Overproduction and rapid purification of the amidase of Streptococcus pneumoniae.
Oligonucleotide-directed mutagenesis of a plasmid containing the lytA gene coding for the pneumococcal amidase has allowed the separation of the coding sequence of the gene. This sequence has been placed in plasmid pIN-III(lppP-5)-A3 downstream from both a modified lipoprotein promoter and the lactose promoter to construct the recombinant plasmid pGL100. When Escherichia coli RB 791 (pGL100) was grown in the presence of lactose, the pneumococcal amidase accounted for 7% of the total protein present in this strain after 18 h incubation at 37 degrees C. The overproduced amidase was purified in a single-step procedure using a choline-Sepharose 6B column taking advantage of the fact that this enzyme was the unique protein with affinity for choline present in extracts obtained from E. coli RB791 (pGL100). The development of the above design opens up the possibility of studying the mechanism that regulates the activity of this important autolysin by using physiochemical techniques that require the availability of high amounts of purified amidase.
García
J L
JL
Centro de Investigaciones Biológicas, C.S.I.C. Velázquez, Madrid, Spain.
García
E
E
López
R
R
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation; D010957:Plasmids; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics
D000581:Amidohydrolases; C019665:amidase