Positive and negative immunomodulation by opioid peptides

The Link module, a 98-amino-acid domain found in hyaluronan binding proteins of human tumor necrosis factor stimulated gene 6 was overexpressed in Escherichia coli. Electrospray ionization mass spectrometry revealed that only 50% of the expressed protein had the expected wild-type molecular weight, with the remaining material having between 1 and 4 arginine to lysine substitutions, arising due to misincorporation at AGA codons. The level of misincorporation was almost completely abolished by mutation of the 4 AGA codons to CGT. This mutation to high-usage arginine codons also increased the level of heterologous protein expression. Refolding of the Link module, which occurred during the purification procedure, gave two species with different disulfide bond organizations that could be separated by high-performance liquid chromatography. One of these had a disulfide bond arrangement consistent with that found in other Link modules and, by nuclear magnetic resonance spectroscopy, was shown to be folded.