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Cloning, Sequencing, Expression and Structural Investigation of Mnemiopsin from Mnemiopsis leidyi: An Attempt Toward Understanding Ca2+-Regulated Photoproteins

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Abstract

A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence identity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27 kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl2. The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.

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Abbreviations

BLAST:

Basic local alignment search tool

BSA:

Bovine serum albumin

EDTA:

Ethylene diamine tetra acetic acid

HLH:

Helix-loop-helix

IPTG:

Isopropyl-β-thiogalactoside

PDB:

Protein data bank

PSQS:

Protein structure quality score

SDS-PAGE:

Sodium dodecyl sulphate polyacryl amide gel electrophoresis

spdb:

Swiss protein data bank

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Authors and Affiliations

  1. Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran

    Mahmoud Reza Aghamaali, Vahab Jafarian, Reyhaneh Sariri, Maryam Molakarimi, Behnam Rasti, Majid Taghdir & Reza Hasan Sajedi

  2. Department of Biochemistry, Tarbiat Modares University, Tehran, Iran

    Reza Hasan Sajedi & Saman Hosseinkhani

Authors
  1. Mahmoud Reza Aghamaali
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  2. Vahab Jafarian
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  3. Reyhaneh Sariri
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  4. Maryam Molakarimi
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  5. Behnam Rasti
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  6. Majid Taghdir
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  7. Reza Hasan Sajedi
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  8. Saman Hosseinkhani
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Correspondence to Reyhaneh Sariri.

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Aghamaali, M.R., Jafarian, V., Sariri, R. et al. Cloning, Sequencing, Expression and Structural Investigation of Mnemiopsin from Mnemiopsis leidyi: An Attempt Toward Understanding Ca2+-Regulated Photoproteins. Protein J 30, 566–574 (2011). https://doi.org/10.1007/s10930-011-9363-8

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  • DOI: https://doi.org/10.1007/s10930-011-9363-8

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