T7 Vectors with a Modified T7lacPromoter for Expression of Proteins inEscherichia coli

Peränen, Johan; Rikkonen, Marja; Hyvönen, Marko; Kääriäinen, Leevi

doi:10.1006/abio.1996.0187

Cited by 270 publications

(222 citation statements)

References 0 publications

Supporting

Mentioning

217

Contrasting

Order By: Relevance

“…The cDNA corresponding to the ORF of DmMANF was cloned into a T7lac vector, expressed in Escherichia coli and purified as previously described (43,44). Rabbits were immunized and the obtained antiserum was affinity purified (43).…”

Section: Methodsmentioning

confidence: 99%

Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons

Palgi

Lindström

Peränen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

127

214

View full text Add to dashboard Cite

In vertebrates the development and function of the nervous system is regulated by neurotrophic factors (NTFs). Despite extensive searches no neurotrophic factors have been found in invertebrates. However, cell ablation studies in Drosophila suggest trophic interaction between neurons and glia. Here we report the invertebrate neurotrophic factor in Drosophila, DmMANF, homologous to mammalian MANF and CDNF. DmMANF is expressed in glia and essential for maintenance of dopamine positive neurites and dopamine levels. The abolishment of both maternal and zygotic DmMANF leads to the degeneration of axonal bundles in the embryonic central nervous system and subsequent nonapoptotic cell death. The rescue experiments confirm DmMANF as a functional ortholog of the human MANF gene thus opening the window for comparative studies of this protein family with potential for the treatment of Parkinson's disease.development ͉ dopamine ͉ Drosophila ͉ glia ͉ neurite

show abstract

Section: Methodsmentioning

confidence: 99%

Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons

Palgi

Lindström

Peränen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

127

214

View full text Add to dashboard Cite

show abstract

“…The Op18 open reading frame was amplified by PCR with Vent DNA polymerase (New England Biolabs) introducing NcoI and BamHI restriction sites at the 5Ј-and 3Ј-end, respectively, and cloned into the pHAT2 vector (30). The resulting protein contains the peptide MSHH-HHHHS fused to the N terminus of human Op18.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Microtubule Destabilizing Activity of Op18/Stathmin Downstream of Rac1

Wittmann

Bokoch

Waterman‐Storer

2004

Journal of Biological Chemistry

217

183

View full text Add to dashboard Cite

In the leading edge of migrating cells, a subset of microtubules exhibits net growth in a Rac1-and p21-activated kinase-dependent manner. Here, we explore the possibility of whether phosphorylation and inactivation of the microtubule-destabilizing protein Op18/ stathmin could be a mechanism regulating microtubule dynamics downstream of Rac1 and p21-activated kinases. We find that, in vitro, Pak1 phosphorylates Op18/ stathmin specifically at serine 16 and inactivates its catastrophe promoting activity in biochemical and time lapse microscopy microtubule assembly assays. Furthermore, phosphorylation of either serine 16 or 63 is sufficient to inhibit Op18/stathmin in vitro. In cells, the microtubule-destabilizing effect of an excess of Op18/ stathmin can be partially overcome by expression of constitutively active Rac1(Q61L), which is dependent on Pak activity, suggesting that the microtubule cytoskeleton can be regulated through inactivation of Op18/ stathmin downstream of Rac1 and Pak in vivo. However, in vivo, Pak1 activity alone is not sufficient to phosphorylate Op18, indicating that additional pathways downstream of Rac1 are required for Op18 regulation.

show abstract

“…Preparation of Recombinant Protein-The coding sequence for Fs1, comprising residues 64 -136 of the mature follistatin gene product fused to a 5Ј-AUG codon, was amplified using a standard PCR protocol and inserted into the multiple cloning sequence of the T7-based expression vector pBAT4 (21). Expression of soluble recombinant protein was subsequently carried out in an Escherichia coli BL21(DE3) host and was induced at 37°C with 400 M isopropyl-1-thio-␤-D-galactopyranoside.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structures of the Heparan Sulfate-binding Domain of Follistatin

Innis

Hyvönen

2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Follistatin associates with transforming growth factor-␤-like growth factors such as activin or bone morphogenetic proteins to form an inactive complex, thereby regulating processes as diverse as embryonic development and cell secretion. Although an interaction between heparan sulfate chains present at the cell surface and follistatin has been recorded, the impact of this binding reaction on the follistatin-mediated inhibition of transforming growth factor-␤-like signaling remains unclear. To gain a structural insight into this interaction, we have solved the crystal structure of the presumed heparan sulfate-binding domain of follistatin, both alone and in complex with the small heparin analogs sucrose octasulfate and D-myo-inositol hexasulfate. In addition, we have confirmed the binding of the sucrose octasulfate and D-myo-inositol hexasulfate molecules to this follistatin domain and determined the association constants and stoichiometries of both interactions in solution using isothermal titration calorimetry. Overall, our results shed light upon the structure of this follistatin domain and reveal a novel conformation for a hinge region connecting epidermal growth factor-like and Kazal-like subdomains compared with the follistatin-like domain found in the extracellular matrix protein BM-40. Moreover, the crystallographic analysis of the two protein-ligand complexes mentioned above leads us to propose a potential location for the heparan sulfate-binding site on the surface of follistatin and to suggest the involvement of residues Asn 80 and Arg 86 in such a follistatin-heparin interaction.The secreted polypeptide follistatin regulates several signaling pathways in a cell-and tissue-specific manner, largely through its ability to inactivate transforming growth factor-␤-like growth factor molecules (1, 2). Although first identified as a factor capable of countering the inducing effects of activin on follicle-stimulating hormone secretion (3, 4), follistatin has now been isolated and characterized from a variety of tissues and organisms, where it has been shown to take part in processes such as cell growth, differentiation, and secretion (5-7). Moreover, the importance of this polypeptide has been demonstrated in follistatin-null mice, which can survive birth but die from multiple skeletal and cutaneous abnormalities within a few hours of delivery (8).Follistatin isoforms sharing the same N terminus but differing in the length of their polypeptide chain and in their glycosylation patterns coexist in vivo, with molecular masses ranging from 31 to 39 kDa (4, 9). Indeed, alternative splicing of the follistatin gene yields the Fs-315 and Fs-288 isoforms, featuring 315 and 288 residues, respectively. An additional variant of follistatin, Fs-303, is thought to arise from the proteolytic cleavage of the Fs-315 C terminus and is likely to be the predominant species in vivo (3, 10, 11). Despite its relatively limited size, follistatin contains a total of 36 cysteine residues, believed to be arranged into nonoverlapping set...

show abstract

T7 Vectors with a Modified T7lacPromoter for Expression of Proteins inEscherichia coli

Cited by 270 publications

References 0 publications

Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons

Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons

Regulation of Microtubule Destabilizing Activity of Op18/Stathmin Downstream of Rac1

Crystal Structures of the Heparan Sulfate-binding Domain of Follistatin

Contact Info

Product

Resources

About