Molecular cloning, expression and purification of L-amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma.
A cDNA encoding LAAO from the Malayan pit viper (Calloselasma rhodostoma) was cloned into an expression vector of the methylotropic yeast Pichia pastoris. The LAAO open reading frame was inserted after the alpha-MF-signal sequence. Upon induction soluble and active LAAO is produced and exported into the culture supernatant at a concentration of up to 0.4 mg/L. Recombinant LAAO was purified from this by ion exchange and molecular sieve chromatography to yield apparently homogeneous protein in quantities of approximately 0.25 mg/L growth medium. Expressed LAAO exhibits the same electrophoretic mobility as native LAAO (62 kDa) and exhibits approximately the same extent of glycosylation as authentic LAAO from snake venom. Catalytic properties and substrate specificity of recombinant LAAO are similar to those of native enzyme.
Kommoju
Phaneeswara Rao
PR
Fachbereich Biologie, Universität Konstanz, Konstanz D-78457, Germany.
Macheroux
Peter
P
Ghisla
Sandro
S
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic; D050830:L-Amino Acid Oxidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017819:Viperidae
D017931:DNA Primers; D011994:Recombinant Proteins; D050830:L-Amino Acid Oxidase