High-throughput Quantitative Comparison Of Specific Activities Of Unpurified Fusion Enzyme/Mutants With Tag In Lysates And Its Application

After the6-His tag and the enzyme mutant fused and expressed, wecan take care of ELISA principle or magnetic separation principle on thebasis of a fixed amount of affinity separation medium by measuring themaximum adsorption capacity of affinity separation medium calculatingthe equivalent amount(Vs) of specific activities under high-throughputmodel to determine the specific activity of enzyme/mutants accuratelyand quantitatively.The method can effectively overcome the effects whichcaused by cloning inoculation quantity, expression of efficiency, cell lysisefficiency difference to measuer the specific activity in cell lysis solution,and avoid the occurrence of a false positive mutant，of course we can takeadvantage of magnetic separation technique to get this method application.Determining the specific activity of unpurified fulse enzyme/mutants inlysates is the most important. Also effectively avoid the challenge oftechnology, cost, and labor in the purification process of six–Histidine-tagged enzyme/mutants. The keys to this method are a suitablespecific activity of six–Histidine-tagged enzyme/mutants as a startingmaterial to design the mutant library, and a suitable tag and affinityseparation medium. In this article, firstly establish a method toquantitatively determine the specific activity of unpurified fulseenzyme/mutants in lysates. 1. The method of quantitatively determining the specific activity ofunpurified fulse enzyme/mutants in lysates.The experiments in wells or tubes use the principle of affinityseparation medium specificly binding with His-Tag of fulseenzyme/mutants.Fix the quantity of affinity separation medium inwells/tubes, the Maximal adsorption of protein is derictly related tobinding capacity. In theory, same medium and same binding capacity canresult to the same quantity of binding protein N. Now, the Vs is as themost valuable datas to compare specific activities of differentenzymes/mutants. When it achives the Maximal adsorption capacity (N),even we increase the quantity of protein in the wells/tubes, V is a fixvalue—Vs. However, it needs a large quantity of enzyme to reach the inreality. Maximal adsorption capacity and reaction is too fast to measure inreactive. Thus, by tracking the relationship of V and Quantity of totalprotein to calculate the specific activies with double reciprocal method andMatlab curve fitting method predicting the N. In experiment, we can useELISA principle or magnetic separation principle operating multiplemutations in multiple wells/tubes, achive high-throughput model. On thebase of balance between tags with affinity separation medium, this articlehas established a new analysis method. The method needn’t the purifiedprotein, the process itself is the purification, separation, determiningreaction speed Vs,that is the specific activity of pure enzyme in wells/tubeswhich is a non-purified cell lysates of the fusion activity of comparisonprovides a new line effective way. 2. Application (1)By total synthesis of the gene and mutants constructed, we get fuseenzyme of Est1/mutants M326Lwith6-His-Tag, the mcAb is as the affinitysparation medium by ELISA principle under High throughput model toinduced expression the enzyme/mutants, determine the proteinconcentration and apparent specific activities to compare the activitiesquantitatively. By using the double reciprocal method to predict the Vs, itcan get the activity ratio of Est1and M326L:3.0±0.2，CV≤10%（n≥5）,comparision of multi batch induced expression and Matlab normalityanalysis, get the ratio of two enzyme:3.0±0.5，CV≥18%（n1=25，n2=11）in250ml TB medium;3.2±1.3，CV≥40%（n1=122,n2=35）in4ml TBmedium. In this application, the quantity of antibody in wells is0.6ug, butthis condition must be fulfilled: the quantity of total protein is above200ug.The selectivity is high, but the lack of affinity, so, for enzyme mutantlibrary screening received the sample volumeand cost constraints. Ofcourse, the sample in this method is cell lysate supernatant; this methodincrease accuracy of the relative, at the same time, also makes protein neednot be purified in the complex process, especially suitable for enzymewhich stability is sensitive with temperature, surfactant metal ion. Even ifthe induced expression efficiency of enzyme/mutants appares more than5times, only if Vmin≥0.090and Vmax≤1.800, mcAb binding rate is above40%, the R2≥0.95of double reciprocal plot, the method can determine theVs accurately and CV≤10%.3. Application (2)In the experiment of the ECAP and mutant R167K as the applicationmodel, the fulse tag is also6-His-Tag. ECAP and mutant R167K afterpurified by Ni-NTA, which is a very useful method, can get the ratio ofECAP and mutant R167K accurately is1.75. By using the method which is established in this aiticle, with the Matlab curve fitting method, it canget the ratio of ECAP and mutant R167K is1.8，CV≤10%(n=3)，R2≥95%,with the ratio of purified tag fusion enzyme and its mutant specificactivity is less than3%. Comparision of multi batch induced expressionand Matlab normality analysis, get the ratio of two enzyme is1.2±0.3,CV≥20%（n1=n2=13）in4ml TB medium. Further proves that the method isheavy meaning to compare specific activity of fulse enzyme/mutants in thecell lysate without purification. In this application, the Ni-NTA magneticbeads have been used as the affinity separation medium. We can base onthe specific activity of enzyme/mutants to adjustment the quantity ofmagnetic beads or the time of ranction to control the reaction speed. Whenthe quantity of magnetic beads is25ug, only if the quantity of total proteinis from1ug to20ug, it can effectively predict the maximum adsorptioncapacity (N) of EP tubes and avoid the effect of adsorption and separationof complex protein. Of course, one point to note is the: double reciprocalplot application condition is n≥20N. In this application, the quantity oftotal protein is too low to meet the double reciprocal method analysis ofthe data requirements, thus replaced by Matlab curve fitting method. It isof great significance. When the Ni-NTA magnetic beads are used as theaffinity separation medium, the whole experiment process is only2h, it’sso short than the mcAb’s26h. Reduce the test time, even more sensitive tothe temperature of the enzyme, can also be used. So it has much strongerposition.The key of applicating the method in establishment andhigh-throughput screening mutant library is designed for the initialenzyme activity of peptide tag fusion enzyme as a mutant library building.According the activity to select fixed amount of affinity medium, sample protein appropriate dilution and the suitable reaction time is extremelyimportant. Due to the much higher affinity of Ni-NTA beads than mcAb,but it is less selectivity than mcAb, so in the experiemental process,protein contents more need careful control. The whole operation,confirmthe adsorption reaction of Ni-NTA beads is more suitable for mutantscreening, but not suitable for the mutant library.In these applications, itcan be effectively overcome the efficiency of different clone inoculationquantity, induced expression efficiency, cell lysis efficiency to determinespecific activity of enzyme/mutants. Try to avoid the false positivemutants can be performed in a high-throughput mode. Provide a fast,efficient, accurate method to obtain high activity mutant and mutantlibrary and screening.