Affinity Purification of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit ϵN-Methyltransferase

P. Wang, M. Royer, R. L. Houtz

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23 Scopus citations


Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit ε{lunate}N-methyltransferase (Protein methylase III, Rubisco LSMT, EC catalyzes methylation of the ε{lunate}-amino group of Lys-14 in the large subunit of Rubisco. In this paper, an affinity purification procedure for pea (Pisum sativum L. cv Laxton′s Progress No. 9) Rubisco LSMT is described and characterized. Spinach (Spinacia oleracea L. cv Melody) Rubisco, a substrate for pea Rubisco LSMT, was immobilized to polyvinylidene fluoride (PVDF) transfer membranes (Immobilon-P) and used as a ligand for the affinity purification of Rubisco LSMT from pea leaf extracts and chloroplast lysates. Pea Rubisco LSMT specifically bound to PVDF-immobilized spinach Rubisco but not to control PVDF membranes which contained immobilized BSA or pea Rubisco. Rubisco LSMT was not eluted by 1 M KCl but was specifically released by S-adenosyl-L-methionine (AdoMet) or spinach Rubisco. Elution of Rubisco LSMT by AdoMet was a result of catalytic methylation of the PVDF-immobilized spinach Rubisco, and was therefore more efficient than elution by the competitive Ligand spinach Rubisco, An increase in the specific activity of Rubisco LSMT of approximately 7000-fold was achieved in one step with this affinity purification technique. Rubisco LSMT is a monomeric protein with a molecular mass of ∼60 kDa.

Original languageEnglish
Pages (from-to)528-536
Number of pages9
JournalProtein Expression and Purification
Issue number4
StatePublished - Aug 1995

ASJC Scopus subject areas

  • Biotechnology


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