High-level expression of human histone H4 in E. coli.

The relationship between structure and function of the nucleosome is an area of exciting and intense research, exemplified by the recent report of the first high-resolution crystallography data (5). The nucleosome, the basic unit of chromatin, consists of DNA wrapped around the core histones H3, H4, H2A and H2B. This structure serves not only to package and organize the two meters of DNA found in a typical human nucleus, but it also plays a role in gene expression and DNA repair (4,10,13,14). Acetylation of the N-terminal tails of histone proteins has long been associated with transcriptionally active chromatin, and recent studies have shown that transcriptional regulators contain histone acetylase and/or deacetylase activities (1,9). To demonstrate the specific effect of the nucleosome at the molecular level, it is essential to obtain a homogeneous population of nucleosomes reconstituted from their individual histone and DNA components. Although expression of histones H2A, H2B and H3 was described by Luger et. al. (6), expression of H4 has been problematic, even as a fusion protein, and has not been achieved without extensive alteration of the cDNA. Here we report for the first time, to our knowledge, a simple method for high-level expression of human histone H4 protein in E. coli without modification of the cDNA. The procedure involves a unique combination of the appropriate expression vector, bacteria host cell line and optimized growth conditions. Cloning. The coding region of human H4 cDNA (pF0002), kindly provided by Drs. Janet Stein and Gary Stein (12), was amplified by the polymerase chain reaction (PCR) using primers containing an NdeI restriction endonuclease site at the start codon and a BamHI site following the stop codon. H4 was subcloned into the pET-11a E. coli expression vector (creating H4: pET11a) in which protein expression is under control of the bacteriophage T7lac promoter sequence (Novagen, Madison, WI, USA). Thus, there is no background induction of expression during routine subcloning procedures in host cells that do not contain T7 RNA polymerase, such as DH5α (Life Technologies, Gaithersburg, MD, USA) and Epicurean coli XL-1 Blue (Stratagene, La Jolla, CA, USA), thus overcoming any direct toxic effects of histone protein. Expression. Once the sequence of H4:pET11a was verified by dideoxy sequencing, the construct was transformed into four bacteria host cell lines: (i) BL21(DE3), deficient in lon and