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Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombin-cleavable fusion protein in Escherichia coli. 1996

K Masuda, and T Kamimura, and M Kanesaki, and K Ishii, and A Imaizumi, and T Sugiyama, and Y Suzuki, and E Ohtsuka
Teijin Institute for Biomedical Research, Tokyo, Japan.

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.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010196 Pancreatic Elastase A protease of broad specificity, obtained from dried pancreas. Molecular weight is approximately 25,000. The enzyme breaks down elastin, the specific protein of elastic fibers, and digests other proteins such as fibrin, hemoglobin, and albumin. EC 3.4.21.36. Elastase,Pancreatopeptidase,Elastase I,Pancreatic Elastase I,Elastase I, Pancreatic,Elastase, Pancreatic
D010446 Peptide Fragments Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques. Peptide Fragment,Fragment, Peptide,Fragments, Peptide
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D011993 Recombinant Fusion Proteins Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes. Fusion Proteins, Recombinant,Recombinant Chimeric Protein,Recombinant Fusion Protein,Recombinant Hybrid Protein,Chimeric Proteins, Recombinant,Hybrid Proteins, Recombinant,Recombinant Chimeric Proteins,Recombinant Hybrid Proteins,Chimeric Protein, Recombinant,Fusion Protein, Recombinant,Hybrid Protein, Recombinant,Protein, Recombinant Chimeric,Protein, Recombinant Fusion,Protein, Recombinant Hybrid,Proteins, Recombinant Chimeric,Proteins, Recombinant Fusion,Proteins, Recombinant Hybrid
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D003545 Cysteine A thiol-containing non-essential amino acid that is oxidized to form CYSTINE. Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005813 Genes, Synthetic Biologically functional sequences of DNA chemically synthesized in vitro. Artificial Genes,Synthetic Genes,Artificial Gene,Gene, Artificial,Gene, Synthetic,Genes, Artificial,Synthetic Gene

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