Expression and radiolabeling of recombinant proteins containing a phosphorylation motif

D. Mohanraj, J. L. Wahlsten, Sundaram Ramakrishnan

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Radiolabeled proteins are useful in basic and clinical research. Current methods available for radiolabeling proteins involve chemical derivatization, resulting in multiple additions of radionuclides at random sites. A method designed to specifically localize the radionuclide to a unique site will offer advantages of control and predictability in radiolabeling. We have studied the usefulness of a prokaryotic expression vector by incorporating the coding sequence of a consensus phosphorylation motif (Kemptide) for the cAMP-dependent protein kinase A immediately upstream to the multiple cloning site. This vector was used to express five different recombinant proteins with a phosphorylation site at the amino terminus. In addition, the phosphorylation motif was introduced into two other proteins and expressed in yeast. The genetically engineered proteins were purified to homogeneity by affinity chromatography and radiolabeled with [γ32P]-ATP in vitro. All seven proteins used in this study could be expressed with the phosphorylation sequence at their amino terminus and specifically labeled without loss of biological activity. This strategy allows the option of labeling proteins to high or low specific radioactivity and holds potential for in vitro binding and in vivo localization studies.

Original languageEnglish (US)
Pages (from-to)175-182
Number of pages8
JournalProtein Expression and Purification
Volume8
Issue number2
DOIs
StatePublished - Jan 1 1996
Externally publishedYes

Fingerprint

Recombinant Proteins
Phosphorylation
Proteins
Cyclic AMP-Dependent Protein Kinases
Radioisotopes
kemptide
Consensus Sequence
Affinity Chromatography
Radioactivity
Organism Cloning
Adenosine Triphosphate
Yeasts
Research

ASJC Scopus subject areas

  • Biotechnology

Cite this

Expression and radiolabeling of recombinant proteins containing a phosphorylation motif. / Mohanraj, D.; Wahlsten, J. L.; Ramakrishnan, Sundaram.

In: Protein Expression and Purification, Vol. 8, No. 2, 01.01.1996, p. 175-182.

Research output: Contribution to journalArticle

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