Penicillin-binding protein 3 of Streptococcus pneumoniae and its application in screening of β-lactams in milk.
The soluble form of penicillin-binding protein 3 (sPBP3(∗)) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP-Amp) to the sPBP3(∗) by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3(∗) exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.
College of Veterinary Medicine, China Agricultural University, Beijing 100094, People's Republic of China; National Reference Laboratories for Veterinary Drug Residue, District Haidian, Beijing 100193, People's Republic of China.
Jing
J
Zhang
Zhanhui
Z
Wang
Kai
K
Wen
Xiao
X
Liang
Jianzhong
J
Shen
D000667:Ampicillin / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods*; D005504:Food Analysis / Q000379:methods*; D005506:Food Contamination / Q000032:analysis; D006735:Horseradish Peroxidase / Q000378:metabolism; D008892:Milk / Q000737:chemistry*; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D013296:Streptococcus pneumoniae; D047090:beta-Lactams / Q000032:analysis* / Q000378:metabolism
D001426:Bacterial Proteins; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams; D000667:Ampicillin; D006735:Horseradish Peroxidase
Cephalosporins; Microplate receptor assay; Milk; Penicillin-binding protein 3; Penicillins; Residue