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Journal of Biochemistry Advance Access originally published online on May 7, 2008
Journal of Biochemistry 2008 144(2):223-233; doi:10.1093/jb/mvn061
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© 2008 The Japanese Biochemical Society

Protein Methyltransferase Activities in Commercial In vitro Translation Systems

Robert B. Denman*

Biochemical Molecular Neurobiology Laboratory, Department of Molecular Biology, New York State Institute for Basic Research, In Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314

*To whom correspondence should be addressed. Tel: 718 494-5199, Fax: 718 494-5905, E-mail: rbdenman{at}yahoo.com

Received April 18, 2008; Accepted April 25, 2008


  Abstract

Protein arginine methylation is a well-known post-translational modification that has been shown to occur in rabbit reticulocyte in vitro translation lysates (RRL); however, it is not known whether this is a general feature of in vitro-produced proteins from other eukaryotic cell-free translation systems, particularly insect-derived lysates (ICL). Because methylation can affect protein localization, RNA binding and protein–protein interactions this may be of great importance as in vitro-produced proteins are often used in assays of protein function. Here, I report the presence of base-stable and base-labile methyltransferase activities in RRL, ICL and wheat germ in vitro extracts (WGE). Indeed, the presence of CARM1 in RRL and ICL and a class II protein arginine methyltransferase activity (PRMT5/7) is documented in all three systems. Additionally, the lysine methyltransferase that modifies eukaryotic elongation factor 1A (eEF-1A) was detected in ICL and WGE. Importantly, using a defined set of substrates under identical conditions I show that all three in vitro systems contain different complements of the various methyltransferases. These data suggest that three systems can be used in a complementary fashion to investigate the effect(s) of post-translational modification on protein function.

Key Words: fragile X mental retardation protein, histone, myelin basic protein, protein arginine methyltransferase, protein lysine methyltransferase

Abbreviations: CARM1/PRMT4, Coactivator of arginine methylation 1; eEF-1A, eucaryotic elongation factor 1A; ICL, insect-derived lysates; MT, methyltransferase; PKMT, protein lysine methyltransferase; PRMT, protein arginine methyltransferase; RRL, rabbit reticulocyte in vitro translation lysates; SAH, S-adenosyl-homocysteine; SAM, S-adenosyl-methionine; WGE, wheat germ in vitro extract


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