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

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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 the cloning vector for gene expression.

Experimental Materials

E. coli LeuRS was purified from highly expressed E. coli strains. The purification process involved multiple chromatographic steps to ensure high purity and activity of the enzyme.

Experimental Procedure

1. Construction of Mutant LeuRS Gene

The gene sequence encoding six variants of LeuRS was amplified using a two-step PCR method, with the wild-type leuS gene from E. coli serving as the template. Primers containing NcoI and HindIII restriction sites were used in the first round of PCR. The amplified products were digested with these enzymes and cloned into the expression vector pTrc-99B. The correctness of each variant was confirmed by DNA sequencing.

2. Expression and Purification of LeuRS and Its Variants

The recombinant plasmids, including LeuRS and its six variants (LeuRS-E292K, LeuRS-E292F, LeuRS-E292S, LeuRS-E292D, LeuRS-E292Q, LeuRS-E292A), were transformed into E. coli strain TG1. After selection on ampicillin-containing LB medium, the correct clones were cultured in 5 mL of liquid medium. Induction with 0.5 mM IPTG was performed to express the proteins. SDS-PAGE was used to analyze the expression levels. For large-scale purification, 500 mL cultures were induced and harvested. The cells were lysed using sonication, and the supernatant was subjected to DEAE-Sepharose™ Fast Flow and HA-Ultrogel column chromatography. The purified protein was concentrated using PEG 20000 and stored at -20°C in 50% glycerol.

3. Protein Concentration Determination

The concentration of crude and purified proteins was measured using the Bradford assay. The purified samples were also quantified using absorbance at 280 nm, where 1 A₂₈₀ corresponds to 1.62 mg/mL.

4. Enzyme Activity Assays

The amino acid activation reaction was carried out under specific conditions, including 100 mM HEPES (pH 7.8), 10 mM KF, 10 mM MgCl₂, 4 mM ATP, 2 mM [³²P]PPi, and 1 mM leucine. The reaction was initiated with 4 nM LeuRS, and samples were taken at different time points to measure the incorporation of radiolabeled ATP. The aminoacylation reaction was analyzed similarly, using tRNA^leu and measuring the formation of leucyl-tRNA. The activities were defined based on the amount of substrate converted 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 using a 0.1 cm path-length cuvette. The protein concentration was 0.2 mg/mL in 10 mM potassium phosphate buffer (pH 6.8). Fluorescence spectra were measured using a Hitachi F-4010 fluorometer. Excitation at 295 nm and emission between 300–400 nm were recorded, with a sample concentration of 0.1 mg/mL in the same buffer. These techniques were used to assess the secondary structure and conformational changes of the protein.