Cytoplasmic expression of a thermostable invertase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis> in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> |
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Authors: | Han Bin Pek Pei Yu Lim Chengcheng Liu Dong-Yup Lee Xuezhi Bi Fong Tian Wong Dave Siak-Wei Ow |
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Institution: | 1.Bioprocessing Technology Institute,A*STAR (Agency for Science, Technology and Research),Singapore,Singapore;2.Department of Chemical and Biomolecular Engineering,National University of Singapore,Singapore,Singapore;3.Molecular Engineering Laboratory, Biomedical Sciences Institutes,A*STAR (Agency for Science, Technology and Research),Singapore,Singapore |
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Abstract: | ObjectivesTo evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis.ResultsThe thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively.ConclusionsInvertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries. |
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