Abstract
Flavohemoglobins, which are widely distributed in prokaryotes and eukaryotes, play key roles in oxygen (O2) transport and nitric oxide (·NO) defense. Hmp is the flavohemoglobin of Escherichia coli, and here we report that the translational fusion of Hmp to the N-terminus of heterologous proteins increases their expression in E. coli. The effect required the fusion of the proteins, and was independent of both the O2-binding and catalytic activity of Hmp. Increased expression was at the translational level, likely to be downstream of initiation, and we observed that as little as the first 100 amino acids of Hmp were sufficient to boost protein production. These data demonstrate the potential of Hmp as an N-terminal fusion tag to increase protein yield, and suggest that the utility of bacterial hemoglobins to biotechnology goes beyond their O2 transport and ·NO detoxification capabilities.
Highlights
Flavohemoglobins are composed of globin domains, which can bind heme, fused to FAD- and NAD(P)-binding domains that facilitate electron transfer [1]
E. coli flavohemoprotein (Hmp) expression in E. coli is largely regulated by NsrR, which represses its transcription in the absence of ·nitric oxide (NO), and under normal growth, Hmp is present in cells at trace levels and is induced in response to nitrosative stress [8,9]
We had observed that translationally fusing Hmp to superfold green fluorescent protein (sfGFP) with a (Gly-Ser-Ser-Gly)3 linker produced an ~3-fold increase in sfGFP under ·NO stress [28]
Summary
Flavohemoglobins are composed of globin domains, which can bind heme, fused to FAD- and NAD(P)-binding domains that facilitate electron transfer [1]. They are widely distributed in prokaryotes and eukaryotes and Hmp is the flavohemoglobin of E. coli, which is arguably the most studied [2]. Hmp is the main ·NO detoxification enzyme for E. coli at dissolved O2 concentrations ranging from fully aerobic down to. Hmp expression in E. coli is largely regulated by NsrR, which represses its transcription in the absence of ·NO, and under normal growth, Hmp is present in cells at trace levels and is induced in response to nitrosative stress [8,9]
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