, 5, 7) E. coli lysate of pET-32-EGF, pET-32-claMP-EGF, and pET-32-EGF-claMP 16 h after IPTG induction. (b) (Lane 1) molecularweight standard, (lane 2) purified EGF-Ni-claMP,(lane 3) purified EGF, and (lane 4) purified Ni-claMP-EGF.
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fig3: Addition of claMP Tag to EGF did not significantlyhinder expression or purification of desired product. Coomassie stained18% tris-tricine gels illustrating the expression of pET-32-EGF, pET-32-claMP-EGF, and pET-32-EGF-claMP and finalpurified EGF products. (a) (Lane 1) molecular weight standard, (lanes2, 4, 6) E. coli lysate of pET-32-EGF,pET-32-claMP-EGF, and pET-32-EGF-claMP before IPTG induction, (lanes 3, 5, 7) E. coli lysate of pET-32-EGF, pET-32-claMP-EGF, and pET-32-EGF-claMP 16 h after IPTG induction. (b) (Lane 1) molecularweight standard, (lane 2) purified EGF-Ni-claMP,(lane 3) purified EGF, and (lane 4) purified Ni-claMP-EGF.
Mentions: SDS-PAGE was used to verify the claMP Tagdidnot adversely affect expression of claMP-Tagged EGF;all EGF variants showed excellent expression and reasonably similarexpression levels (Figure 3a). The molecularweight of each fusion protein was expected to be approximately 24kDa. As shown in Figure 3a, no band is presentat this size in the preinduction samples (lanes 2, 4, and 6), buta band appears in the postinduction sample for each of the variousconstructs (lanes 3, 5, and 7), confirming successful expression.Native EGF was used as a standard to establish relative expressionof the claMP-Tagged forms. N-terminal and C-terminalplacement led to a 14% and 29% decrease in EGF expression yield, respectively.Differences in expression among the variants were on the same orderas batch-to-batch variation among replicates of the same protein.Therefore, insertion of the claMP Tag into the proteinsequence has a negligible effect on protein expression, as determinedby densitometric analysis of whole cell lysates.

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