Efficient production of a folded and functional, highly disulfide-bonded beta-helix antifreeze protein in bacteria.
The Tenebrio molitor thermal hysteresis protein has a cysteine content of 19%. This 84-residue protein folds as a compact beta-helix, with eight disulfide bonds buried in its core. Exposed on one face of the protein is an array of threonine residues, which constitutes the ice-binding face. Previous protocols for expression of this protein in recombinant expression systems resulted in inclusion bodies or soluble but largely inactive material. A long and laborious refolding procedure was performed to increase the fraction of active protein and isolate it from inactive fractions. We present a new protocol for production of fully folded and active T. molitor thermal hysteresis protein in bacteria, without the need for in vitro refolding. The protein coding sequence was fused to those of various carrier proteins and expressed at low temperature in a bacterial strain specially suited for production of disulfide-bonded proteins. The product, after a simple and robust purification procedure, was analyzed spectroscopically and functionally and was found to compare favorably to previously published data on refolded protein and protein obtained from its native source.
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
Maya
M
Bar
Roy
R
Bar-Ziv
Tali
T
Scherf
Deborah
D
Fass
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013712:Tenebrio / Q000737:chemistry* / Q000235:genetics
D021301:Antifreeze Proteins; D004220:Disulfides