Thermostable and Long-Circulating Albumin-Conjugated Arthrobacter globiformis Urate Oxidase

Abstract

Urate oxidase derived from Aspergillus flavus has been investigated as a treatment for tumor lysis syndrome, hyperuricemia, and gout. However, its long-term use is limited owing to potential immunogenicity, low thermostability, and short circulation time in vivo. Recently, urate oxidase isolated from Arthrobacter globiformis (AgUox) has been reported to be thermostable and less immunogenic than the Aspergillus-derived urate oxidase. Conjugation of human serum albumin (HSA) to therapeutic proteins has become a promising strategy to prolong circulation time in vivo. To develop a thermostable and long-circulating urate oxidase, we investigated the site-specific conjugation of HSA to AgUox based on site-specific incorporation of a clickable non-natural amino acid (frTet) and an inverse electron demand Diels–Alder reaction. We selected 14 sites for frTet incorporation using the ROSETTA design, a computational stability prediction program, among which AgUox containing frTet at position 196 (Ag12) exhibited enzymatic activity and thermostability comparable to those of wild-type AgUox. Furthermore, Ag12 exhibited a high HSA conjugation yield without compromising the enzymatic activity, generating well-defined HSA-conjugated AgUox (Ag12-HSA). In mice, the serum half-life of Ag12-HSA was approximately 29 h, which was roughly 17-fold longer than that of wild-type AgUox. Altogether, this novel formulated AgUox may hold enhanced therapeutic efficacy for several diseases.

Chemical Modification of Cysteine with 3-Arylpropriolonitrile Improves the In Vivo Stability of Albumin-Conjugated Urate Oxidase Therapeutic Protein

“…To express AgUox-frTet, C321delA.exp [pDule_C11] [pBAD_AgUox-196Amb] Escherichia coli cells containing the optimized MjtRNA Tyr /MjTyrRS toward frTet were prepared and used as previously reported [36]. E. coli cells cultured in Luria broth (LB) medium containing ampicillin (100 µg/mL) and tetracycline (10 µg/mL) at 37 • C overnight with shaking were inoculated into 2× YT medium containing conditions identical to those of the LB medium.…”

“…As a model therapeutic protein, we chose AgUox, a homotetrameric enzyme consisting of identical 34 kDa subunits with high thermostability and high expression level in E. coli, and thus has great potential to treat hyperuricemia conditions/gout [30]. First, we prepared AgUox containing frTet (Figure 1) by adapting expression and purification methods previously described except that AgUox was used instead of Aspergillus flavus Uox [36]. Among the non-natural amino acids containing the tetrazine group for the IEDDA reaction, frTet, whose reactivity has been well defined [32,36,40], was successfully incorporated into the protein.…”

“…First, we prepared AgUox containing frTet (Figure 1) by adapting expression and purification methods previously described except that AgUox was used instead of Aspergillus flavus Uox [36]. Among the non-natural amino acids containing the tetrazine group for the IEDDA reaction, frTet, whose reactivity has been well defined [32,36,40], was successfully incorporated into the protein.…”

“…We chose Uox as a model therapeutic protein, since it is convenient to produce from E. coli expression hosts, and its activity can be determined by a simple biochemical assay. In previous studies, the urate oxidase derived from Aspergillus flavus (AfUox) was used [30,36]. However, since AfUox has a low thermostability, it is not ideal to evaluate the in vivo stability of thiol-APN reaction products over a long period [37].…”

“…However, since AfUox has a low thermostability, it is not ideal to evaluate the in vivo stability of thiol-APN reaction products over a long period [37]. Therefore, we chose a thermostable Uox derived from Arthrobacter globiformis (AgUox) in this study [30,36,38,39]. Then, a hetero-bifunctional crosslinker containing a trans-cylooctene (TCO) group and either maleimide or APN was conjugated to the Cys34 of HSA to prepare MAL-HSA or APN-HSA, respectively (Figure 1).…”