Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombin-cleavable fusion protein in Escherichia coli.
We have developed a high-level production system for the C-terminal domain of secretory leukoprotease inhibitor (SLPI) to investigate its pharmacological activities. A gene for the C-terminal domain of SLPI, (Asn55-Ala 107)SLPI, was constructed from chemically synthesized deoxyoligonucleotides. It was fused to a gene for the N-terminal portion of human growth hormone via a DNA sequence encoding Leu-Val-Pro-Arg, which can be cleaved by thrombin. The fused gene was expressed in Escherichia coli under the control of a trp promoter, and the fusion protein was obtained as an inclusion body. After sulfonation of the cysteine residues, the sulfonated fusion protein was cleaved at the desired site by thrombin. Sulfonated (Asn55-Ala107) SLPI was refolded in Tris buffer containing reduced and oxidized glutathione. The resulting (Asn55-Ala107) SLPI was purified by cation-exchange chromatography and reverse-phase high performance liquid chromatography. The final yield was 50 mg/I culture. (Asn55-Ala107) SLPI was as active against elastase as, but had less trypsin inhibitory activity than, native SLPI. This system is suitable for the large-scale production of the C-terminal domain of SLPI, which is an elastase-specific inhibitor.
Masuda
K
K
Teijin Institute for Biomedical Research, Tokyo, Japan.
Kamimura
T
T
Kanesaki
M
M
Ishii
K
K
Imaizumi
A
A
Sugiyama
T
T
Suzuki
Y
Y
Ohtsuka
E
E
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005813:Genes, Synthetic; D005978:Glutathione / Q000494:pharmacology; D019382:Human Growth Hormone / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D014176:Protein Biosynthesis; D017510:Protein Folding; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D053481:Secretory Leukocyte Peptidase Inhibitor; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
D000596:Amino Acids; D010446:Peptide Fragments; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D011993:Recombinant Fusion Proteins; C506814:SLPI protein, human; D053481:Secretory Leukocyte Peptidase Inhibitor; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D019382:Human Growth Hormone; C011119:S-sulphocysteine; D010196:Pancreatic Elastase; D013917:Thrombin; D005978:Glutathione; D003545:Cysteine