Overexpression, purification, and refolding of link module from human TSG-6 in Escherichia coli: effect of temperature, media, and mutagenesis on lysine misincorporation at arginine AGA codons.
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.
Day
A J
AJ
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.
Aplin
R T
RT
Willis
A C
AC
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D016326:Extracellular Matrix Proteins; D005347:Fibroblasts; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011509:Proteoglycans; D017421:Sequence Analysis; D013696:Temperature; D000099249:Proteoglycan Link Protein
D001120:Arginine; D015815:Cell Adhesion Molecules; D004220:Disulfides; D016326:Extracellular Matrix Proteins; D008239:Lysine; D011506:Proteins; D011509:Proteoglycans; D000099249:Proteoglycan Link Protein; C072273:TNFAIP6 protein, human