Expression of chicken linker histones in E. 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 deuteration and replacement of methionines with selenomethionine as well as expression of specific domains of histones. We show that full-length H5 cannot be expressed in E. coli. We have determined that the problem 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 increasing amounts of the tail, but at the point where over two-thirds of this region is transcribed, the protein ceases to be made. Surprisingly, full-length H1 is made. In vitro studies show that the H5 gene can 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 eukaryotic and prokaryotic ribosomes to translate this message. The expression of the globular domains of H5 and H1 posed a different problem. There was little or no expression of some of the constructs, even though they were fragments of larger constructs that were well made. Replacement of the first five codons downstream of the initiating ATG codon with those optimized for E. coli, and which were AT rich, restored expression. This may have general implications for expression of eukaryotic proteins in E. coli.
Gerchman
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Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
Graziano
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Ramakrishnan
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D006657:Histones / Q000096:biosynthesis* / Q000302:isolation & purification; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D016364:Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic
D003062:Codon; D006657:Histones; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine