Recombinant Expression Of Uricase From Bacillus Fastidiosus Atcc29604 And Its Preliminary Sequence-activity Relationship

Uricase from Bacillus fastidiosus ATCC 29604 can be used as an analytical tool to determine serum uric acid by a kinetic uricase method, and it also has promise as a biodrug for the treatment of hyperuricemia. The preliminary characterizations and most of the coding sequence of the intracellular Bacillus fastidiosus uricase was abtained, but its N-terminus sequence was not completely revealed. The N-terminus sequence for this intracellular uricase was A(Q)E(Q)RTMFYGKGDV by Edman degradation, but the first two amino acid residues at the N-terminus were still uncertain after 5'-RACE was performed. Herein, the cloned sequence for uricase was examined firstly, and then a series of N-terminus variants were constructed and studied, to explore the N-terminus for desirable properties.1. Recombinant expression of the uricase variantsA pair of primers was designed according to the known coding region of uricase from Bacillus fastidiosus (FJ393559), then the sequence was cloned and inserted into pET28a. Different Nterminus variants were designed. After the induction for about 20 h at 18℃with 1.0 mmol/L of IPTG, soluble uricase was obtained and accounted for more than 50% of total soluble proteins. Among these variants, the yield was about 1 kU/L in TB medium for that with the N-terminus of AERTMFYGK (the first methionine was expected to be removed). After the purification by DEAE-cellulose 52 chromatography and preparative gel electrophoresis, the specific activity of this variant reached up to 11.5U/mg, closed to that of the natural uricase; MALDI-TOF-MS analysis gave a primary peak of 35.779 kDa, which showed only a 10-Da deviation from the calculated molecular weight after the removal of the first methionine. But the molecular weight of the natural intracellular uricase from Bacillus fastidiosus ATCC 29604 was 204-Da larger than that of variant with the N-terminus of AERTMFYGK, and 147-Da larger than that of the variant with the N-terminus of QERTMFYGK. Therefore, the coding sequence of the natural intracellular uricase was still not revealed, and other modification(s) on the natural intracellular uricase may account for the differences in its molecular weight from expected candidates.2. Characterization of the recombinant variant with a known sequenceAt 25℃, pH 9.2, the Km for the uricase was (0.29±0.03) mmol/L (n = 5), and Ki of xanthine was (33±4)μmol/L (n = 3), which showed a high resistance against xanthine. The optimum temperature was 25-30℃, and the optimum pH was 8.9-9.5. The activity was activated by some cations, among which magnesium ions in the 0.1M Tris-HCl pH8.9 buffer solution showed the strongest activation. The pH value and temperature also had apparent effects on the uricase. AT pH7.4, Km of uricase was 91±5μmol/L (n = 3), 228±15μmol/L (n = 3), 892±40μmol/L (n = 3) in 10℃, 25℃, 40℃, respectively, while that of the enzyme at pH9.2 was 180±6μmol/L (n = 4), 290±30μmol/L (n = 5), 1010±50μmol/L (n = 3) in 10℃, 25℃, 40℃, respectively. The maximum reaction rates were obtained by the conversion of initial rates according to single substrate Michaelis-Menten kinetics, and according to Arrenius experience formula the activation energy of the uricase at pH7.4 and pH9.2 was estimated as 31 kJ/mol and 16 kJ/mol, respectively, which was much lower than the activation energy for acetylcholinesterase reaction. The catalytic activity of this variant is clearly much lower than that of acetylcholinesterase. Therefore, Arrenius empirical formula does not apply to the situation described in this article.3. Relationship between sequences and activitiesIn a preparation of the variant with N-terminus of AERTMFYGK that had only one polypeptide of about 35 KDa by SDS-PAGE, there were two components with mobility consistent with those of tetramer and dimmer, respectively, analyzed by non-denaturating PAGE. Moreover, with the decrease of specific activities, the abundance of the stained band of tetramer decreased accordingly, indicating that tetramer is the highly active form of the enzyme as presumed. Experimental results showed that C-terminus of His-tag has almost negiligible effect on the specific activity of the enzyme, while the N-terminus affected specific activity significantly: the highest specific activity of the variant with the N-terminus of AERTMFYGK was 11.5 U/mg, while that of the variant with the N-terminus of GFYGK was only 0.1 U/mg, which had almost only one polypeptide of dimer by non-denaturating PAGE analysis. The comparison of five variants of the uricase also demonstrated that their Ki, Km, and the optimum temperature and pH were not significantly affected by their N-terminus sequence or their C-terminus of His-tag. But His-tag on their C-terminus enhanced their sensitivity to metal ion activation and pH value, and improved the stability at 40℃. However, either of the two variants with His-tag on their C-terminus could not be purified by Ni+ affinity chromatography, suggesting that the His-tag might be buried.4. Homology modeling and preliminary analysis of relationship between sequence and functionA model of the variant with N-terminus of AERTMFYGK was built, taking uricase from Bacillus sp.TB-90 (1J2G.pdb) as the template (sequence identities above 50%, more than 70% similarity). It was found that the amino residue--R in the N-terminus was very close to the residues of ED in the vicinity of the 310th residues at C-terminus of another subunit, which may have a very strong electrostatic interaction. This may be the structural basis of the dissociation of tetramer upon dialysis and cation activation effect; and such interactions may affect the fine process of the assembly dissociation of the subunits in the tetramer.In summary, the characterization of the variant with the N-terminus of AERTMFYGK was very similar to that of natural uricase, and the first amino residue of N-terminus -- methionine was lost; the complete sequence of the natural intracellular uricase was still not revealed, and the possibility of other modifications on natural uricase existed; its third amino residue of N-terminus -- arginine might play an important role on the formation of the active site. In future, the N-terminus may be modified to enhance the the specific activity and thermostability.