claMP Tag: A Versatile Inline Metal-Binding Platform Based on the Metal Abstraction Peptide

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.

Thioredoxin fusion construct enables high-yield production of soluble, active matrix metalloproteinase-8 (MMP-8) in Escherichia coli

“…Two constructs of MMP-8 were prepared, one containing solely MMP-8 and one bearing an additional N-terminal cla MP Tag (Asn-Cys-Cys). 27,29 The primers contained a region matching the pET-32Xa/LIC vector (underlined), a portion matching the MMP-8 catalytic domain (italics), the cla MP Tag (bold) and an inserted linker (bold and underlined) between the cla MP Tag and MMP-8. 5’- GGT ATT GAG GGT CGC AAT CCA GGA AAC CCC AAG TG -3’ (forward primer no cla MP Tag), 5’- TGC GGC TCT TCT GGC ATT GAG GGT CGC AAC CCC AAG TGG GAA -3’ (first forward primer for cla MP-link insertion), 5’- GGT ATT GAG GGT CGC CCA GAT CTG GGT AAC TGC TGC GGC TCT TCT GGC -3’ (second forward primer for cla MP-link insertion), and 5’- AGA GGA GAG TTA GAG CCT TAT CCA TAG ATG GCC TG-3’ (reverse primer for both constructs).…”

“…27 As previously reported, UV-vis absorbance spectroscopy at 425 nm was used to assess metal occupancy of the cla MP Tag in the fusion construct. 27 The molar extinction coefficient for 425 nm is 350 M −1 cm −1 .…”

“…27 As previously reported, UV-vis absorbance spectroscopy at 425 nm was used to assess metal occupancy of the cla MP Tag in the fusion construct. 27 The molar extinction coefficient for 425 nm is 350 M −1 cm −1 . Following purification, at 0.5 mM, the highest concentration of the fusion protein, the A425 indicates complete nickel insertion into the cla MP Tag has been achieved.…”

“…26 Genetic engineering of MAP into a plasmid creates, the cla MP Tag, a linker-less carrier for many transition metals with utility in healthcare applications. 27 The cla MP Tag is a tripeptide consisting of the amino acid sequence Asn-Cys-Cys, which scavenges small transition metals from chelating agents. 28 Because of the uniquely beneficial properties of the cla MP Tag, which includes extraordinarily tight binding, resistance to metal release upon dilution at serum pH and specific release in acidic conditions as in endosomes, the cla MP Tag is being investigated as a method for targeted delivery of metals for therapeutic and diagnostic applications by designing targeting proteins to contain the metal bound tripeptide.…”

“…28 Because of the uniquely beneficial properties of the cla MP Tag, which includes extraordinarily tight binding, resistance to metal release upon dilution at serum pH and specific release in acidic conditions as in endosomes, the cla MP Tag is being investigated as a method for targeted delivery of metals for therapeutic and diagnostic applications by designing targeting proteins to contain the metal bound tripeptide. 27 The cla MP Tag has not been investigated previously in a system, such as matrix metalloproteinases, in which a structural and/or catalytic metalbinding site is present. Analysis of the activity and stability of these two fusion proteins and their cleaved products is presented herein.…”