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.

Keywords: Arthrobacter globiformis; gout; half-life extension; inverse electron demand Diels-Alder reaction; site-specific albumin conjugation; thermostability; urate oxidase.

Figure 1

(a) Crystal structure of AgUox (PDB code: 2YZE) showing the selected sites for frTet incorporation. (b) The frTet incorporation sites and corresponding AgUox variants containing frTet are indicated in the table.

Figure 2

Expression and purification of AgUox-frTet variants. (a) Coomassie blue-stained protein gels of AgUox-WT and AgUox-frTet (Ag1–14) variants. Lanes: MW, molecular weight marker; BI, before induction; AI, after induction. (b) Image of Coomassie blue-stained protein gels of AgUox-WT and AgUox-frTet variants after purification.

Figure 3

Thermostability assessment of AgUox-WT and AgUox-frTet variants. (a) Relative enzyme activity of AgUox-WT and AgUox-frTet (Ag1–14) variants in PBS monitored at 0 and 120 h. Red line indicates the 50% enzymatic activity of AgUox-WT. (b) Relative enzymatic activity of AgUox-WT and five AgUox-frTet variants (Ag1, 6, 8, 10, and 12) monitored at 0, 120, and 240 h. The relative activity of AgUox-frTet variants was normalized against the enzymatic activity of AgUox-WT.

Figure 4

Incorporation of frTet into AgUox. Fluorescence (illumination λex = 302 nm, with wavelengths at 510 and 610 nm in Chemidoc XRS+ system) and Coomassie blue-stained protein gel for reaction mixture of TCO-Cy3 with AgUox-WT or AgUox-frTet (Ag) variants.

Figure 5

SDS-PAGE analysis of AgUox-HSA conjugate variants. The protein gel was visualized using Coomassie blue staining. Lanes: MW, molecular weight marker; 1, AgUox-HSA from Ag1 variant; 2, AgUox-HSA from Ag6 variant; 3, AgUox-HSA from Ag8 variant; 4, AgUox-HSA from Ag10 variant; 5, AgUox-HSA from Ag12 variant.

Figure 6

Purification of HSA-conjugated Ag12 variant (Ag12-HSA). Size-exclusion chromatography of Ag12-HSA conjugate mixture (right) and SDS-PAGE analysis of the eluted fractions (F1–F3) of Ag12-HSA (left). The protein gel was stained by Coomassie blue for the visualization of protein bands. Lanes: MW, molecular weight marker.

Figure 7

Specific activity of AgUox-WT, Ag12, and Ag12-HSA. Experiments were performed in quadruplicate, and error bars indicate the standard deviation.

Figure 8

Pharmacokinetic studies of AgUox-WT and Ag12-HSA in mice. Enzymatic activity of residual AgUox-WT and Ag12-HSA conjugate samples was measured at 15 min; at 3, 6, and 12 h for AgUox-WT; and 15 min and 12, 24, 48, and 72 h for Ag12-HSA. The samples for pharmacokinetic studies were intravenously injected into BALB/c female mice (n = 4). Error bars indicate the standard deviation.