High-level expression of active human cystatin C in Escherichia coli.
Expression of the human cysteine proteinase inhibitor, cystatin C (CysC) in the cytoplasm of Escherichia coli was studied using a cDNA fragment encoding the cysteine proteinase inhibitor controlled by the phage lambda pR/cI857 system. The yield of CysC was low, probably due to proteolytic degradation. By fusing the cysC cDNA to a DNA fragment encoding the signal peptide of the E. coli outer membrane protein A, it was possible to produce a substantial amount of CysC in the periplasm. The processing of the signal peptide was shown to be quantitative and to result in CysC with the correct N-terminal amino acid. Yields higher than 1000 micrograms CysC/ml can be obtained by initiating the product formation at a moderate temperature (40 degrees C) late in an optimized fermentation process. A method that gives selective extraction of the periplasmic proteins and at the same time stabilizes CysC has been used.
Dalbøge
H
H
Nordisk Gentofte, A/S, DK-2820, Denmark.
Jensen
E B
EB
Tøttrup
H
H
Grubb
A
A
Abrahamson
M
M
Olafsson
I
I
Carlsen
S
S
D010582:Bacteriophage lambda / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D055316:Cystatin C; D015891:Cystatins / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic
C525030:CST3 protein, human; D055316:Cystatin C; D015891:Cystatins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D004247:DNA; D003545:Cysteine