Legoux R, Delpech B, Dumont X, Guillemot J C, Ramond P, Shire D, Caput D, Ferrara P, Loison G
Sanofi Elf BioRecherches, Labège, France.
J Biol Chem. 1992 Apr 25;267(12):8565-70.
Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.
对黄曲霉尿酸氧化酶（尿酸酶）进行蛋白水解后得到的肽段进行氨基酸测序，从而设计出寡脱氧核苷酸探针，这些探针分别用于从黄曲霉cDNA文库和基因组文库中获取1.2千碱基对和5千碱基对的DNA片段。该cDNA片段包含尿酸酶的完整编码区，与基因组片段比较后发现该基因的编码区存在两个短内含子。黄曲霉尿酸酶与高等生物的尿酸酶总体上有40%的一致性，但有许多保守氨基酸。迄今在其他尿酸酶中发现的高度保守的共有模式在黄曲霉酶中被发现有改变，特别是在第150位附近的序列Val-Leu-Lys-Thr-Thr-Gln-Ser，在丝状真菌中它独特地被改变为Val-Leu-Lys-Ser-Thr-Asn-Ser。通过盒式诱变在编码序列的5'末端附近引入沉默突变，以符合大肠杆菌的密码子使用情况，并且尿酸酶以完全可溶的、具有生物活性的形式在大肠杆菌细胞质中表达。
