磁珠固定多抗预测最大吸附活性识别阳性突变体

Prediction of maximum absorption activities with polyclonal antibodies immobilized on magnetic submicron particles to recognize positive mutants

目的考察磁珠固定多抗吸附细胞裂解液中酶/突变体,分析吸附酶活性对样品蛋白量响应曲线预测最大吸附活性(Vs)用于比较识别阳性突变体的可靠性。方法纯化重组表达大肠杆菌碱性磷酸酶(ECAP)及绿脓杆菌芳香硫酸酯酶(PAAS)为免疫原;兔抗血清用33%硫酸铵分级和DEAE-纤维素层析纯化得多抗。磁珠羧基活化后固定多抗;用对硝基酚显色底物,碱终止后测定405nm吸收增量反映酶活性;分析吸附响应曲线预测Vs。结果多抗磁珠吸附酶的容量约2.0mg/g磁珠。ECAP比活性超过PAAS比活性70倍。ECAP突变体R168K较野生型比活性提高约50%,用2.5μg多抗磁珠反应10min即可预测Vs识别此阳性突变体;用PAAS突变体G138S为起始酶,用10μg多抗磁珠测定吸附酶反应20min后吸收预测Vs,能识别活性提高20%以上的PAAS弱阳性突变体。结论只要被吸附酶活性能可靠测定,优化磁珠固定多抗的用量能预测低活性酶/突变体Vs进行比较识别阳性突变体。

Objective To observe polyclonal antibodies immobilized on magnetic submicron particles （MSP） as affinity adsorbents and test the reliability of predicted maximum adsorption activity of an enzyme/mutant from a cell lysate （Vs） in recognizing positive mutants. Methods Escherichia coli alkaline phosphatase （ECAP） and Pseudomonas Aeruginosa arylsulfatase （PAAS） were purified by affinity chromatography to serve as immunogens for the preparation of their antisera, which after fractionation by 33% ammonium sulfate and DEAE-cellulose chromatography yielded the respective polyclonal antibodies. After activation of COOH on MSP, polyclonal antibodies of each enzyme were immobilized to give MSP-polyAb. Activities of an adsorbed enzyme were measured with a chromogenic substrate of 4-nitrphenol by determining absorbance at 405 nm after the termination of reaction by alkali. Based on the response curve of activities of the adsorbed enzyme to protein quantities of a lysate, Vs was predicted for comparison. Results The maximum adsorption quantity of ECAP or PAAS on the respective MSP- polyAb was about 2.0 mg/g. Specific activity of ECAP after affinity purification was about 70-fold of that of PAAS. ECAP mutant R168K showing about 50% activity improvement versus ECAP was recognized by comparison of Vs predicted with only 2.5 μg of MSP-polyAb; with PAAS mutant G138S as the starting one, the use of 10.0 μg of MSP-polyAb to predict Vs recognized the mutants bearing more than 20% activity improvement. Conclusion With an optimized quantity of MSP-polyAb to predict Vs, weak positive mutants of an enzyme of low activity can be recognized when activities of the adsorbed enzyme/mutant are reliably measured.