Cloning, Expression, Purification And Characterization Of Arginine Kinase From Locust

Arginine kinase (AK, ATP: L-arginine N-phosphotransferase; EC 2.7.3.3) is a member of what appears to be a highly conserved family of phosphotransferases that reversibly catalyze the transfer of phosphate from phosphoarginine to ADP, yielding ATP. AK is distributed in invertebrates and its function is similar to creatine kinase (CK) in vertebrates. Phosphoarginine plays a critical role as an energy reserve because it can be transferred to ATP when energy is needed.In the paper, the gene of AK was frist cloned, expressed, purified. And then, the characterization, enzymatic mechanism,substrate synergism, the transcription regulatory model, the function of promoter and the influence of single mutant on the activity, structure and stability of AK were studied so that we could provide clues to the control of insect pests. The following results were achieved:1. The cloning of AK gene. Two degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from locust muscle according to the homologous sequences from other invertebrates. The middle fragment of interested cDNA was obtained by RT-PCR. The 5'and 3'fragment of the cDNA was isolated by 5'and 3'RACE. The clone, which named LmmAK (Accession Number: DQ513322), contains 2014 bp nucleotides with an open reading frame (ORF) of 1068 bp comprising 355 amino acid residues with the predicted molecular mass of 40 kD. Amino acid sequence alignment revealed that arthropod AKs contained two domains. One is the conserved GS region, in which Y75 and P272 represent two important conserved amino acids of AK playing important role in substrate synergism, the other one is signature sequence including"C-P-(S/T)-N-(I/L)-G-T", conserved amino acids of ATP-guanidino phosphotransferases.2. The over-expression and purification of AK. The gene encoding Locusta migratoria manilensis AK was expressed in Escherichia coli by two prokaryotic expression plasmids, pET-30a and pET-28a. The recombinant protein was expressed as inclusion bodies using pET-30a. After denaturation, the recombinant AK was successfully renatured and confirmed to be enzymatically active. Addition of Tween-20 and SDS to the dilution system led to higher renaturation efficiency. Using another expression plasmid, pET-28a, and changing the expression conditions resulted in a soluble and functional form of AK, which was purified by an improved method using Sephadex G-75 chromotography to a final yield of 90 %.3. Properties of recombinant AK. Some parameters for the renatured and soluble forms of AK, including Km, Kd, specific activity, electrophoretic mobility and isoelectric focusing, were identical with those of AK obtained directly from L. migratoria manilensis leg muscle. Comparison of kinetic constants with those of AKs from other sources indicated that L. migratoria manilensis AKs have the highest kcat and stronger synergistic substrate binding.4. Amino-acid residues those are responsible for substrate synergism of AK. A series of mutants were constructed to investigate the amino-acid residues responsible for the synergism in substrate binding of arginine kinase (AK). The Y75F and Y75D mutants showed strong synergism (Kd/Km = 6.2-13.4), while in single mutants, P272G and P272R, and a double mutant, Y75F/P272G, the synergism was almost completely lost (Kd/Km = 1.1-1.4). Another double mutant, Y75D/P272R, had characteristics similar to those of the wild-type enzyme. All these results suggest that the amino-acid residues 75 and 272 play an important role in regulating the synergism in substrate binding of AK. All the results provided direct evidence that there is a subtle relationship between the synergism in substrate binding and the conformational change.5. Amino acid residue P272 is involved in AK activity, structure and stability. Our studies revealed that this residue is related to the structural stability and activity, though it is not located in the active site. When the structure of AK was impaired by mutation, AK was in a partially unfolded state with more hydrophobic exposure, which was prone to aggregate under environmental stresses. Mutation at this position influences transition from the molten globule intermediate to the native state in folding process. The results provided herein may suggest that some residues near the active site may play a relatively important role in keeping AK activity and structural stability6. Expression regulatory model of AK. The level of enzyme protein, arginine kinase-specific activity and mRNA was evaluated by western blot analysis, enzyme activity assay and northern blot analysis. The results indicate that enzyme activity and enzymatic protein levels increase continuously during the growth, the enzyme-specific activity remains constant, mRNA levels show minor changes indicating a post-transcriptional regulation. The results suggest that the regulation of this gene expression in the locust is mostly exerted at the post-transcriptional level, including mRNA maturation, stability and translation.