Test reagent
1. L-Leucine, ATP, DTT, tetrasodium pyrophosphate and HA-Ultrogel were purchased from Sigma, USA.
2. [32p]-Sodium pyrophosphate was purchased from DuPont, USA.
3. L-[I4C]-leucine and DEAE-SepharoseTM Fast Flow were purchased from Amersham, UK.
4. Restriction enzymes and T4 DNA ligase were purchased from MBI Ferments, Lithuania.
5. Plasmid pTrc-99B.
Experimental Materials
E. coli LeuRS was purified from highly expressed E. coli strains
Experimental procedure
1. Construction of mutant LeuRS gene
The gene sequence of six LeuRS variant enzymes was amplified by a two-step PCR reaction using the constructed gene leuS encoding E. coli LeuRS as a template.
5' and 3' primers for one round of PCR reactions: the upstream and downstream primers have NcoI and HindIII cleavage sites, respectively, and the primers used to construct each mutation are omitted. The PCR reaction product was digested with NcoI and HindIII, and inserted into the incision between the two restriction sites of NcoI and HindIII of the expression vector pTrc-99B. The correctness of the obtained LeuRS 6 variant enzyme genes was confirmed by DNA sequencing.
2. Expression and purification of LeuRS and its variant enzyme genes
A recombinant plasmid containing the gene of LeuRS and its six variants, LeuRS-E292K, LeuRS-E292F, LeuRS-E292S, LeuRS-E292D, LeuRS-E292Q, LeuRS-E292A, was transformed into E. coli strain TG1, and the correct plasmid was selected. The transformants were cultured in 5 mL of ampicillin-LB liquid medium, and the transformants were cultured at 37 ° C, shaking at 300 rpm to logarithmic growth phase, induced with 0.5 mM IPTG for 6 hours, 20 ul of the culture solution was taken out, and an equal volume of SDS-PAGE was added. The sample buffer was boiled at 100 ° C for 10 minutes, and the supernatant was centrifuged for SDS-PAGE to examine the expression of each variant enzyme gene in the transformant. 5 mL of high expression transformant was transferred to 500 mL of ampicillin-LB liquid medium at 37 ° C, and cultured at 300 rpm to logarithmic growth phase, and induced by 0.5 mM IPTG for 8 hours, and the cells were collected. The wet cells were suspended in 20 ml of 0 ° C pre-cooled destructurization buffer (50 mM potassium phosphate buffer, pH 6.8, containing 10 mM β-mercaptoethanol, 2 mM PMSF, 10% glycerol), ice bath Ultrasonic bacteria. The bacterial suspension was centrifuged at 12,000 g for 30 minutes at 4 ° C, and then continuously centrifuged at 1 ° C for 1 hour at 4 ° C. The supernatant was purified by DEAE-SepharoseTM Fast Flow and HA-Ultrogel two-step column chromatography. The linear elution gradient used in DEAE-Sephorose CL-6B fast flow purification was 50 mM to 500 mM potassium phosphate buffer, pH 6.8. The linear elution gradient used in the purification of HA-Ultrogel was a potassium phosphate buffer pH 6.8 of 20 mM to 300 mM. The collected elution peaks containing LeuRS and the variant enzyme were concentrated with PEG 20000 and dialyzed against 10 mM potassium phosphate buffer pH 6.8. Add 50% 0 ° C pre-cooled glycerol and store at -20 °C.
3. Determination of protein concentration
The crude pump protein concentration was determined by the Bradford method. The concentration of the purified LeuRS and the variant enzyme can be calculated by the following formula: 1 A280 = 1.62 mg/ml.
4. Determination of enzyme activity
The conditions for the amino acid activation reaction were as follows: 35 ul of the reaction solution containing 100 mM HEPES (pH 7.8), 10 mM KF, 10 mM MgCl 2 , 4 mM ATP, 2 mM [32P] PPi and 1 mM leucine, and incubated at 37 ° C. Start reaction at about 4 nmol/L LeuRS. At different times, 15 ul of the reaction solution was taken out, and 200 ul of quenching solution (3.5% perchloric acid, 2% activated carbon and 0.05 M sodium pyrophosphate) was added to stop the reaction in the quenching solution. Add 5 ml of 10 mM sodium pyrophosphate solution, filter the above solution with Whatman GF/C filter paper, collect the activated carbon powder adsorbing 32P-ATP, and wash it with 10 mM sodium pyrophosphate (2x5 ml) and 95% ethanol (2x5 ml). After draining and drying, the scintillation liquid is added, and the scintillation liquid is 0.6% 2-(4-tert-butylbenzene-5-(4-bisphenyl) 1,3,4 diazole [solvent is ethylene glycol methyl ether /toluene (volume ratio of 6/4)], the amount of 32P-ATP adsorbed by activated carbon powder is counted. The unit of amino acid activation reaction activity is defined as the amount of enzyme required to catalyze the formation of ATP per minute of PPi per minute under the measurement conditions. The conditions of the aminopurification reaction are as follows: 35 ul of the reaction solution containing 100 mM Tris-HCl (pH 7.8), 30 mM KCl, 12 mMMgCl2, 4 mM ATP, 0.1 mM EDTA, 0.5 mM DTT 20 uM tRNAleu, 0.1 mMC leucine was incubated at 37 ° C, and the reaction was started with about 5 nM LeuRS. 15 ul of the reaction droplets were taken on Whatman 3# filter paper at different times ((1.0 cm × 1.0 cm), after standing for 5 seconds, The paper was put into 5% trichloroacetic acid (containing 5 mM leucine), soaked at 0 ° C for 10 minutes, replaced with 5% trichloroacetic acid solution twice, and then soaked twice with 95% ethanol to dry the filter paper. PPO/POPOP scintillation fluid ((5 g PPO0.25 g POPOP dissolved in 1 L toluene), liquid scintillation counting. Amino deuteration reaction unit is defined as: 1 nanomolar leucine-tRNAleu per minute catalyzed under the conditions of the assay. The amount of enzyme required.
5. Determination of CD spectra and fluorescence spectra
The instrument used to determine the CD spectrum was a Jasco J-715 circular dichroism meter. The concentration of the protein sample in a 10 mM potassium phosphate solution (pH 6.8) was 0.2 mg/ml, the temperature was measured at room temperature, and the diameter of the cuvette used was 0.1 cm. The fluorescence spectrum was measured in Japanese Hitachi F-4010 fluorescence. The spectrophotometer was used to measure the emission spectrum with an excitation wavelength of 295 nm, a slit of 3 nm, an emission spectrum of 300-400 nm, and a fluorescent cup with an optical path length of 1 cm. The protein sample was in 10 mM potassium phosphate solution. The concentration in (pH 6.8) was 0.1 mg/ml.
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