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Purification of a PHA-Like Chitin-binding Protein from Acacia farnesiana Seeds: A Time-dependent Oligomerization Protein

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Abstract

A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI = 4.0 ± 0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.

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Abbreviations

AFAL:

Acacia farnesiana lectin-like protein

VL:

vinorama lectin

GlcNac:

N-acetyl-glucosamine

PPL-1:

Parkia platycephala lectin

PPL-2:

Parkia platycephala chimerolectin

bCFL:

Cratylia floribunda lectin

BSA:

bovine serum albumin

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

HPLC:

high-pressure liquid chromatography

TFA:

trifluoroacetic acid

EDTA:

ethylenediaminetetracetic acid

DTT:

dithiothreitol

PTH:

phenylthiohydantoin

PHAL:

Phaseolus vulgaris leukocyte agglutinin

DBL:

Dolichos biflorus lectin

PDL:

Parkia discolor lectin

PSL:

Parkia speciosa lectin

PJL:

Parkia javanica lectin

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Acknowledgments

This work was partly financed by Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Laboratório de Espectrometria de Massas/LNLS, Campinas—Brazil, and FAPESP (SMOLBNet, 01/07532-0). BSC and AHS are senior investigators of CNPq. We also thank Dr. A. Leyva for English language editing of the manuscript.

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Authors and Affiliations

  1. Departamento de Biologia, Universidade Federal da Paraíba, João Pessoa, Brazil

    T. Santi-Gadelha, B. A. M. Rocha, C. A. A. Gadelha & B. S. Cavada

  2. BioMol Lab, Depto de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, Brazil

    C. C. Oliveira, K. S. Aragão, E. S. Marinho & C. S. Nagano

  3. Campus Litoral Paulista, Universidade Estadual Paulista, São Vicente, São Paulo, Brazil

    M. H. Toyama

  4. Faculdade de Medicina de Sobral, Universidade Federal do Ceará, Sobral, Brazil

    V. P. T. Pinto

  5. Departamento de Ciências Físicas e Biológicas, Universidade Regional do Cariri, Crato, Brazil

    B. A. M. Rocha & P. Delatorre

  6. IQG, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil

    J. L. Martins & F. R. Galvani

  7. Departamento de Engenharia de Pesca, Universidade Federal do Ceará, Fortaleza, Brazil

    A. H. Sampaio

  8. Université des Sciences et Technologies de Lille, Lille, France

    H. Debray

Authors
  1. T. Santi-Gadelha
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  2. B. A. M. Rocha
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  3. C. C. Oliveira
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  4. K. S. Aragão
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  5. E. S. Marinho
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  6. C. A. A. Gadelha
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  8. V. P. T. Pinto
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  9. C. S. Nagano
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  10. P. Delatorre
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  11. J. L. Martins
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  13. A. H. Sampaio
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  14. H. Debray
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  15. B. S. Cavada
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Corresponding author

Correspondence to B. S. Cavada.

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Santi-Gadelha, T., Rocha, B.A.M., Oliveira, C.C. et al. Purification of a PHA-Like Chitin-binding Protein from Acacia farnesiana Seeds: A Time-dependent Oligomerization Protein. Appl Biochem Biotechnol 150, 97–111 (2008). https://doi.org/10.1007/s12010-008-8144-0

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