High-level production and purification of Escherichia coli N-acetylneuraminic acid aldolase (EC 4.1.3.3).
The Escherichia coli gene which encodes N-acetylneuraminic acid aldolase was isolated by the polymerase chain reaction, cloned into the inducible expression vector pTTQ18, and overexpressed in E. coli. The high yield of aldolase was achieved through both optimum growth of cells and efficient expression of the aldolase gene (20-30% soluble cellular protein). The recombinant enzyme was purified to homogeneity with an activity of 1.2-2.2 U/mg, which compared favorably with that of commercial preparations of E. coli aldolase (1.1 U/mg) and Clostridium perfringens aldolase (0.4 U/mg). The cloning strategy, fermentation conditions, purification protocol, and activity assay are described.
Lilley
G G
GG
C.S.I.R.O. Division of Biomolecular Engineering, Parkville, Australia.
von Itzstein
M
M
Ivancic
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D002244:Carbon / Q000737:chemistry; D003001:Cloning, Molecular; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D011994:Recombinant Proteins; D002244:Carbon; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase