Abstract
Anabaena variabilis double mutant (C503S/C565S) phenylalanine ammonia-lyase (PAL) is an appealing enzyme in the enzyme-replacement therapy of phenylketonuria. Yet abundant production of this enzyme has been of concern for industrial production. In this study, we have characterized 1175 bacterial signal peptides (SPs) and identified the most efficient ones for the excretory production of mutant AvPAL. Analysis by SignalP 4.1 revealed that more than 61% of SPs had a D-score greater than 0.7, denoting to be highly efficient. The optimum length of a bacterial SP was 25–30. The preferable net positive charge and the second residue of N-region were + 2 and Lys/Arg, respectively. Highly efficient SPs possessed 3–5 Leus in their H-region and A/L/VXA-FF cleavage site. Highly efficient SPs were from Escherichia coli, corroborating the necessity of an agreement between SPs and the host. Physiochemical characterization of mutant AvPAL conjugates via ProtParam and PROSOII, revealed that ~ 99.5% of proteins would not be entraped in inclusion bodies. Secretory pathways were identified by EffectiveDB and more than 98% of SPs were cleavable. Chimeras were modeled using the I-TASSER program, being evaluated by the Ramachandran plots. The mRNA secondary structure of mutant AvPAL upon linkage to SPs was assessed using the mfold program. It was shown that the linkage of a SP does not affect mutant AvPAL’s stability at the protein or mRNA level. AllergenFP tool demonstrated that chimeras were not allergen. SPs, including FMF4_ECOLX, E2BB_ECOLX, and LPTA_ECOLI exhibited the highest propensity for secretion and appropriate features in all analyses.
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Abbreviations
- Phe:
-
Phenylalanine
- PAL:
-
Phenylalanine Ammonia Lyase
- PKU:
-
Phenylketonuria
- t-CA:
-
Trans-cinnamic acid
- LNAA:
-
Large neutral amino acid
- SP:
-
Signal peptide
- HMM:
-
Hidden Markov model
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Authors would like to thank Shiraz University of Medical Sciences, Shiraz, IRAN for the grant number: 1396-01-36-16684.
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Owji, H., Hemmati, S. A comprehensive in silico characterization of bacterial signal peptides for the excretory production of Anabaena variabilis phenylalanine ammonia lyase in Escherichia coli. 3 Biotech 8, 488 (2018). https://doi.org/10.1007/s13205-018-1517-3
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DOI: https://doi.org/10.1007/s13205-018-1517-3