Data for high-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins

Data in this article are associated with the research article “Highthroughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins” (Yang et al., 2017) [1]. This article provided data on how to develop an immunoturbidimetric assay (ITA) of enzyme/mutants as proteins in cell lysates in high-throughput (HTP) mode together with HTP assay of their activities to derive their specific activities in cell lysates for comparison, with Pseudomonas aeruginosa arylsulfatase (PAAS) and Bacillus fastidious uricase (BFU) plus their mutants as models. Data were made publicly available for further analyses.


Specifications
graph, figure How data was acquired Biotek ELX 800 and BIOTEK EON microplate readers to record the adsorption for activity assay, and to record the extinction of ITA complex for selective quantification of protein in 96-well plates Data format Raw and analyzed Experimental factors Interferences found from denaturated proteins in medium, which are eliminated through filtration of cell lysates through 0.22 μm membrane Experimental features Specific activities of enzyme/mutants in cell lysates based on ITA of their proteins with one of the purified enzyme/mutants as the reference protein were compared with those of purified enzyme/mutants by directed assay Data source location Chongqing Medical University, Chongqing 400016, China Data accessibility Data are available with this article

Value of the data
Supporting the validity of ITA of a group of enzyme/mutants as proteins in cell lysates to derive their specific activities for comparison.
Supporting much higher reliability to recognize a positive mutant of 50% higher activity by the comparison of specific activities based on ITA of enzyme/mutants as proteins in cell lysates than the comparison of other activity indices.
Supporting the incomparable advantage of cost and labor for the elucidation of sequence-activity relationship of an enzyme.

Data
The data in this article provides information on how to develop an experimental system to determine specific activities of enzyme/mutants in cell lysates in HTP mode based on ITA of enzyme/ mutants as protein and HTP assay of activities (Figs. 1-7 and Tables 1-25). Data were provided for the validity of the proposed strategy, the performance to recognize the positive mutant in each random pair of PAAS/mutants during HTP screening, and the efficacy to elucidate sequence-activity relationships of both BFU and PAAS in HTP mode (Fig. 8, Fig. 9 and Table 26). Purification of polyclonal antibodies analyzed by SDS-PAGE. 1. proteins in antisera; 2. proteins in the precipitate by 33% ammonia sulfate at 4°C, yield 20%; 3. the dissolved precipitate after DEAE-cellulose chromatography at pH 6.5, yield 25%; 4. BSA; 5. Proteins in the supernatant of 33% ammonia sulfate (application data in Table 12).    Table 13, standard error of estimate was about 0.013 for PAAS from 0.2 to 2.4 μg (a), but was as large as about 0.056 for PAAS from 0.2 to 4.0 μg (b)).  Table 14, standard error of estimate was about 0.003 for fitting with a quadratic function (a), but was about 0.004 for fitting with a linear function (b), for BFU from 0.20 to 3.0 μg).

Experimental design
The proposed strategy requires consistent accessible epitopes among enzyme/mutants for the use of just one reference [1].
The actions of PAAS/mutants and BFU/mutants do not alter cell growth and they should have consistent abundance among intracellular proteins after induced expression under the same conditions. They served as two models to test the validity of the proposed strategy.
The abundance of an enzyme/mutant based on ITA was the ratio of its protein quantity determined by ITA to that of total proteins; the abundance of an enzyme/mutant by activity was the ratio of its apparent specific activity to its specific activity after careful purification.
For a group of PAAS/mutants or BFU/mutants, they should have consistent accessible epitopes when their abundance based on ITA with just one reference is consistent with each other and further consistent with those by activities.

