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Cloning, heterologous expression and structural characterization of an alkaline serine protease from sea water haloalkaliphilic bacterium

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Abstract

Cloning and functional attributes of a serine protease gene from haloalkaliphilic bacteria are described. The protease gene of ∼1,600 bp amplified from the genomic DNA was cloned into TA vector followed by the sub-cloning into pUC19 for expression. Growth of the organism and gene expression was studied at 30 and 37 °C in the presence of 0.5–2.0 mM IPTG. Sequencing of the gene and homology search of the sequence revealed that the gene encoded an extracellular alkaline serine protease belonging to superfamily subtilisin-like hydrolases. The amino acid sequence alignment resulted from the BLAST search of the subtilisin exhibited high sequence homology with the Bacillus subtilis ssp. subtilis strain 168 and subtilisins of other Bacillus sp., B. subtilis and B. mojavensis. The deduced amino acid sequence exhibited a mature protease of a 419 amino acid, single-chained monomeric peptide with the large number of the positively charged amino acids suggesting its hydrophilic nature.

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Acknowledgments

R.V. is grateful to DBT, New Delhi for Junior and Senior Research fellowships. This work was supported by DBT, New Delhi, as a Multi Institutional Project involving Saurashtra University, Delhi University South Campus and IIT Delhi. We are grateful to Mr. Rajendrakumar D. Joshi and Mr. Harendra Jha, Department of Pharmacology Molecular Biology Division, Piramal Life Sciences Ltd. Mumbai for providing facilities, plasmids and strains for the molecular biology work. The authors acknowledge Dr. Rupal Joshi for her inputs in the isolation of various halophilic and haloalkaliphilic bacteria during her research at Saurashtra University, Rajkot. Infrastructural facilities provided under the DST-FIST Programme to the Department of Biosciences, Saurashtra University, Rajkot are gratefully acknowledged. The facilities and support provided by the Saurashtra University and UGC, New Delhi are also acknowledged.

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Author notes

  1. Chirantan M. Rawal

    Present address: Microbiology Division, Silvassa College, Naroli, Dadara and Nagar Haveli (UT), Silvassa, India

Authors and Affiliations

  1. UGC-Centre of Advanced Studies, Department of Biosciences, Saurashtra University, Rajkot, 360005, Gujarat, India

    Vikram H. Raval, Chirantan M. Rawal, Sandeep Pandey, Hitarth B. Bhatt, Bharat R. Dahima & Satya P. Singh

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  1. Vikram H. Raval
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Correspondence to Satya P. Singh.

Electronic supplementary material

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Table S1

(DOC 109 kb)

Fig. S1

Multiple sequence alignment of Ve2-20-91 clone sequence with the known proteases from the database using Clustal X program (DOC 1259 kb)

Fig. S2

InterProscan result showing domain analysis of protein (DOC 609 kb)

Fig. S3

Swiss model work space results showing Q-mean score of query protein (DOC 294 kb)

Fig. S4

Swiss model work space results showing Q-mean score comparison with non-redundant PDB structures (DOC 404 kb)

Fig. S5

PROSA result showing Z-score model quality. The plot displays z-scores of all experimentally determined protein chains in current PDB. In this plot, groups of structures from different sources X-ray (light blue), NMR (dark blue) are distinguished by different colors. It can be used to check whether the Z-score of the input structure is within the range of scores typically found for native proteins of similar size (DOC 609 kb)

Fig. S6

PROSA result showing local model quality by plot of energies as a function of amino acid sequence position i. The positive values correspond to problematic or erroneous parts of the input structure (DOC 436 kb)

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Raval, V.H., Rawal, C.M., Pandey, S. et al. Cloning, heterologous expression and structural characterization of an alkaline serine protease from sea water haloalkaliphilic bacterium. Ann Microbiol 65, 371–381 (2015). https://doi.org/10.1007/s13213-014-0869-0

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  • DOI: https://doi.org/10.1007/s13213-014-0869-0

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