A new method for the purification of bioactive insulin-like growth factor-binding protein-3

Pircher, Haymo; Matscheski, Andrea; Laich, Andreas; Hermann, Michael; Moser, Barbara; Viertler, Hans-Peter; Mičutková, Lucia; Lindner, Herbert; Sarg, Bettina; Zwerschke, Werner; Jansen‐Dürr, Pidder

doi:10.1016/j.pep.2010.02.005

Cited by 4 publications

(11 citation statements)

References 33 publications

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“…Designation of lanes is as in panel a . Please note that elution from the heparin affinity column reveals several distinct forms of IGFBP-6, probably differing in the extent of glycosylation, similar to previous findings with IGFBP-3 (Pircher et al 2010)…”

Section: Resultssupporting

confidence: 89%

“…Purified IGFBP-3 (Pircher et al 2010) was labeled in the same way und used as a control. While fluorescent labeled IGFBP-3 was readily internalized after 2 h, accumulating in vesicular structures, no uptake was observed for IGFBP-6 within this time frame (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The production of IGFBP‐6 by 24 isolated single clones was monitored by ELISA, and the clone with the highest production rate was selected for further analysis. To avoid possible complications during purification due to the presence of serum, as observed with IGFBP‐3 (Pircher et al 2010), IGFBP‐6 expressing cells were washed and subsequently grown in serum‐free medium for 3 days. The conditioned medium was harvested and 7 times replaced by serum‐free medium.…”

Section: Resultsmentioning

confidence: 99%

“…On a larger scale, purification has been done from supernatants of recombinant Pichia pastoris strains expressing and secreting hIGFBP‐6, using ion exchange chromatography and HIC (Chen et al 2007). To provide a gentle and efficient procedure to obtain mg quantities of recombinant hIGFBP‐6 secreted from human cells, we have explored the biosynthetic capacities of human osteosarcoma cells (U‐2OS) which were successfully used for highly efficient ectopic production of other human proteins, including IGFBP‐3 (Pircher et al 2010). In this communication, we used an efficient purification procedure independent of the IGF‐II binding capacity of hIGFBP‐6 to produce highly pure IGFBP‐6 protein.…”

Section: Introductionmentioning

confidence: 99%

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Purification and characterization of native human insulin-like growth factor binding protein-6

Taferner

Mičutková

Hermann

et al. 2011

J. Cell Commun. Signal.

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Insulin-like growth factor binding proteins (IGFBPs) are key regulators of insulin-like growth factor (IGF) mediated signal transduction and thereby can profoundly influence cellular phenotypes and cell fate. Whereas IGFBPs are extracellular proteins, intracellular activities were described for several IGFBP family members, such as IGFBP-3, which can be reinternalized by endocytosis and reaches the nucleus through routes that remain to be fully established. Within the family of IGFBPs, IGFBP-6 is unique for its specific binding to IGF-II. IGFBP-6 was described to possess additional IGF-independent activities, which have in part been attributed to its translocation to the nucleus; however, cellular uptake of IGFBP-6 was not described. To further explore IGFBP-6 functions, we developed a new method for the purification of native human IGFBP-6 from cell culture supernatants, involving a four-step affinity purification procedure, which yields highly enriched IGFBP-6. Whereas protein purified in this way retained the capacity to interact with IGF-II and modulate IGF-dependent signal transduction, our data suggest that, unlike IGFBP-3, human IGFBP-6 is not readily internalized by human tumor cells. To summarize, this work describes a novel and efficient method for the purification of native human insulin-like growth factor binding protein 6 (IGFBP-6) from human cell culture supernatants, applying a four-step chromatography procedure. Intactness of purified IGFBP-6 was confirmed by IGF ligand Western blot and ability to modulate IGF-dependent signal transduction. Cellular uptake studies were performed to further characterize the purified protein, showing no short-term uptake of IGFBP-6, in contrast to IGFBP-3.Electronic supplementary materialThe online version of this article (doi:10.1007/s12079-011-0126-2) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Purification and characterization of native human insulin-like growth factor binding protein-6

Taferner

Mičutková

Hermann

et al. 2011

J. Cell Commun. Signal.

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“…Native human IGFBP‐3 was produced in U‐2OS cells transfected with an IGFBP‐3 expression vector, and the protein was isolated from the conditioned supernatant, as described previously 21. A purification control was performed by subjecting supernatant of mock‐transfected U‐2OS to the same purification procedure as unlabeled IGFBP‐3.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the cellular uptake and nuclear delivery of insulin‐like growth factor binding protein‐3 in human osteosarcoma cells

Mičutková

Hermann

Offterdinger

et al. 2011

Intl Journal of Cancer

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Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) regulates cell proliferation and survival by extracellular interaction and inactivation of the growth factor IGF-I. Beyond that, IGF-independent actions mediated by intracellular IGFBP-3 including nuclear-IGFBP-3, have also been described. We here show, using both confocal and electron microscopy and cell fractionation, that the extracellular addition of IGFBP-3 to living cells results in rapid uptake and nuclear delivery of IGFBP-3, by yet partly unknown mechanisms. IGFBP-3 is internalized through a dynamin-dependent pathway, traffics through endocytic compartments and is finally delivered into the nucleus. We observed docking of IGFBP-3 containing structures to the nuclear envelope and found IGFBP-3 containing dot-like structures to permeate the nuclear envelope. In summary, our findings establish the pathway by which this tumor suppressor protein is delivered from extracellular space to the nucleus.

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Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells

Wanscher

Ebersole

Streicher

et al. 2015

Protein Expression and Purification

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A new method for the purification of bioactive insulin-like growth factor-binding protein-3

Cited by 4 publications

References 33 publications

Purification and characterization of native human insulin-like growth factor binding protein-6

Purification and characterization of native human insulin-like growth factor binding protein-6

Analysis of the cellular uptake and nuclear delivery of insulin‐like growth factor binding protein‐3 in human osteosarcoma cells

Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells

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