The production of soluble and correctly folded recombinant bovine beta-lactoglobulin variants A and B in Escherichia coli for NMR studies.
The production of soluble and correctly folded eukaryotic proteins in prokaryotic systems has always been hampered by the difference in or lack of cell machinery responsible for folding, post-translation modification and secretion of the proteins involved. In the case of bovine beta-lactoglobulin (BLG), a major cow's milk allergen and a protein widely used for protein folding studies, a eukaryotic yeast expression system has been the preferred choice of many researchers, particularly for the production of isotopically labeled protein required for NMR studies. Although this system yields high amounts of recombinant protein, the BLG produced is usually associated with extracellular polysaccharides, which is problematic for NMR analysis. In our study we show that when co-expressed with the signal-sequence-less disulfide bond isomerase (Delta ssDsbC) in the dual expression vector, pETDUET-1, both BLG A and BLG B can be reproducibly produced in a soluble form. Expression was carried out in Escherichia coli Origami(DE3), a trxB/gor mutant for thioredoxin- and glutathione reductase, which allows for proper formation of disulfide bonds in the cytoplasm. The protein was purified by anion exchange chromatography followed by salting-out at low pH and size exclusion chromatography. Our expression system is able to consistently produce milligram quantities of correctly folded BLG A and B with no additional amino acid residues at the N-terminus, except for a methionine. (15)N-labeled BLG A and B, prepared and purified using this method, produced HSQC spectra typical of native bovine BLG.
Ponniah
Komala
K
Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Loo
Trevor S
TS
Edwards
Patrick J B
PJ
Pascal
Steven M
SM
Jameson
Geoffrey B
GB
Norris
Gillian E
GE
D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007782:Lactoglobulins / Q000096:biosynthesis* / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012995:Solubility
D004220:Disulfides; D007782:Lactoglobulins; D011994:Recombinant Proteins