Document Detail


Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.
MedLine Citation:
PMID:  1314544     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.
Authors:
G K Reddy; S Gunwar; C B Green; D T Fei; A B Chen; S C Kwok
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Archives of biochemistry and biophysics     Volume:  294     ISSN:  0003-9861     ISO Abbreviation:  Arch. Biochem. Biophys.     Publication Date:  1992 May 
Date Detail:
Created Date:  1992-05-15     Completed Date:  1992-05-15     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0372430     Medline TA:  Arch Biochem Biophys     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  579-85     Citation Subset:  IM    
Affiliation:
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Blotting, Western
Chromatography, High Pressure Liquid
Chromatography, Ion Exchange
Cloning, Molecular / methods
Cyclic AMP / metabolism
Endometrium / drug effects,  metabolism
Endopeptidases / metabolism
Escherichia coli / genetics*
Female
Humans
Molecular Sequence Data
Plasmids
Protein Precursors / isolation & purification*,  metabolism,  pharmacology
Recombinant Proteins / isolation & purification*,  pharmacology
Relaxin / isolation & purification*,  metabolism,  pharmacology
Restriction Mapping
Swine
Grant Support
ID/Acronym/Agency:
1 S10RR05554-01/RR/NCRR NIH HHS; HD24599/HD/NICHD NIH HHS
Chemical
Reg. No./Substance:
0/Protein Precursors; 0/Recombinant Proteins; 60-92-4/Cyclic AMP; 87004-01-1/prorelaxin; 9002-69-1/Relaxin; EC 3.4.-/Endopeptidases

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