Stability Analysis of an Inline Peptide-based Conjugate for Metal Delivery: Nickel(II)-claMP Tag Epidermal Growth Factor as a Model System

Abstract

Metals are a key component of many diagnostic imaging and biotechnology applications, and the majority of cancer patients receive a platinum-based drug as part of their treatment. Significant effort has been devoted to developing tight binding synthetic chelators to enable effective targeted delivery of metal-based conjugates, with most successes involving lanthanides rather than transition metals for diagnostic imaging. Chemical conjugation modifies the protein’s properties and generates a heterogeneous mixture of products. Chelator attachment is typically done by converting the amino group on lysines to an amide, which can impact the stability and solubility of the targeting protein and these properties vary among the set of individual conjugate species. Site-specific attachment is sought to reduce complexity and control stability. Here, the metal abstraction peptide (MAP) technology was applied to create the claMP Tag, an inline platform for generating site-specific conjugates involving transition metals. The claMP Tag was genetically encoded into epidermal growth factor (EGF) and loaded with nickel(II) as a model system to demonstrate that the tag within the homogeneous inline conjugate presents sufficient solution stability to enable biotechnology applications. The structure and disulfide network of the protein and chemical stability of the claMP Tag and EGF components were characterized.

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

“…The cla MP Tag is an extremely stable, inline module for targeted metal delivery. 27,30 It consists of the amino acid sequence Asn-Cys-Cys and rapidly and quantitatively binds metal at basic pH, 30,31 here during purification upon exposure to the Ni-IMAC resin. The recognition sequence of Factor Xa follows this cla MP-link spacer in the cla MP-link-MMP-8 construct to permit cleavage of the additional tag and release of the same MMP-8 product.…”

“…The cla MP Tag binds metal extraordinarily tightly at basic pH and this fusion system becomes occupied with nickel during IMAC purification. 30 The metal-free cla MP Tag has been shown to inhibit MMP-8 activity and is being investigated as a potential regulator of the activity of MMP-8. 29 cla MP-link-MMP-8 was originally designed to contain a linker peptide sequence that would allow the cla MP Tag to interact with the active site zinc to potentially inhibit the activity of the enzyme as a pro-moiety that is releasable by adjusting the pH to below neutral or by inserting another metal into the tag.…”

Metal-bound claMP Tag inhibits proteolytic cleavage

“…In previous studies, it was determined that the MAP chemistry is compatible with recombinant production of difficult to express proteins having intramolecular disulfide bonds because the engineered claMP Tag does not interfere with proper folding and protein structure (Mills et al, 2014;Mills and Laurence, 2015). In the preparation of proteins used in these studies, however, slow or incomplete cleavage of the fusion partners was observed for some constructs.…”

“…Our lab discovered the unique metal abstraction peptide (MAP) chemistry and developed the inline claMP Tag, which is currently being investigated as a targeted delivery method of transition metals for therapeutic and diagnostic applications. The tag consists of the amino acid sequence Asn-Cys-Cys and binds transition metals extraordinarily tightly (Laurence et al, 2012;Mills et al, 2014;Mills and Laurence, 2015), making it possible to increase effective circulation time and target-specific tissue accumulation for transition metal-based imaging agents. The metal-claMP complex has been shown to not disrupt the overall protein structure, including proteins that depend on formation of multiple, complex disulfide bonds for proper folding and function (Mills et al, 2014;Mills and Laurence, 2015).…”

“…The tag consists of the amino acid sequence Asn-Cys-Cys and binds transition metals extraordinarily tightly (Laurence et al, 2012;Mills et al, 2014;Mills and Laurence, 2015), making it possible to increase effective circulation time and target-specific tissue accumulation for transition metal-based imaging agents. The metal-claMP complex has been shown to not disrupt the overall protein structure, including proteins that depend on formation of multiple, complex disulfide bonds for proper folding and function (Mills et al, 2014;Mills and Laurence, 2015). To investigate susceptibility of the metal-bound claMP Tag to proteolytic cleavage and determine viability of using this tag in targeted imaging applications, an in vitro proof of concept study of a fusion protein was carried out by engineering the claMP Tag adjacent to a known recognition site for the serum protease Factor Xa.…”