PAAS As A Label Enzyme For SDESA-ELISA And Its Directed Evolution System

ELISA is widely used in clinical laboratory analysis, health inspection and biomedical researches, but each time classical ELISA couLd only measure one-component. Through the swift switch of measurement wavelength, two-wavelength absorbance can be measured for spectrophotometric-dual-enzyme-simuLtaneous-assay (SDESA). ELISA via SDESA was denoted SDESA-ELISA, showing doubled analysis efficiency. In microplate readers, standard filters enbale the assay of absorbance at 405 and 450 nm; the suitable pair of label enzymes and their proper pair of chromogenic substrates shouLd meet the following prerequisites:(1) the maximum isoabsorption wavelength of substrate A under the action of enzyme A was about 405nm while its maximal difference absorption peak was close to 450nm, the maximum difference absorption peak of product B under the action of enzyme B was about 405nm; (2) the paired label enzymes have compatible buffers and sufficiently high specific activity; (3) The paired label enzymes have high stability; (4) the interference with enzymatic activities by substrates and products involved was negligible. After Screening, 4-nitro-1-naphthylphosphate (4NNPP) served as substrate A. The activity and stability of Escherichia coli alkaline phosphatase (ECAP) made it suitable as enzyme A. A suitable enzyme B is mandatory. Limited by the optimum pH, only acetylcholine esterase (AChE) and Pseudomonas aeruginosa arylsuLphatase (PAAS) were candidates for enzyme B. In this project, the two pairs of enzymes with ECAP were compared for SDESA, followed by the development of a directed evolution system for molecuLe engineering of PAAS.1. Characterization of SDESA of ECAP and AChEThe maximum difference absorption peak of 4-Nitro-l-naphthyl (4NNP) as 4NNPP product was about 450nm and the maximum isoabsorption wavelength was about 405nm. AChE catalyzes acetylthiocholine chloride (ATCh) hydrolysis to release 2-nitrobenzoic acid (NTB) from 5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB); the maximum difference absorption peak of NTB was about 410nm. Specific activities and stability of ECAP and AChE in the same basic Tris-HCl buffer were suitable. For ECAP and AChE, SDESA showing sensitivity, detection limit, linear range consistent with separate assay. However, ATCh has poor stability, which prevents the production of open-to-use reagents for clinical laboratory analysis.2. Characterization of SDESA of ECAP and PAASThe maximum difference absorption peak of the product of PAAS action on potassium 4-nitrophenylsuLfate (4PNS) was about 405nm. ECAP and PAAS in alkaline DEA-HCl buffer exhibited optimum specific activity. For ECAP and PAAS, SDESA showing sensitivity, detection limit, linear range consistent with separate assay. Their chromogenic substrates had better ATCh of spectral properties and stronger stability. The highest specific activity of the wild type PAAS was only 39 kU/g, and its half-life at 37℃ was only four days in Tris-HCl buffer. Activities of PAAS mutants need to be improved by more than 20-fold and more than 85% of their activities shouLd be reserved after 15 days at 37℃. MolecuLar engineering of PAAS is mandatory for its application to for SDESA-ELISA.3. A directed evolution system of PAASAccording the reported sequence, the encoding gene was inserted into pET28a vector for induced expression in Escherichia coli BL21 (DE3). Mutagenesis of the C-terminus of PAAS was performed to develop a directed evolution system via error-prone PCR (ep-PCR). Optimizations of the concentrations of Mg2、Mn2+、Tm and so on, for ep-PCR followed by ligation, and transformation, those mutated PASS were induced for expression in E. coli to give a library of PAAS mutants. Accordingly, the high-throughput screening system was developed as follows.(1) 48-well plates for high-throughput induced expression. The intra and inter-assay coefficient of variation were about 20%, mainly due to the unavoidable difference in the growth of bacteria on LB plates;(2) Cells were lyzed with an alkaline buffer. The main component of the lysis buffer was Tris-HCl (pH9.0 1.0M), the auxiliary components included Tween-20 (1/1000) for destruction of cells and 4-amino benzamidine (2u M) for limitation of degradation. The cell suspension after induced expression and the alkaline lysis buffer was mixed at 8:1 ratio for lysis at room temperature for 3 to 6 hours.(3) Enzyme activities of the alkaline lysates were measured with a microplate reader in the high-throughput 96-well mode;(4) A threshold for a positive mutant was proposed, by receiver operator curve analysis (ROC) of the two-fold difference between activities of PAAS mutants M72Q and G138S. According to the area under the curve (AUC), the cutoff corresponding to 90% sensitivity was used as the threshold for positive mutants.