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Preparation of Recombinant Metallo-β-lactamase and Development of Its Rapid Test Strip
Author: HanZuo
Tutor: XuZhiNan
School: Zhejiang University
Course: Biological Engineering
Keywords: metallo-β-lactamases CcrA enzyme activity assay fast test paper
CLC: O629.8
Type: Master's thesis
Year: 2014
Downloads: 4
Quote: 0
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Abstract
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Metallo-B-lactamases (MBLs) require metal ions such as Zn(Ⅱ) to hydrolyze the B-lactam ring of all currently available B-lactam-based antibiotics, resulting the lose of drug activity. However, the enzymatic analyses of MBLs so far lack the pertinence; the general methods applied right now are referred from the iodimetry analysis for the enzymatic determination of penicillinase.Objective To establish the expression system of CcrA in E.coli BL21and subsequent purification method. After achieving the high pure MBL-CcrA, its activity has been first evaluated via the iodimetry method, then the hydroxylamine method was applied and optimized to determine the activity of MBL-CcrA, and finally a simple, convenient and stable fast test paper for MBLs activity could be designed.Methods The plasmid pET21a/d-CcrA was transformed into E.coli BL21to construct the recombinant expressing strain. After over-expression via induction of IPTG and breaking cells via ultrasonication, the resulting protein was purified using Q-Sepharose Column and dialyzed. The purified enzyme was identified by SDS-PAGE analysis and its activity was assayed by iodimetry using different batches of protein as the parallel. Then the activity of MBL-CcrA was also determined through the hydroxylamine method. The effects of various factors on the experimental results were investigated and the reaction conditions were optimized. Based on these results, the fast test paper for MBLs activity as well as the related colorimetric card were designed.Results The MBL-CcrA was successfully expressed in E.coli BL21. By using the gradient NaCl solution from0to500mM, the expressed protein was eluted from the Q-Sepharose Column with high purity, which was further identified via SDS-PAGE analysis. After ultrafilitration, the51.4Kda MBL-CcrA was respectively analyzed by the iodometry and hydroxylamine methods. The comparison indicated that the bias of iodometry was more obvious, and the limitation of such method also including the troublesome operation, time consuming (about5h), lacking the specificity and possessing various interferences(keeping away from light, the titration end determination rely on personal judgment, etc.). On the contrary, the results measured by hydroxylamine within less than1.5H was more reliable (R2=0.9976), and using meropenem as the substrate greatly increased the specificity of method, avoiding many influence factors. Moreover, the definition of MβLs activity was redefined in the study, which shall be more intuitive and simple. According to the relationship between the results of color reactions and the enzyme activity derived from the hydroxylamine method, the fast test paper for MβLs activity as well as the related colorimetric card were designed, which make the operation more convenient and could facilitate the practical applications.Conclusions1, the expression system of MβL-CcrA and related purification method were established, and the pure enzyme with high concentration and activity was obtained. The intuitive and simple definition of MβLs activity was redefined in the study.2、The practical and simple fast test paper for semi-quantitative determination of MβLs activity via the hydroxylamine method was designed in this study.
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