Effect of E292 of Escherichia coli leucine-tRNA synthetase on aminopurification activity - Master's thesis - Dissertation

Probe domestic high current needle, 150 head diameter, 3.0 thread needle, high current needle total

Experimental Reagent

1. L-Leucine, ATP, DTT, tetrasodium pyrophosphate, and HA-Ultrogel were purchased from Sigma, USA.

2. [³²P]-Sodium pyrophosphate was obtained from DuPont, USA.

3. L-[¹⁴C]-leucine and DEAE-Sepharose™ Fast Flow were sourced from Amersham, England.

4. Restriction enzymes and T4 DNA ligase were acquired from MBI Ferments, Lithuania.

5. Plasmid pTrc-99B was used as a vector for gene cloning and expression.

Experimental Materials

E. coli LeuRS was purified from highly expressed E. coli strains.

Experimental Procedure

1. Construction of the Mutant LeuRS Gene

The gene sequences of six LeuRS variants were amplified using a two-step PCR method with the wild-type leuS gene encoding E. coli LeuRS as the template. The forward and reverse primers contained NcoI and HindIII restriction sites, respectively, to facilitate cloning into the expression vector pTrc-99B. After digestion with NcoI and HindIII, the PCR products were inserted into the corresponding sites in the vector. The resulting plasmids were verified by DNA sequencing to confirm the correct mutation.

2. Expression and Purification of LeuRS and Its Variants

The recombinant plasmids containing the LeuRS gene and its six variants (LeuRS-E292K, LeuRS-E292F, LeuRS-E292S, LeuRS-E292D, LeuRS-E292Q, LeuRS-E292A) were transformed into E. coli strain TG1. Transformed cells were grown in ampicillin-LB medium, induced with IPTG, and then harvested. The proteins were extracted, centrifuged, and purified using DEAE-Sepharose™ Fast Flow and HA-Ultrogel column chromatography. The purified proteins were concentrated, dialyzed, and stored at -20°C for further analysis.

3. Protein Concentration Determination

The crude protein concentration was determined using the Bradford assay. For purified samples, the concentration was calculated based on A₂₈₀ absorbance: 1 A₂₈₀ = 1.62 mg/ml.

4. Enzyme Activity Assay

Enzymatic activity was measured under specific conditions. For amino acid activation, the reaction mixture included HEPES buffer, MgClâ‚‚, ATP, and radioactive PPi. The reaction was stopped at different time points, and the amount of incorporated radiolabeled ATP was quantified using scintillation counting. For aminoacylation, tRNA and leucine were used, and the formation of leucyl-tRNA was monitored using filter paper and liquid scintillation counting. The enzyme activity was defined as the amount of product formed per minute per nanomolar of enzyme.

5. CD and Fluorescence Spectroscopy

Circular dichroism (CD) spectra were recorded on a Jasco J-715 spectrometer at room temperature. Fluorescence emission spectra were measured using a Hitachi F-4010 fluorometer, with excitation at 295 nm. These techniques provided insights into the secondary structure and conformational changes of the protein during the enzymatic process.