Analytical Development and Characterization of the Metal-binding claMP Tag for Generation of Inline Bioconjugates to Enable Targeted Metal Delivery: Evaluation of Tag Placement, Metal Insertion and Retention, and Stability of Ni(II)-claMP-Tagged Protein Variants

Abstract

Metal-based protein conjugates have become the primary means of achieving site- specific delivery of metal ions for use in clinical applications. Small, organic chelators are the primary entities used for metal delivery, but they require chemical conjugation to be attached to the protein surface. Conjugation using this method requires extensive optimization and generates a highly heterogeneous product. Therefore, metal-binding peptide tags capable of being engineered into a predetermined position, inline within a protein sequence are highly advantageous. The metal-binding claMP Tag is capable of being inserted inline within a protein sequence to generate a "linker-less" bioconjugate. The claMP Tag module is based upon the metal abstraction peptide (MAP) sequence, NCC (Asn-Cys-Cys), which has been shown to bind transition metals extremely tightly and to retain metal binding in the presence of longer polypeptide sequences, thus allowing for use of this tag as an inline metal-binding agent for the generation of bioconjugates. The primary focus of this work was to characterize the effects of claMP Tag addition on various protein systems to establish its applicability as a metal-delivery platform. Ni(II) was inserted into the tag in these studies because the Ni-claMP complex had previously been extensively characterized using small peptides and it was determined to be highly stable and to have a number of advantageous properties to enable quantitative analysis. The properties of this unique metal-peptide module suggested that it would be amenable to use in a larger protein construct, where retention of the metal would be enabling to targeted delivery. Herein, the unique charge, spectroscopic, and catalytic properties of the Ni-claMP complex were used as the basis for establishing methods to evaluate the structure and stability of claMP Tag- based inline metal-protein conjugates. Two model protein systems were chosen for analytical development that are different in size, composition, and structure to assess the applicability of the claMP Tag within unrelated proteins and using different methods of purification. The placement of the claMP Tag within the protein sequence was varied to determine if tag position differentially affects metal insertion. Because the claMP Tag itself contains two cysteine residues, which may interfere with native disulfide bond formation, the first protein investigated for expression, correct folding and higher order structure, and stability was epidermal growth factor (EGF). EGF is a small, difficult to express, disulfide-containing protein, which is capable of being produced recombinantly in E. coli when fused to thioredoxin, a thiol-modulating protein. The study described herein demonstrates addition of the claMP Tag to either terminus of EGF does not negatively affect the expression or function of the protein. NMR analysis was used to confirm that the disulfide bonds are formed correctly and that the tertiary structure of the protein is well maintained in the metal-claMP Tag conjugate. The unique absorption spectrum and characteristic 2- charge of the properly formed metal-claMP structure were utilized to quantitatively assess both metal incorporation and stability of the conjugate. The data show that addition of the cysteine- containing claMP Tag to a thiol- and disulfide-containing protein does not negatively impact the critical attributes or protein properties, making it amenable to use as an inline metal carrier. A polyhistidine tag was engineered into EGF to enable facile purification using immobilized metal affinity chromatography (IMAC), but this approach leads to concomitant uptake of Ni(II) into the claMP Tag. To evaluate the ability to purify the tagged protein prior to metal insertion as well as different approaches to metal incorporation, maltose-binding protein (MBP) was examined. This second protein system allows for decoupling of the metal insertion and purification steps because amylose resin, which does not rely on metal in any way, enablesaffinity purification of MBP. This allowed for metal insertion efficiencies to be determined before and after purification. It also demonstrated that the claMP Tag may be successfully applied to larger protein systems, because MBP is approximately six-times larger than EFG. Finally, because of its larger size, retention of metal in the claMP Tag within the MBP variant could be evaluated more easily in the presence of a large excess of a potentially competitive high-affinity chelator. This study was used to establish that the unique chemistry that the claMP tag undergoes to bind metal generates a highly stable product.