Structural and functional characterization of a recombinant leucine aminopeptidase

• Optimization of the folding process of a recombinant leucine aminopeptidase.
• Analytical techniques were employed for understanding the folding of the enzyme.
• Proficient buffer conditions were established to improve the structure of the enzyme.
• Folding presumably facilitated the coupling of zinc to the enzyme catalytic site.
• Folding process structurally stabilized the recombinant leucine aminopeptidase.

The function of proteins, such as the catalytic enzyme activity, depends on the interaction of their active sites with their specific substrates and the environment conditions that affect the stability of those sites. This study presents a structure-to-function characterization of the folding process of a recombinant 6×-His tag leucine aminopeptidase (rLAP) based on a platform of analytical techniques. The results demonstrated an increase up to 31 U/mg in the activity of the enzyme after folding as revealed by circular dichroism, intrinsic fluorescence, differential scanning calorimetry, and free thiol analysis. Collectively, these techniques revealed a larger number of covalent and non-covalent bonds within the protein seen as an increase in the chemical and thermal stability, while exhibited a lower level of non-bonded cysteines after the protein was folded. Mass spectrometry analysis showed the maintenance of the distribution of the enzyme isoforms related to N-terminal histidine residues after folding, which confirmed that the enzymatic activity of rLAP depends on its three-dimensional structure rather than N-terminal self-processing activity. In summary, the studied attributes allow a better understanding of the structure-to-function relationship of rLAP, that permit a more proficient manufacturing of the enzyme that would improve the bioprocesses in which is employed.

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