EXPRESSION OF CHICKEN LINKER HISTONES IN ESCHERICHIA-COLI - SOURCES OF PROBLEMS AND METHODS FOR OVERCOMING SOME OF THE DIFFICULTIES

Expression of histones in Escherichia coli is important in structural studies on chromatin, because it allows isotopic labeling such as deut eration and replacement of methionines with selenomethionine as well a s expression of specific domains of histones. We show that full-length H5 cannot be expressed in E. coli. We have determined that the proble m is translational rather than transcriptional. Pulse-labeling studies show that protein turnover is not the reason for lack of accumulation . On dissecting the gene, we find that the problem lies in expressing the highly charged C-terminal tail of H5. We can make progressively in creasing amounts of the tail, but at the point where over two-thirds o f this region is transcribed, the protein ceases to be made. Surprisin gly, full-length H1 is made. In vitro studies show that the H5 gene ca n be translated in a rabbit reticulocyte system but not in an E. coli system, suggesting that there may be a difference in the ability of eu karyotic and prokaryotic ribosomes to translate this message. The expr ession of the globular domains of H5 and H1 posed a different problem. There was little or no expression of some of the constructs, even tho ugh they were fragments of larger constructs that were well made. Repl acement of the first five codons downstream of the initiating ATG codo n with those optimized for E. coli, and which were AT rich, restored e xpression. This may have general implications for expression of eukary otic proteins in E. coli. (C) 1994 Academic Press, Inc.