Other Prokaryotic Expression Systems

Although bacterial hosts can be useful for protein production, E. coli and other prokaryotic expression systems have some limitations. For example, they are incapable of posttranslational modifications such as glycosylation and as a consequence many eukaryotic proteins produced in prokaryotic expression systems are nonfunctional (Rai and Padh, 2001). However, in the case of most peptides, including self-assembling peptides, such modifications are currently not required. Of course, this limitation may have implications for future... 

Copper ion homeostasis in prokaryotes involves Cu ion efflux and sequestration. The proteins involved in these processes are regulated in their biosynthesis by the cellular Cu ion status. The best studied bacterial Cu metalloregulation system is found in the gram-positive bacterium Enterococcus hirae. Cellular Cu levels in this bacterium control the expression of two P-type ATPases critical for Cu homeostasis (Odermatt and Solioz, 1995). The CopA ATPase functions in Cu ion uptake, whereas the CopB ATPase is a Cu(I) efflux pump (Solioz and Odermatt, 1995). The biosynthesis of both ATPases is regulated by a Cu-responsive transcription factor, CopY (Harrison et al., 2000). In low ambient Cu levels Cop Y represses transcription of the two ATPase genes. On exposure to Cu(I), CopY dissociates from promoter/operator sites on DNA with a for Cu of 20 jlM (Strausak and Solioz, 1997). Transcription of copA and copB proceeds after dissociation of CuCopY. The only other metal ions that induce CopY dissociation from DNA in vitro are Ag(I) and Cd(II), although the in vivo activation of copA and copB is specihc to Cu salts. The CuCopY complex is dimeric with two Cu(I) ions binding per monomer (C. T. Dameron, personal communication). The structural basis for the Cu-induced dissociation of CopY is unknown. Curiously, CopY is also activated in Cu-dehcient cells, but the mechanism is distinct from the described Cu-induced dissociation from DNA (Wunderh-Ye and Solioz, 1999). 

This report describes the presence of significant amounts of e-N-acetyllysine in rpST and rbST, eukaryotic proteins expressed in a prokaryotic system. Initial work from our laboratory has also demonstrated the presence of this modified amino acid in two other recombinant eukaryotic proteins expressed in E. coli, bovine placental lactogen and human tissue factor pathway inhibitor (16). ESMS, amino acid sequencing and amino acid analyses were utilized to demonstrate the presence of e-N-acetyllysine in these two recombinant proteins. These data established that this modified amino acid is present in several distinct recombinant eukaryotic proteins expressed in E. coli. 

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