Cell Permeable Cocaine Esterases Constructed by Chemical Conjugation and Genetic Recombination

Abstract

Cocaine esterase (CocE) is the most efficient cocaine-metabolizing enzyme tested in vivo to date, displaying a rapid clearance of cocaine and a robust protection against cocaine’s toxicity. Two potential obstacles to the clinical application of CocE, however, lie in its proteolytic degradation and induced immune response. To minimize these potential obstacles, we attempted non-disruptive cell encapsulation by creating a cell permeable form of CocE, which was achieved by covalently linking a thermally stable CocE mutant (dmCocE) with cell penetrating peptides (CPPs). Two types of CPPs, Tat and the low molecular weight protamine (LMWP), were used in this study. Two types of disulfide-bridged chemical conjugates, Tat-S-S-dmCocE and LMWP-S-S-dmCocE, were synthesized and then purified by heparin affinity chromatography. In addition, four recombinant CPP-dmCocE fusion proteins, Tat-N-dmCocE, LMWP-N-dmCocE, dmCocE-C-Tat, and dmCocE-C-LMWP, were constructed, expressed in E. coli and purified as soluble proteins. Among these six CPP-dmCocE variants, LMWP-S-S-dmCocE showed the highest cocaine-hydrolyzing activity, and dmCocE-C-Tat had the highest production yield. To evaluate their cellular uptake behavior, a covalently-linked fluorophore (FITC) was utilized to visualize the cellular uptake of all six CPP-dmCocE variants in living HeLa cells. All the six variants exhibited cellular uptake, but their intracellular distribution phenotypes differed. While the chemical conjugates showed primarily cytoplasmic distribution, which was likely due to the reduction of the disulfide linkage between CPP and dmCocE, all the other four recombinant fusion proteins displayed both nuclear and cytoplasmic localization, with dmCocE-C-CPP exhibiting higher cytoplasmic distribution during cellular uptake. Based on a balanced consideration of essentials for clinical application, including parameters such as high cocaine-hydrolyzing efficiency, large production yield, major cytoplasmic distribution, etc., the dmCocE-C-Tat fusion protein seems to be the best candidate from this investigation. Further in vivo studies of the cell-encapsulated dmCocE-C-Tat in hydrolyzing cocaine and alleviating immunogenicity and proteolytic degradation in established, clinically relevant mouse models are currently underway in our laboratories. Findings from this research are not only useful for developing other new CPP-CocE constructs, but also valuable for establishing a non-disruptive cell-encapsulation technology for other protein therapeutics that are known to be immunogenic for direct clinical application.

Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity

“…4A). This result was in consistent with the phenomenon described as the report that of the recombinant mutated cocaine esterase fused with a CPP (TAT or LMWP) [36]. The main reason that the fusion protein expressions reduced obviously could be ascribed to the unique amino acid compositions of CPPs consisting of a cluster of arginine residues that are encoded in one of the rarest codons and translated much slower than other amino acids in E. coli (Table 2) [36,37].…”

Chemically and biologically synthesized CPP-modified gelonin for enhanced anti-tumor activity

“…Despite that rGel was successfully expressed using the pET-Gel vector, there was almost no expression of the rG-L under tested conditions. This poor expression of rG-L was likely due to inefficient translation of the LMWP gene caused by codon usage bias, a finding previously reported by Lee and other investigators [33, 41]. Indeed, LMWP was known to consist of abundant arginine residues that was translated by the rarest codons in E. coli [33], hence severely limiting its expression level.…”

“…This poor expression of rG-L was likely due to inefficient translation of the LMWP gene caused by codon usage bias, a finding previously reported by Lee and other investigators [33, 41]. Indeed, LMWP was known to consist of abundant arginine residues that was translated by the rarest codons in E. coli [33], hence severely limiting its expression level. To this regard, the poor translation of LMWP appeared to significantly impair the overall expression of the ultimate rG-L. On the other hand, the use of the BL21-CodonPlus E. coli strain, which contained extra copies of genes encoding the tRNAs for rare amino acids, also did not provide any enhancement on rG-L expression.…”

Preparation and Characterization of Gelonin-Melittin Fusion Biotoxin for Synergistically Enhanced Anti-Tumor Activity

“…The final products of cGel-Mel and rGel-Mel were examined by SDS-PAGE, and western blot assay was further carried out using mouse anti-6×His tag antibody (Abcam, Cambridge, MA) to confirm the expression of rGel-Mel, following the previously reported procedures (24, 25). The final cGel-Mel and rGel-Mel products were quantified by BCA assay, and the purity was determined by densitometry analysis (ImageJ software, National Institutes of Health, Bethesda, MD).…”