Expression and Radiolabeling of Recombinant Proteins Containing a Phosphorylation Motif

Mohanraj, D.; Wahlsten, Jennifer L.; Ramakrishnan, Sundaram

doi:10.1006/prep.1996.0090

Cited by 13 publications

(9 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Human IL‐11 labeling with 125 I has been reported [36], but our numerous attempts to iodinate hIL‐11 were unsuccessful due to a loss of bioactivity after labeling. As it had been shown that the incorporation of a phosphorylation site into several proteins, such as IFN‐α and diphtheria toxin, resulted in a high specific radioactivity after 32 P‐labeling and had no significant effect on their biological activity [37–39], we therefore decided to adopt a similar strategy for IL‐11. This strategy is illustrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

Engineering and use of ³²P‐labeled human recombinant interleukin‐11 for receptor binding studies

Wang¹,

Wilkin²,

Boisteau³

et al. 2002

European Journal of Biochemistry

View full text Add to dashboard Cite

Human interleukin-11 (hIL-11) is a pleiotropic cytokine that is involved in numerous biological activities such as hematopoiesis, osteoclastogenesis, neurogenesis and female fertility. IL-11 is obviously a key reagent to study the IL-11 receptors. However, conventional radio-iodination techniques lead to a loss of IL-11 bioactivity. Here, we report the construction and the production of a new recombinant human . In this molecule, a speci®c phosphorylation site (RRASVA) has been introduced at the N-terminus of rhIL-11. It can be speci®cally phosphorylated by bovine heart protein kinase and accordingly, easily radiolabeled with 32 P. A high radiological speci®c activity (250 000 c.p.m.Áng )1 of protein) was obtained with the retention of full biological activity of the protein. The binding of 32 P-labeled FPDIL-11 to Ba/F3 cells stably transfected with plasmids encoding human IL-11 receptors a and b chains (IL-11Ra and gp130) was speci®c and saturable with a high a nity as determined from Scatchard plot analysis. Availability of this new ligand should prompt further studies on IL-11R structure, expression and regulation.

show abstract

Section: Resultsmentioning

confidence: 99%

Engineering and use of ³²P‐labeled human recombinant interleukin‐11 for receptor binding studies

Wang¹,

Wilkin²,

Boisteau³

et al. 2002

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The cysteine-toserine mutants and the alanine mutants in mVEGF-B 167 pSG5 as well as mVEGF-B exon 1-5 mutant containing a C-terminal Kemptide motif (29), VEGF-B kEx1-5 pSG5, were generated by M13-based in vitro single-stranded mutagenesis employing the helper phage M13KO7 (30) and the dut Ϫ ung Ϫ Escherichia coli strain RZ1032 (31). The primers 5Ј-ACGTAGATCTCCTG-TGTCCCAG-3Ј and 5Ј-ACGTGAATTCTCAGCTGTCTG-GCTTCAC -3Ј (introducing a stop codon after exon 5) were used to PCR-amplify the alanine mutants, which were subcloned in pMT͞Bip͞V5-HisC (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells

Olofsson

Korpelainen

Pepper

et al. 1998

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

462

290

View full text Add to dashboard Cite

show abstract

“…The diphtheria toxin polypeptide used in the present study was mutagenized by DNA-PCR to include a free cysteine residue at the carboxyl terminus of the truncated diphtheria toxin (DT385, residues 1-385 of the intact toxin molecule) to facilitate chemical conjugation. Expression and purification of the toxin polypeptide was performed as previously described (Mohanraj et al, 1996).…”

Section: Expression and Purification Of Vegf121 And Dt385mentioning

confidence: 99%

Inhibition of angiogenesis and tumour growth by VEGF121–toxin conjugate: differential effect on proliferating endothelial cells

Wild¹,

Mohanraj

Olson

et al. 2000

Br J Cancer

View full text Add to dashboard Cite

Vascular endothelial growth factor (VEGF) plays an important role in tumour angiogenesis. VEGF binds to tyrosine kinase receptors, which are expressed almost exclusively on tumour endothelium. Therefore, VEGF can be used to target toxin molecules to tumour vessels for anti-angiogenic therapy. However, recent evidence suggests that VEGF can also bind in an isoform-specific fashion to a newly identified neuropilin-1 (NP-1) receptor. NP-1 is widely expressed in normal tissue and presents a potential target for unwanted toxicity. As a consequence, we investigated whether the VEGF121 isoform, which lacks the NP-1 binding domain, could be used to target toxin polypeptides to tumour vasculature. Treatment of endothelial cells with a VEGF121–diphtheria toxin (DT385) conjugate selectively inhibited proliferating endothelial cells, whereas confluent cultures were completely resistant to the construct. In addition, VEGF121–DT385 conjugate treatment completely prevented tumour cell induced angiogenesis in vivo. Most importantly, the conjugate inhibited tumour growth in athymic mice and induced tumour-specific vascular damage. There was also no apparent toxicity associated with the treatment. Our results suggest that proliferating endothelial cells are highly sensitive to VEGF121–toxin conjugates and that the binding to NP-1 receptors is not necessary for efficient inhibition of tumour growth. © 2000 Cancer Research Campaign

show abstract

Expression and Radiolabeling of Recombinant Proteins Containing a Phosphorylation Motif

Cited by 13 publications

References 0 publications

Engineering and use of ³²P‐labeled human recombinant interleukin‐11 for receptor binding studies

Engineering and use of ³²P‐labeled human recombinant interleukin‐11 for receptor binding studies

Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells

Inhibition of angiogenesis and tumour growth by VEGF121–toxin conjugate: differential effect on proliferating endothelial cells

Contact Info

Product

Resources

About

Expression and Radiolabeling of Recombinant Proteins Containing a Phosphorylation Motif

Cited by 13 publications

References 0 publications

Engineering and use of 32P‐labeled human recombinant interleukin‐11 for receptor binding studies

Engineering and use of 32P‐labeled human recombinant interleukin‐11 for receptor binding studies

Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells

Inhibition of angiogenesis and tumour growth by VEGF121–toxin conjugate: differential effect on proliferating endothelial cells

Contact Info

Product

Resources

About

Engineering and use of ³²P‐labeled human recombinant interleukin‐11 for receptor binding studies

Engineering and use of ³²P‐labeled human recombinant interleukin‐11 for receptor binding studies