Penicillin-binding protein 2x of Streptococcus pneumoniae. Expression in Escherichia coli and purification of a soluble enzymatically active derivative.
A 2.5-kb DNA fragment including the structural gene coding for the penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae has been cloned into the vector pJDC9 and expressed in Escherichia coli. Mapping of RNA polymerase binding sites by electron microscopy indicated that the pbpX promoter is well recognized by the E. coli enzyme. However, high-level expression occurred mainly under the control of the lac promoter upstream of the pJDC9 multiple cloning site. After induction with isopropyl beta-d-thiogalactopyranoside, PBP 2x was expressed as one of the major cellular proteins. PBP 2x produced in E. coli corresponded to the pneumococcal PBP 2x in terms of electrophoretic mobility, fractionation with the cytoplasmic membrane, and penicillin-binding capacity. Deletion of 30 hydrophobic N-terminal amino acid residues at positions 19-48 resulted in high-level expression of a cytoplasmic, soluble PBP 2x derivative (PBP 2x*) which still retained full beta-lactam-binding activity. A two-step procedure involving dye affinity chromatography was established for obtaining large amounts of highly purified enzymatically active PBP 2x*.
Laible
G
G
Max-Planck Institut für molekulare Genetik, Berlin, Federal Republic of Germany.
Keck
W
W
Lurz
R
R
Mottl
H
H
Frère
J M
JM
Jamin
M
M
Hakenbeck
R
R
D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002458:Cell Fractionation; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006602:Hexosyltransferases; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013296:Streptococcus pneumoniae / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D012321:DNA-Directed RNA Polymerases; D002267:Muramoylpentapeptide Carboxypeptidase