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Structure-function analysis of insuline-like growth factor I interactions
KTH, Superseded Departments, Biochemistry and Biotechnology.
1997 (English)Doctoral thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH , 1997. , 69 p.
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-2490ISBN: 91-7170-150-8 (print)OAI: oai:DiVA.org:kth-2490DiVA: diva2:8080
Public defence
1997-04-01, 00:00
Note
QC 20100618Available from: 2000-01-01 Created: 2000-01-01 Last updated: 2011-11-08Bibliographically approved
List of papers
1. High-level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli.
Open this publication in new window or tab >>High-level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli.
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1996 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 7, no 2, 131-41 p.Article in journal (Refereed) Published
Abstract [en]

An approach to produce 13C- and 15N-enriched proteins is described. The concept is based on intracellular production of the recombinant proteins in Escherichia coli as fusions to an IgG-binding domain, Z, derived from staphylococcal protein A. The production method provides yields of 40-200 mg/l of isotope-enriched fusion proteins in defined minimal media. In addition, the Z fusion partner facilitates the first purification step by IgG affinity chromatography. The production system is applied to isotope enrichment of human insulin-like growth factor II (IGF-II), bovine pancreatic trypsin inhibitor (BPTI), and Z itself. High levels of protein production are achieved in shaker flasks using totally defined minimal medium supplemented with 13C(6)-glucose and (15NH4)2SO4 as the only carbon and nitrogen sources. Growth conditions were optimized to obtain high protein production levels and high levels of isotope incorporation, while minimizing 13C(6)-glucose usage. Incorporation levels of 13C and/or 15N isotopes in purifies IGF-II, BPTI, and Z were confirmed using mass spectrometry and NMR spectroscopy. More than 99% of total isotope enrichment was obtained using a defined isotope-enriched minimal medium. The optimized systems provide reliable, high-level production of isotope-enriched fusion proteins. They can be used to produce 20-40 mg/l of properly folded Z and BPTI proteins. The production system of recombinant BPTI is state-of-the-art and provides the highest known yield of native refolded BPTI.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-13472 (URN)8616269 (PubMedID)
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2017-12-12Bibliographically approved
2. Structural changes in insulin-like growth factor (IGF) I mutant proteins affecting binding kinetic rates to IGF binding protein 1 and IGF-I receptor.
Open this publication in new window or tab >>Structural changes in insulin-like growth factor (IGF) I mutant proteins affecting binding kinetic rates to IGF binding protein 1 and IGF-I receptor.
1997 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, no 14, 4108-4117 p.Article in journal (Refereed) Published
Abstract [en]

Ligand binding properties of five single amino acid substituted variants (V11A, D12A, Q15A, Q15E, and F16A) of human insulin-like growth factor I (IGF-I) were analyzed with respect to their binding affinities and binding kinetics to recombinant IGF binding protein 1 (IGFBP-1) and a soluble form of the IGF type I receptor (sIGF-I(R)), respectively. Side chains of the substituted residues are all predicted to be the most surface exposed in the alpha-helical portion of the B-region of the IGF-I molecule. The IGF-I variants were produced as fusion proteins to a IgG(Fc) binding protein domain, Z. Ligand binding kinetic rates were determined using BIAcore biosensor interaction analysis technology. All IGF-I variants showed altered binding affinities to both IGFBP- I and sIGF-I(R). Secondary structure content of the IGF-I variants was estimated using far-UV circular dichroism spectroscopy, followed by variable selection secondary structure calculations. The amount of calculated alpha-helicity is reduced for all the mutants, most predominantly for IGF-I(V11A) and IGF-I(F16A) proteins. Surprisingly, most of the effects of reduced binding affinities to both target proteins are attributed to lowered on-rates of binding, and these are correlated with the amount of alpha-helicity in each IGF-I variant. In addition, in some of the IGF-I variants, lowered off-rates of binding are observed. From the results, we propose that IGF-I is unusually sensitive to structural changes by surface amino acid substitutions in the B-region of the molecule. Therefore, biochemical or biological properties of amino acid substituted variants of IGF-I cannot be used in a straightforward way to dissect the direct involvement in binding of individual amino acid residues since structural changes may be involved.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-13474 (URN)10.1021/bi961553i (DOI)9100004 (PubMedID)
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2017-12-12Bibliographically approved
3. Characterization of ligand binding of a soluble human insulin-like growth factor I receptor variant suggests a ligand-induced conformational change.
Open this publication in new window or tab >>Characterization of ligand binding of a soluble human insulin-like growth factor I receptor variant suggests a ligand-induced conformational change.
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1997 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 13, 8189-8197 p.Article in journal (Refereed) Published
Abstract [en]

