Structure-function characterization of a recombinant aspartic proteinase from Arabidopsis thaliana



Mazorra-Manzano, Miguel Angel

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University of Guelph


An investigation of the expression, purification and structure-function characterization of the recombinant aspartic proteinase AtAP A1 from ' Arabidopsis thaliana' was undertaken. The cDNA encoding the precursor of AtAP A1 was cloned into the expression vector pPICZ[alpha]A and the recombinant enzyme expressed using the yeast 'Pichia pastoris.' Purification involved ionic exchange chromatography (DEAE and Mono Q), acid incubation and size exclusion chromatography. The mature form of the rAtAP A1 was found to be a heterodimeric protein with a molecular mass of 47 kDa and consisted of a heavy chain and a light chain (approx. 32 and 16 kDa, respectively) linked via disulfide bonds. The protein also included a plant specific insert (PSI), a saposin-like protein domain, which was glycosylated and resulted in the microheterogeneity of the light chain. N-terminal sequence analysis of the heavy and light chain revealed that the mature form of rAtAP A1 retained most of the PSI in the mature form which contributed to a higher content of [alpha]-helix, as determined via circular dichroism spectroscopy, than reported for other aspartic proteinases lacking of this insert. The catalytic properties of the rAtAP A1 were similar to other plant aspartic proteinases with activity in acid pH range with a maximum at pH 4.0 and 45-60°C. Using the synthetic substrate Lys-Pro-Ala-Glu-Phe-Phe(NO2)-Ala-Leu (SS1), a ' Km' of 44 [mu]M and a 'kcat' of 55 s-1 were observed. The enzyme was completely inhibited by pepstatin A. Using oxidized insulin [beta]-chain, rAtAP A1 was found to have a high preference for the hydrophobic bond Leu15-Tyr 16 followed by Phe24-Phe25 and Phe25-Tyr 26. The enzyme was most stable at pH 5-6 and retained 70-80% activity after one month storage at 37°C. Temperature stability was pH dependent and showed the highest stability at pH 5.3. DSC confirmed that melting temperature of rAtAP A1 at pH 5.3 was higher (79.6°C) than at pH 3.5 (72.9°C) or pH 7.0 (60.8°C). A mutant (N83PSIS) lacking of the consensus N-glycosylation site indicated that glycosylation was required for correct folding since the mutant was not able to autoactivate and no activity against the SS1 was observed.



Structure-function, Characterization, Recombinant aspartic proteinase, Arabidopsis thaliana, AtAP A1