Materials and methods
All experiments involving rabbits were approved by the Animal Care and Use Committee of Chongqing Medical University (China).
For each enzyme/mutant, a individual clone was amplified at 37°C and 180 rpm, for 12 h with 0.50 mL of the medium in 48-well microplates, but for 4.0 h with 4.0 mL or 250 mL of the medium in glass bottles. Each enzyme/mutant was induced by 1.0 mM isopropyl-β-D-thiogalactoside at 16°C and 110 rpm for 20 h. Cells were broken by sonication treatment in 20 mM Tris-HCl at pH 8.0; 0.50 mL of the cell lysate after centrifugation at 10,000 rpm for 15 min was filtered through 0.22-μm membrane to serve as a sample for both ITA and activity assay [1,2].
With PAAS/mutants, 2.0 mM potassium 4-nitrophenylsulfate was used in 1.0 mM Tris-HCl at pH 9.0 to measure the absorbance at 405 nm with Biotek ELX 800 [1,3]. With BFU/mutants, 0.14 mM uric acid was employed in 0.20 M sodium borate at pH 9.2 to measure absorbance at 293 nm with Biotek EON [1,4]. Their initial rates were derived from absorbance change from 10.0 to 15.0 min since reaction initiation [1]. The apparent specific activity was the ratio of the activity to the quantity of total proteins in an unpurified sample.
ITA quantified the difference of the extinction at 370 and 700 nm for reaction mixture of 0.20 mL in 96-well microplates with Biotek EON.
Ratios of their specific activities were calculated with the specific activities of 14.6, 4.3, 3.4, 2.3 kU/ g for PAAS, M72Q, G138S and M72D, respectively. The four enzymes after affinity purification once were concurrently assumed to have the purity of 48%, to correct the effects of purity on their specific activities. BIOTEK ELX800 reader (data from 10 to 15 min).

Table 2
Deviations between systems for M72D activity assay and the correction of its specific activity.  Table 3 Deviations between systems for M72Q activity assay and the correction of its specific activity.

M72Q
MAPADA UV 1600 spectrophotometer (data within 1.0 min) MAPADA UV 1600 spectrophotometer (data from 10 to 15 min) The ratio of rate within 1.0 min to that from 10 to 15 min, all by MAPADA UV 1600 spectrophotometer Biotek ELX 800 microplate reader (data from 10 to 15 min)  Table 4 Deviations between systems for G138S activity assay and the correction of its specific activity.

G138S
MAPADA UV 1600 spectrophotometer (data within 1.0 min) MAPADA UV 1600 spectrophotometer (data from 10 to 15 min) The ratio of rate within 1.0 min to that from 10 to 15 min, all by MAPADA UV 1600 spectrophotometer Biotek ELX 800 microplate reader (data from 10 to 15 min)  The enzyme was purified by Ni 2 þ -NTA column and buffer was pre-incubated at room temperature for 30 min prior to use. Specific activity by BIOTEK ELX800 microplate reader with just 0.20 mL reaction mixture at room temperature; initial rate was determined with data from 10 to 15 min after agitation for 5 min.

Table 7
Correction of the specific activities of BFU/mutants.
After correction of purity Before correction of purity Specific activity of BFU was assumed to 9.0 kU/g to approximate its purity after DEAE-cellulose chromatography twice and such purity was assigned to that of other mutants for the correction of their specific activities. Activities were determined with BioTek Eon by absorbance of uric acid at 293 nm.   There were about additional 14 μg host proteins with the sample of 3% abundance of PAAS in comparison of that with the sample of 50% abundance. The background with lysates of untransformed cells gave A 340-700 of 0.473. And t-test indicated insignificant differences for the same quantities of PAAS but different abundance in artificial cell lysates, as indicated as *. There were additional 14 μg host proteins with the sample of 3% abundance of PAAS in comparison of that with the sample of 50% abundance of PAAS. The background with lysates of untransformed cells gave A 340-700 of 0.310. And t-test indicated insignificant differences for the same quantity of PAAS but different abundance in artificial cell lysates, which was indicated as *.               a Number in parenthesis indicated independent lysates, and all samples suitable for ITA were analyzed. For each enzyme/ mutant, paired t-test comparison supported no difference for the abundance derived from activity and by ITA. There were no differences among abundance derived from activity or by ITA in each group of enzyme/mutants under stated conditions. b Each specific activity after purification was expressed in kU/g, with CVo 15% from three independent preparations of each enzyme. c All the cell lysates prepared from 4.0 or 250 mL medium for the amplification of cells and induced expression were included for analyses.

Table 25
Associations of specific activities based on ITA with those determined after purification.