Details of the signal transduction mechanisms of the tyrosine kinase family of growth factor receptors remain elusive. In this work, we describe an extensive study of kinetic and thermodynamic aspects of growth factor binding to a soluble extracellular human insulin-like growth factor-I receptor (sIGF-IR) variant. The extracellular receptor domains were produced fused to an IgG-binding protein domain (Z) in transfected human 293 cells as a correctly processed secreted alpha-beta'-Z dimer. The receptor was purified using IgG affinity chromatography, rendering a pure and homogenous protein in yields from 1 to 5 mg/liter of conditioned cell media. Biosensor technology (BIAcore) was applied to measure the insulin-like growth factor-I (IGF-I), des(1-3)IGF-I, insulin-like growth factor-II, and insulin ligand binding rate constants to the immobilized IGF-IR-Z. The association equilibrium constant, Ka, for the IGF-I interaction is determined to 2.8 x 10(8) M-1 (25 degrees C). Microcalorimetric titrations on IGF-I/IGF-IR-Z were performed at three different temperatures (15, 25, and 37 degrees C) and in two different buffer systems at 25 degrees C. From these measurements, equilibrium constants for the 1:1 (IGF-I:(alpha-beta'-Z)2) receptor complex in solution are deduced to 0.96 x 10(8) M-1 (25 degrees C). The determined heat capacity change for the process is large and negative, -0.51 kcal (K mol)-1. Further, the entropy change (DeltaS) at 25 degrees C is large and negative. Far- and near-UV circular dichroism measurements display significant changes over the entire wavelength range upon binding of IGF-I to IGF-IR-Z. These data are all consistent with a significant change in structure of the system upon IGF-I binding.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13475 (URN)9079636 (PubMedID)
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2017-12-12Bibliographically approved
4. Binding affinities of insulin-like growth factor-I (IGF-I) fusion proteins to IGF binding protein 1 and IGF-I receptor are not correlated with mitogenic activity.
Open this publication in new window or tab >>Binding affinities of insulin-like growth factor-I (IGF-I) fusion proteins to IGF binding protein 1 and IGF-I receptor are not correlated with mitogenic activity.
1997 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 416, no 3, 259-264 p.Article in journal (Refereed) Published
Abstract [en]

In this report, comparisons between molecular affinities and cellular proliferation activities have been made for insulin-like growth factor-I (IGF-I) and two IGF-I fusion proteins in order to evaluate fusion proteins as tools for receptor binding studies. Binding affinities and growth promoting effects of the N-terminal fusion Z-IGF-I and the C-terminal fusion IGF-I-Z, and native recombinant human IGF-I, were analyzed. Binding kinetic properties of the three IGF-I variants were analyzed using BIAcore kinetic interaction analysis testing for binding to both human IGF binding protein 1 (IGFBP-1) and a soluble form of the human IGF type I receptor extracellular domains (sIGF-IR). The growth promoting effects on SaOS-2 human osteosarcoma cells of the different fusion proteins were analyzed. A comparison of receptor binding affinities and growth promoting effects shows that the fusion protein receptor affinity does not correlate with proliferative potential. The IGF-I-Z fusion, with the lowest receptor affinity, shows similar proliferative potential to native IGF-I. However, the Z-IGF-I fusion protein, with twice the receptor affinity of IGF-I-Z, displays only about 70% of the IGF-I-Z growth promoting activity. Both IGF-I fusion proteins possess similar affinity to IGFBP-1. These results indicate that determinants other than the receptor affinity could be involved in the regulation of IGF-I proliferative action. This study demonstrates that ligand fusion proteins may be useful to study mechanisms of ligand induced receptor activation.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13476 (URN)9373165 (PubMedID)
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2017-12-12Bibliographically approved
5. The IGFBP-1 binding epitope of IGF-1 probed by heteronuclear NMR and mutational analysis
Open this publication in new window or tab >>The IGFBP-1 binding epitope of IGF-1 probed by heteronuclear NMR and mutational analysis
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(English)Manuscript (preprint) (Other academic)
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-13478 (URN)
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2011-11-08Bibliographically approved

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