High-level expression of a gene encoding the human complement factor C5a in Escherichia coli.
The synthetic C5a gene was initially found to be expressed poorly in Escherichia coli. We undertook studies to determine the reasons for poor expression and to increase expression. The work was focused on the role of the mRNA structure in C5a expression and stability of its product in E. coli. We present data on the effects of varying the sequence at the 5' end of mRNA as well as different ribosome-binding sites on expression. Evaluation of the stability of C5a showed rapid degradation of C5a in wild-type E. coli (half-life 3-5 min). Screening of several protease-deficient strains of E. coli showed that C5a was much more stable in an htpR strain carrying a mutation in the sigma subunit of RNA polymerase that is specific for heat shock promoters. The mutation is associated with a proteolytic deficiency. The half-life of C5a was increased to 20 min. By manipulating the expression vector, the regulatory region for the C5a gene, the host strain, growth conditions and methods for recovering the protein, C5a levels were increased 300-fold over previously reported amounts to about 3% of total cellular protein.
D001483:Base Sequence; D003001:Cloning, Molecular; D003182:Complement C5 / Q000235:genetics* / Q000378:metabolism; D015936:Complement C5a; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D005838:Genotype; D006801:Humans; D007700:Kinetics; D010957:Plasmids; D012270:Ribosomes / Q000378:metabolism; D013045:Species Specificity; D014158:Transcription, Genetic
D003182:Complement C5; D004274:DNA, Recombinant; D015936:Complement C5a