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Human neutrophil peptide 3 could be functionally expressed in Rhodobacter sphaeroides

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Nie X., Zhang L., Hu Z., Liu Y., Zhao Z.

2015

vol. 62

issue 2

259-263

Human neutrophil peptides (HNPs) possess high antimicrobial activities against a broad spectrum of microorganisms. Rhodobacter sphaeroides is the best-characterized photosynthetic bacterium and exhibits potential as a novel expression system. Up to date, no literature has been reported regarding expression of HNP3 in Rb. sphaeroides. In the present study, the HNP3 gene fragment was amplified by SOE PCR and ligated into photosynthetic bacteria light-harvesting complex 2 (LH2) expression vector leading to HNP3 fusion protein expression vector. The HNP3 fusion protein was successfully expressed as rapidly evaluated by the LH2 characteristic peaks at ~800 nm and ~850 nm before purification and SDS/PAGE. Subsequently, the HNP3 fusion protein was purified by one-step affinity chromatography, and could be rapidly detected by the color and the spectral absorption at ~800 nm and ~850 nm before SDS/PAGE. Antimicrobial activity assay suggested that the HNP3 fusion protein exhibited high antimicrobial activity towards E. coli. The present study may supply an insight into employing the novel Rb. sphaeroides expression system, exhibiting dramatic advantages over currently used commercial expression system, to heterologously express human neutrophil peptides.

Thermostable Pyrococcus woesei β-D-galactosidase - high level expression, purification and biochemical properties

100%

Wanarska M., Kur J., Pladzyk R., Turkiewicz M.

2005

vol. 52

issue 4

781-787

The gene encoding β-D-galactosidase from Pyrococcus woesei was PCR amplified, cloned, expressed in Escherichia coli under the control of an inducible T7 promoter, purified and characterized. The expression system was developed by the construction of recombinant plasmid, based on the high copy number pUET1 vector, giving four times more efficient expression of P. woesei β-D-galactosidase (20 mg of enzyme from 1 liter of culture) than that obtained from a previously constructed one. The recombinant enzymes were purified in a two-step procedure: double heat-denaturation of E. coli cell proteins and affinity chromatography on p-aminobenzyl 1-thio-β-D-galactopyranoside-agarose. To achieve efficient purification of P. woesei β-D-galactosidase by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the N- or the C-terminal of the coding sequence. The obtained fusion proteins revealed the same specific activity of approximately 5400 U/mg, which was 10 times lower than the wild-type β-D-galactosidase (51100 U/mg). The activity of P. woesei β-D-galactosidase was enhanced by thiol compounds, Mg2+ ions and D-galactose, and was inhibited by heavy metal ions and D-glucose, while Ca2+ ions had no effect.

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2 Acta Biochimica Polonica

1 2015

1 2005

1 Hu Z.

1 Kur J.

1 Liu Y.

1 Nie X.

1 Pladzyk R.

1 Turkiewicz M.

1 Wanarska M.

1 Zhang L.

1 Zhao Z.

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Human neutrophil peptide 3 could be functionally expressed in Rhodobacter sphaeroides

Thermostable Pyrococcus woesei β-D-galactosidase - high level expression, purification and biochemical properties