Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.
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.
Reddy
G K
GK
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160.
Gunwar
S
S
Green
C B
CB
Fei
D T
DT
Chen
A B
AB
Kwok
S C
SC
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D000242:Cyclic AMP / Q000378:metabolism; D004717:Endometrium / Q000187:drug effects / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011498:Protein Precursors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D012065:Relaxin / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D013552:Swine
D011498:Protein Precursors; D011994:Recombinant Proteins; C038656:prorelaxin; D012065:Relaxin; D000242:Cyclic AMP; D010450:Endopeptidases