Soluble Expression, Purification Of Penicillin-binding Protein3and Developing Of Testing Kit For Screening Of β-lactam Antibiotics In Milk

Beta-lactam antibiotics are among the most widely used antibiotics for the treatment of bacterialinfections in man and animals and are the preferred drugs for the treatment of clinical mastitis in dairycows. Beta-lactams are also administered to animals in feed for growth promotion and for collectiveprophylactic treatment. Consequently, beta-lactams constitute the major source of antibiotic residues inmilk. In order to control the abuse of antibiotics, slow down the formation of antibiotic-resistantbacterial strains and protect the health of consumers, the monitoring of beta-lactam residues in edibletissue and milk is important. In addition, beta-lactam antibiotics residues in raw milk seriously interferewith production of fermented dairy in dairy processing which affects the up leaven of cheese, butter andfermented milk and the formation of later flavor. Therefore, maximum residue limits have beenspecified for food products and milk to try and control the levels of these antibiotics reaching theconsumer. For example, China’s ministry of agriculture released the pollution-free food--raw milkindustry standards (NY/T5045-2001) in2001, which clearly prescribed antibiotics may not be detected.This standard has been replaced by NY5045-2008, which established the the maximum residue limit(MRL) for ampicillin in milk is10ug/kg and penicillin is zero. In the EU Council Regulation2377/90,the maximum residue limit(MRL) for both ampicillin and amoxicillin in milk is4μg/kg, forcefquinome20μg/kg, and for cefacetril in milk125μg/kg. It is urgent to develop a simple, quick,sensitive and low-cost detection method for beta-lactam antibiotics in milk to fulfill the requirements ofenterprises and customers.Nowadays, there are numerous screening tests commercially available which are able to detectbeta-lactam antibiotics in milk and indicate the presence at or above the maximum residue limits. Thesescreening tests are microbial growth inhibition assay, enzyme-linked immunosorbent assay (ELISA) andreceptor binding assays. However, microbiological methods have the lengthy incubation step necessary,a low detection limit, a large impact on human factors and it’s difficult to standardize quantitativedetection. Immunoassays has become the fastest growing and most widespread screening method for itssimplicity, rapidity, strong specificity, high sensitivity and large capability. There are several advantagesto replace antibodies with receptor proteins. Receptor binding assay can use one kit to detect multipleresidues while ELISA needs more kits. Only the active, intact beta-lactam structure is recognized byreceptor proteins, whereas most antibodies detect both active and inactive forms. And it’s easier toprepare large scale receptor proteins than antibodies. This paper utilizes a new receptor protein--PBP3from Streptococcus pneumoniae R6to develop a novel receptor/enzymatic based microplate assay forthe detection and determination of penicillins and cephalosporins with intact bata-lactam structure inmilk and provides the basis for establishing a simple, large capable, high sensitive and low-cost rapiddetection method.To study the structure and application of penicillin-binding protein3(PBP3), the pbp3gene ofStreptococcus pneumonia R6was cloned and the recombinant protein of PBP3was expressed in Escherichia coli with expression vector pGEX-6p-pbp3*. GST-PBP3*fusion protein was purified withGlutathione-Sepharose4B column and firstly digested by PreScission Protease for cutting off the GSTtag, and then passed through the Glutathione-Sepharose4B column for getting the purified PBP3protein. The MW of PBP3*is about43.6kD and from1L culture medium we can get6.6g wet netbacteria and at last23mg/L purified PBP3*protein with a high yield about0.35%. The PBP3activitywas identified by its binding activity to β-lactam antibiotics, such as cefquinome of which the IC50valueis6.2μg/kg. The pI of PBP3*was determined at4.5to avoid precipitation caused by improper pHbuffers during operation. The truncated pbp3gene was verified by sequence analysis and thepGEX-6p-pbp3*expression plasmid was constructed successfully. The active PBP3was successfullyobtained from prokaryotic expression system, and this laid a firm foundation for further study of thestructure and application of PBP3.Then we utilized PBP3*to develop the receptor/enzymatic based microplate assay for thedetection and determination of penicillins and cephalosporins with intact bata-lactam structure in milk.In the assay, the receptor protein is immobilised to a microplate in the first step. Beta-lactams, if presentin the standard and sample, compete with horseradish peroxidase labelled beta-lactam (HRP-Amp) for alimited number of acceptor sites on the microplate. After incubation at37℃incubator to allow acompetition reaction to take place, the microplate is then washed to remove excess reagents. Theenzyme substrate is added. After an incubation period to allow maximum colour development, thecolour reaction is stopped by addition of acid. This produces a color change from blue to yellow, and theabsorbance are read at450nm. A standard curve is then conducted to determine IC50and thebeta-lactam concentration in the standard and sample. Different IC50values means that PBP3*hasdifferent affinities to different beta-lactam antibiotics. The cross-reactivity rates proved PBP3*couldonly recognize beta-lactam antibiotics and it shows higher affinity with cephalosporins than penicillins.The recovery in spiked milk samples was not very high and there is room to improve. The precisionexperiment exposed the existing problems of this method, like bad repeatability or precision, whichsuggests it needs further optimization of reaction conditions to get into the market.. Considering thereceptor binding assays have clear advantages and the PBP3*has a higher affinity to beta-lactams thanPBP2x more commonly used, further research is promising.