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The recombinant expression and activity detection of MAF-1 fusion protein.

Fu P, Wu J, Gao S, Guo G, Zhang Y, Liu J - Sci Rep (2015)

Bottom Line: This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure.The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1.The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Pathogen Biology Laboratory, Guizhou Medical University, No 9 Beijing Road, Guiyang, Guizhou Province, 550004, P.R.China.

ABSTRACT
This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

No MeSH data available.


Constructed MAF-1 gene expression vector and analyzed the fusion protein.(a) The result of PCR for MAF-1. (Lane 1, DL2000 DNA Marker. Lane 2, MAF-1 gene). (b) Electrophoresis of digesting recombinant plasmid pET-28a(+)-MAF-1. (Lane 1, DL2000 DNA Marker. Lane 2, EcoRI and Hind III digestion of recombinant plasmid pET-28a(+)-MAF-1). (c) The amino acids sequence of MAF-1 fusion protein. (___, The insert amino acid sequence). d:The secondary structure prediction for MAF-1 fusion protein.
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f1: Constructed MAF-1 gene expression vector and analyzed the fusion protein.(a) The result of PCR for MAF-1. (Lane 1, DL2000 DNA Marker. Lane 2, MAF-1 gene). (b) Electrophoresis of digesting recombinant plasmid pET-28a(+)-MAF-1. (Lane 1, DL2000 DNA Marker. Lane 2, EcoRI and Hind III digestion of recombinant plasmid pET-28a(+)-MAF-1). (c) The amino acids sequence of MAF-1 fusion protein. (___, The insert amino acid sequence). d:The secondary structure prediction for MAF-1 fusion protein.

Mentions: Taking the 3rd instars of Musca domestica larvae RNA as a template and amplifying the mature peptide fragment of MAF-1 (468 bp) through PCR, a distinctive stripe near visible 500 bp was identified (Fig. 1a) when 1% agarose gel electrophoresis was performed. The digestion products from the double digestion with EcoR I and Hind III to pET-28a(+)-MAF-1 recombinant plasmid was analyzed with 1% AGE. It was shown that there is a clear stripe visible near 500 bp (Fig. 1b), which was about the size of target gene. In addition, the sequencing report of the recombinant plasmid insertion sequence agrees with theoretical sequence, which shows successful establishment of recombinant plasmid. Analyzed with DNAman software it was shown that the recombinant MAF-1 protein sequence was a 156 N-terminals amino acid sequence of the MAF-1 mature peptide with an added 35 amino acid residue, which contained the His label sequence (Fig. 1c). The secondary structure of recombinant MAF-1 fusion protein, analysed with SOPMA tools of ExPASy, demonstrated that the introduction of the His label retained the secondary structural nature of the MAF-1 mature peptide (Fig. 1d).


The recombinant expression and activity detection of MAF-1 fusion protein.

Fu P, Wu J, Gao S, Guo G, Zhang Y, Liu J - Sci Rep (2015)

Constructed MAF-1 gene expression vector and analyzed the fusion protein.(a) The result of PCR for MAF-1. (Lane 1, DL2000 DNA Marker. Lane 2, MAF-1 gene). (b) Electrophoresis of digesting recombinant plasmid pET-28a(+)-MAF-1. (Lane 1, DL2000 DNA Marker. Lane 2, EcoRI and Hind III digestion of recombinant plasmid pET-28a(+)-MAF-1). (c) The amino acids sequence of MAF-1 fusion protein. (___, The insert amino acid sequence). d:The secondary structure prediction for MAF-1 fusion protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589688&req=5

f1: Constructed MAF-1 gene expression vector and analyzed the fusion protein.(a) The result of PCR for MAF-1. (Lane 1, DL2000 DNA Marker. Lane 2, MAF-1 gene). (b) Electrophoresis of digesting recombinant plasmid pET-28a(+)-MAF-1. (Lane 1, DL2000 DNA Marker. Lane 2, EcoRI and Hind III digestion of recombinant plasmid pET-28a(+)-MAF-1). (c) The amino acids sequence of MAF-1 fusion protein. (___, The insert amino acid sequence). d:The secondary structure prediction for MAF-1 fusion protein.
Mentions: Taking the 3rd instars of Musca domestica larvae RNA as a template and amplifying the mature peptide fragment of MAF-1 (468 bp) through PCR, a distinctive stripe near visible 500 bp was identified (Fig. 1a) when 1% agarose gel electrophoresis was performed. The digestion products from the double digestion with EcoR I and Hind III to pET-28a(+)-MAF-1 recombinant plasmid was analyzed with 1% AGE. It was shown that there is a clear stripe visible near 500 bp (Fig. 1b), which was about the size of target gene. In addition, the sequencing report of the recombinant plasmid insertion sequence agrees with theoretical sequence, which shows successful establishment of recombinant plasmid. Analyzed with DNAman software it was shown that the recombinant MAF-1 protein sequence was a 156 N-terminals amino acid sequence of the MAF-1 mature peptide with an added 35 amino acid residue, which contained the His label sequence (Fig. 1c). The secondary structure of recombinant MAF-1 fusion protein, analysed with SOPMA tools of ExPASy, demonstrated that the introduction of the His label retained the secondary structural nature of the MAF-1 mature peptide (Fig. 1d).

Bottom Line: This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure.The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1.The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Pathogen Biology Laboratory, Guizhou Medical University, No 9 Beijing Road, Guiyang, Guizhou Province, 550004, P.R.China.

ABSTRACT
This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

No MeSH data available.