Preparation of Recombinant Histone H3 as a Substrate for Protein Kinase Assays

✍ Scribed by Sunke Himpel; Hans-Georg Joost; Walter Becker

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 297 KB
• Volume: 274
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4205

✦ Synopsis

Histones from calf thymus are widely utilized as in vitro substrates for protein kinases [e.g., cAMP-dependent protein kinase, protein kinase C, insulin receptor kinase (1-3)]. We have recently used histone H3 as an in vitro substrate for protein kinases of the DYRK family (4). However, even highly purified histones from eukaryotic sources are not ideally suitable for enzymatic analyses as they are necessarily heterogeneous due to posttranslational modifications and due to the presence of isoforms with minor sequence differences (5,6). We reasoned that this problem can be avoided by the use of recombinant histones. The only published method to prepare recombinant histones (7) takes considerable time and effort, since the intended use of the histones in this study (in vitro reconstitution of nucleosomes) required extremely pure proteins. We have therefore developed a faster and simpler protocol that allows us to prepare bacterially expressed recombinant histone H3 as a well-defined, homogeneous substrate for enzymatic studies of protein kinases.

Construction of the expression plasmid. The coding region of histone H3 was amplified by PCR from random-primed rat lung cDNA with the forward primer 5Ј-TCATATGACCATGGCCCGTACNAA-3Ј, which contained an endogenous NcoI recognition site at the start codon (underlined), and the reverse primer 5Ј-CCCTCTCGAGACGAATGCG-3Ј, which created a new XhoI site preceding the stop codon. The PCR product was cloned with blunt ends into the SmaI site of pUC18. The engineered restriction sites were used to subclone the cDNA into the T7 RNA polymerase highexpression system vector pET-28a(ϩ) (Novagen), gen-