Expression, purification and cellular uptake of a S100A4 protein » książka
Expression, purification and cellular uptake of a S100A4 protein
ISBN-13: 9783639173062 / Angielski / Miękka / 2009 / 152 str.
A recombinant S100A4 protein was produced in Pichia pastoris (Pichia). The expression vector contained parts of the AOX1 promoter and also a secretion signal, making it possible to purify the protein from the yeast media after methanol induction. Attempts were made to purify the protein from the media both by hydrophobic interaction chromatography (HIC) and by anionic ion exchange chromatography (IEC). The binding capacity to the hydrophobic material was rather low, but the eluted protein did however show an acceptable purity for utilization in downstream functional assays. The protein eluted from the hydrophobic column was labelled with the fluorescent dye (Cy3) and used for internalization studies in the human cell line HeLa. Similar to a mouse rS100A4 produced in bacterial cells, the Pichia produced protein was also found internalized into the HeLa cells. The biological significance of these findings is at present not elucidated and will be further studied.
A recombinant S100A4 protein was produced in Pichia pastoris (Pichia). The expression vector contained parts of the AOX1 promoter and also a secretion signal, making it possible to purify the protein from the yeast media after methanolinduction. Attempts were made to purify the protein from the media both by hydrophobic interactionchromatography (HIC) and by anionic ion exchange chromatography (IEC). The binding capacity tothe hydrophobic material was rather low, but the eluted protein did however show an acceptable purityfor utilization in downstream functional assays. The protein eluted from the hydrophobic column was labelled with the fluorescent dye (Cy3) and used for internalization studies in the human cell line HeLa. Similar to a mouse rS100A4 produced in bacterial cells, the Pichia produced protein was also found internalized into the HeLa cells. The biological significance of these findings is at present not elucidated and will be furtherstudied.
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