Polyclonal Antibodies Used To Compare The Specific Activities Of Enzyme/Mutants In Lysates

Molecular engineering of enzymes is a focus in biotechnology and primarily utilizes rational design of mutants and directed evolution.In general,rational design of mutants is technically challenging and requires huge computation resource.Differently,directed evolution through the generation of mutant libraries with a starting enzyme is more practical,but tolerates heavy burden on the screening of mutants to recognize positive ones.In theory,comparison of specific activities after purification is plausible to recognize positive mutants,but which prevented by the cost and time for purification of mutants.It seem feasible to compare activities or apparent specific activities of mutants in cell lysates after induced expression,which suffers large uncertainly due to the dynamic abundance of mutant proteins among intracellular proteins after induced expression.Clearly,new strategies for reliable recognition of positive mutants are needed.In fact,anti-6His monoclonal antibody immobilized on microplate wells react with carboxylesterase(Est1)and its mutants,as well as the Ni2+-NTA magnetic particles react with E.coli alkaline phosphatase(ECAP)and its mutant R168 K,have been shown to be effective in predicting Vs to identify positive mutants,which has been patented in China..Unfortunately,microplate immobilized monoclonal antibodies have a narrow range of applications and insufficient for predicting Vs of 6His-carboxyesterase and its mutants.In addition,Ni2+-NTA magnetic particles can inhibit the activity of Pseudomonas aeruginosa arylsulfatase(PAAS)and mutants,and have higher requirements for abundance in cell lysates.Predicting Vs requires the affinity and selectivity of the affinity adsorbent for the starting enzyme is sufficiently high,do not interfere with the determination of the activity,and do not depend on the expression of the fusion enzyme.Therefore,the polyclonal antibody as an affinity adsorbent has been improved in terms of binding affinity,application range,cost and time-consuming.1.Use of microplate-immobilized polyclonal antibodies to predict maximum activities of adsorbed enzyme/mutants from cell lysates for recognition of positive mutantsIn the experiment of ECAP and its mutant R168 K as the application model,the anti-ECAP rabbit antiserum precipitated with 33% ammonium sulfate to get crude polyclonal antibody,purified polyclonal antibody was called when DEAE cellulose column purified.According to the aforementioned patented method,crude polyclonal antibody immobilized on microplate wells;ECAP and mutant R168 K cell lysates as samples,the active concentrations and protein concentrations are measured and then activities of adsorbed enzymes and their mutants can be compared.In sample lysates after induced in 4.0 ml culture medium,the activity of ECAP reached 19.7 ± 5.7 KU/g(n = 12)while that of mutant R167 K was 36.7 ± 15.1 KU/g(n = 12),giving a ratio of(1.9 ± 0.3).This ratio of activity was much larger than that after affinity purification twice(P< 0.01).With the same batch of the crude polyclonal antibodies against ECAP,the ratio of Vs for ECAP and mutant R168 K estimated by fitting with Matalab was 1.6 ± 0.1(n=2),while reached 1.5 ± 0.1(n=2)by regression analysis.The ratios of Vs for ECAP and its mutant R168 K by the two approaches for data processing were consistent with each other,and further consistent with that after affinity purification or by using Ni2+-NTA to predict Vs,Which supported the reliability of Polyclonal antibodies as affinity adsorbents to predict Vs.2.Use of MSP with polyclonal antibodies to predict specific activities of adsorbed enzyme/mutants from cell lysates6His-tagged ECAP and 6His-tagged PAAS were take as model,their antiseras were fractionated by 33% ammonium sulfate before DEAE-cellulose chromatography to yield purified polyclonal antibodies.After the activation of COOH on MSP,the conjugation of polyclonal antibodies of each enzyme to MSP gave MSP-polyAb for the adsorption of ECAP/mutants or PAAS/mutants.Activities of the adsorbed ECAP/mutant were measured with a chromogenic substrate of 4-nitrphenol to determine absorbance at 405 nm after the termination of reaction by alkali.From the response curve of activities of the adsorbed enzyme to protein quantities of a sample lysate,Vs was predicted for comparison.assuming the consistent activity of the adsorbed ECAP and free ECAP,the maximum adsorption quantity of ECAP on the respective MSP-polyAb reached about 2.0 mg/g.ECAP after affinity purification had specific activity of about 70-fold of that of PAAS.ECAP mutant R168 K showing about 50% improvement of activity to ECAP was recognized by predicting Vs with just 2.5 μg;MSP-polyAb;with PAAS mutant G138 S bearing about 25% activity of PAAS as the starting one,the use of 10.0 μg MSP-polyAb to predict Vs successfully recognized the respective mutants bearing more than 20% improvement of activity.With MSP-polyAb as the affinity adsorbents to predict Vs,it was practical to recognize weak positive mutants of an enzyme bearing low activity,when activities of the adsorbed enzyme were measurable.3.Immunoturbidimetric assay of an enzyme and its mutants in cell lysates for facile estimation of their specific activitiesWith PAAS and Bacillus fastidious uricase(BFU)plus their mutants as models,the derivation of specific activities of an enzyme and its mutants in cell lysates based on immunoturbidimetric assay(ITA)of their proteins was tested.With a microplate reader to measure the extinction at 340 and 700 nm of 0.20 mL reaction mixtures containing 0.75 mg anti-serum,ITA quantified PAAS or BFU from 0.6 to 2.4μg according to calibration curve.There was consistent abundance of PAAS/mutants in cell lysates after induced expression under the same conditions,supporting their consistent immunological reaction to the same anti-serum;so was the abundance of BFU/mutants.Specific activities of PAAS/mutants or BFU/mutants in cell lysates based on ITA of proteins showed excellent proportionality to those routinely determined after purification.To recognize the one of 50% higher activity in a pair of PAAS/mutant(s),ROC analysis of specific activities in cell lysates based on ITA of proteins after induced expression in 48-well microplates gave area-under-curve much higher than those by ROC analyses of other activity index.ITA of enzyme/mutants in cell lysates was thus promising to derive their specific activities for verifying positive mutants and elucidating sequence-activity relationship.