Genetic construction of a phosphorylation site in ricin a chain: Specific radiolabeling of recombinant proteins for localization and degradation studies

D. Fryxell; B. Y. Li; D. Mohanraj; B. Johnson; S. Ramakrishnan

doi:10.1006/bbrc.1995.1654

Genetic construction of a phosphorylation site in ricin a chain: Specific radiolabeling of recombinant proteins for localization and degradation studies

D. Fryxell, B. Y. Li, D. Mohanraj, B. Johnson, S. Ramakrishnan

Pharmacology

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

Abstract

Ricin A chain was modified by the addition of the heptapeptide LRRASLG (Kemptide) and a histidine rag for bacterial expression. The mutagenized toxin was purified by nickel column and could be phosphorylated in vitro by protein kinase A as demonstrated by labeling with [γ-³²P] ATP. Kemptide-A chain could be labeled even after reassociation with ricin B chain or disulfide linkage to antibody to form an immunotoxin. The ³²P label in all cases was associated only with the A chain; ricin B chain and antibody were not kinase substrates alone or after conjugation. Kemptide-immunotoxin was tested in cytotoxicity assays and used to monitor internalization of the toxin moiety after [³²P] phosphorylation.

Original language	English (US)
Pages (from-to)	253-259
Number of pages	7
Journal	Biochemical and Biophysical Research Communications
Volume	210
Issue number	2
DOIs	https://doi.org/10.1006/bbrc.1995.1654
State	Published - May 16 1995

Access

10.1006/bbrc.1995.1654

Fingerprint Dive into the research topics of 'Genetic construction of a phosphorylation site in ricin a chain: Specific radiolabeling of recombinant proteins for localization and degradation studies'. Together they form a unique fingerprint.

Cite this

Genetic construction of a phosphorylation site in ricin a chain : Specific radiolabeling of recombinant proteins for localization and degradation studies. / Fryxell, D.; Li, B. Y.; Mohanraj, D.; Johnson, B.; Ramakrishnan, S.

In: Biochemical and Biophysical Research Communications, Vol. 210, No. 2, 16.05.1995, p. 253-259.

Research output: Contribution to journal › Article › peer-review

@article{7e3a863d615142a0bec288bbc8f7593b,

title = "Genetic construction of a phosphorylation site in ricin a chain: Specific radiolabeling of recombinant proteins for localization and degradation studies",

abstract = "Ricin A chain was modified by the addition of the heptapeptide LRRASLG (Kemptide) and a histidine rag for bacterial expression. The mutagenized toxin was purified by nickel column and could be phosphorylated in vitro by protein kinase A as demonstrated by labeling with [γ-32P] ATP. Kemptide-A chain could be labeled even after reassociation with ricin B chain or disulfide linkage to antibody to form an immunotoxin. The 32P label in all cases was associated only with the A chain; ricin B chain and antibody were not kinase substrates alone or after conjugation. Kemptide-immunotoxin was tested in cytotoxicity assays and used to monitor internalization of the toxin moiety after [32P] phosphorylation.",

author = "D. Fryxell and Li, {B. Y.} and D. Mohanraj and B. Johnson and S. Ramakrishnan",

year = "1995",

month = may,

day = "16",

doi = "10.1006/bbrc.1995.1654",

language = "English (US)",

volume = "210",

pages = "253--259",

journal = "Biochemical and Biophysical Research Communications",

issn = "0006-291X",

publisher = "Academic Press Inc.",

number = "2",

}

TY - JOUR

T1 - Genetic construction of a phosphorylation site in ricin a chain

T2 - Specific radiolabeling of recombinant proteins for localization and degradation studies

AU - Fryxell, D.

AU - Li, B. Y.

AU - Mohanraj, D.

AU - Johnson, B.

AU - Ramakrishnan, S.

PY - 1995/5/16

Y1 - 1995/5/16

N2 - Ricin A chain was modified by the addition of the heptapeptide LRRASLG (Kemptide) and a histidine rag for bacterial expression. The mutagenized toxin was purified by nickel column and could be phosphorylated in vitro by protein kinase A as demonstrated by labeling with [γ-32P] ATP. Kemptide-A chain could be labeled even after reassociation with ricin B chain or disulfide linkage to antibody to form an immunotoxin. The 32P label in all cases was associated only with the A chain; ricin B chain and antibody were not kinase substrates alone or after conjugation. Kemptide-immunotoxin was tested in cytotoxicity assays and used to monitor internalization of the toxin moiety after [32P] phosphorylation.

AB - Ricin A chain was modified by the addition of the heptapeptide LRRASLG (Kemptide) and a histidine rag for bacterial expression. The mutagenized toxin was purified by nickel column and could be phosphorylated in vitro by protein kinase A as demonstrated by labeling with [γ-32P] ATP. Kemptide-A chain could be labeled even after reassociation with ricin B chain or disulfide linkage to antibody to form an immunotoxin. The 32P label in all cases was associated only with the A chain; ricin B chain and antibody were not kinase substrates alone or after conjugation. Kemptide-immunotoxin was tested in cytotoxicity assays and used to monitor internalization of the toxin moiety after [32P] phosphorylation.

UR - http://www.scopus.com/inward/record.url?scp=0029043688&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029043688&partnerID=8YFLogxK

U2 - 10.1006/bbrc.1995.1654

DO - 10.1006/bbrc.1995.1654

M3 - Article

C2 - 7755598

AN - SCOPUS:0029043688

VL - 210

SP - 253

EP - 259

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 2

ER -

Genetic construction of a phosphorylation site in ricin a chain: Specific radiolabeling of recombinant proteins for localization and degradation studies

Abstract

Access

Other files and links

Cite this