Dargis M, Malouin F
Département de Microbiologie, Université Laval, Québec, Canada.
Antimicrob Agents Chemother. 1994 May;38(5):973-80. doi: 10.1128/AAC.38.5.973.
A new reagent for the detection of penicillin-binding proteins (PBPs) was developed. An N-hydroxysuccinimide ester of biotin was used to tag beta-lactam antibiotics with free side chain amino groups such as ampicillin (BIO-AMP), 6-aminopenicillanic acid (BIO-APA), and 7-aminocephalosporanic acid (BIO-ACA). Bacterial PBPs from cells or isolated cytoplasmic membranes of Escherichia coli, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae were labeled with BIO-AMP, subjected to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and transferred onto nitrocellulose membranes. Electrophoretic PBP profiles were detected on blots, using colorimetric or chemiluminescence systems, on the basis of the interaction of BIO-AMP-PBP complexes and a streptavidin-peroxidase conjugate. The chemiluminescent reaction permitted a high sensitivity of detection, and PBP profiles could be determined within seconds. All PBP profiles were similar to those obtained with a traditional PBP labeling technique with 125I-labeled penicillin V, except that an additional unidentified PBP (approximately 55,000 Da) was labeled with BIO-AMP in E. coli and H. influenzae. Differences in the intensities of labeling for specific PBPs were observed between the chemiluminescent and radioactive labeling agents and were attributed to the differences in their affinities for PBPs. Similarly, BIO-AMP, BIO-APA, and BIO-ACA produced different PBP profiles. We also investigated the use of BIO-AMP in PBP purification. BIO-AMP-PBP complexes from a mixture of H. influenzae proteins were allowed to bind to avidin immobilized on an agarose support in a microcentrifuge tube. After several washes in the presence of salts, PBPs were eluted by boiling and treatment with SDS. The eluted proteins were separated by electrophoresis on SDS-polyacrylamide gels, and biotinylated proteins were identified on blots by a chemiluminescence reaction. Biotinylation of beta-lactams is rapid, safe, and inexpensive. Our results demonstrate the feasibility of using biotinylated beta-lactams as nonradioactive reagents for the study of PBPs and for the purification of these proteins.
开发了一种用于检测青霉素结合蛋白（PBPs）的新试剂。生物素的N-羟基琥珀酰亚胺酯用于标记具有游离侧链氨基的β-内酰胺抗生素，如氨苄西林（生物素-氨苄西林，BIO-AMP）、6-氨基青霉烷酸（生物素-6-氨基青霉烷酸，BIO-APA）和7-氨基头孢烷酸（生物素-7-氨基头孢烷酸，BIO-ACA）。来自大肠杆菌、流感嗜血杆菌、金黄色葡萄球菌和肺炎链球菌细胞或分离的细胞质膜的细菌PBPs用BIO-AMP进行标记，在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶上进行电泳，然后转移到硝酸纤维素膜上。基于BIO-AMP-PBP复合物与链霉亲和素-过氧化物酶偶联物的相互作用，使用比色或化学发光系统在印迹上检测电泳PBP图谱。化学发光反应具有高检测灵敏度，几秒钟内即可确定PBP图谱。所有PBP图谱与用传统的125I标记青霉素V的PBP标记技术获得的图谱相似，只是在大肠杆菌和流感嗜血杆菌中，一种额外的未鉴定PBP（约55,000 Da）被BIO-AMP标记。在化学发光和放射性标记剂之间观察到特定PBPs标记强度的差异，这归因于它们对PBPs亲和力的差异。同样，BIO-AMP、BIO-APA和BIO-ACA产生不同的PBP图谱。我们还研究了BIO-AMP在PBP纯化中的应用。使来自流感嗜血杆菌蛋白质混合物的BIO-AMP-PBP复合物与固定在微量离心管中琼脂糖支持物上的抗生物素蛋白结合。在有盐存在的情况下进行几次洗涤后，通过煮沸并用SDS处理洗脱PBPs。洗脱的蛋白质在SDS-聚丙烯酰胺凝胶上进行电泳分离，通过化学发光反应在印迹上鉴定生物素化的蛋白质。β-内酰胺的生物素化快速、安全且廉价。我们的结果证明了使用生物素化β-内酰胺作为非放射性试剂研究PBPs及其纯化这些蛋白质的可行性。
