Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. 131 44

The existence of rat 18-kilodalton (kDa) prorelaxin, which has been postulated from the coding sequence of cloned cDNA and the results of cell-free translation studies, has been directly demonstrated in rat ovaries with antibodies against bacterially expressed rat prorelaxin. The peptide expressed in E. coli from a rat prorelaxin cDNA construct was comprised of the B- and A-chains of relaxin and a 105-amino acid connecting region. Immunoreactive bands of 18 and 16.5 kDa were shown in ovaries from day 20 pregnant rats. Partial amino acid sequence analysis of both peptides revealed that they had identical N-terminal sequences, corresponding to rat prorelaxin. Both 18- and 16.5-kDa bands were present only from midpregnancy until near term, when they declined sharply. These changes in the concentration of 18-kDa prorelaxin match changes in preprorelaxin mRNA levels, suggesting that relaxin synthesis is regulated at the transcriptional level and not by protein processing. Prorelaxin was transiently secreted by COS-1 cells transfected with preprorelaxin cDNA. Treatment of culture medium with trypsin resulted in the appearance of material corresponding in size to mature relaxin. Thus, correctly folded prorelaxin appears to be a suitable precursor for relaxin. The combined concentrations of 18- and 16.5-kDa peptides in ovaries on day 20 of pregnancy were considerably more than 30 times greater than that of relaxin, however, suggesting that prorelaxin might also be more than a precursor per se.
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PMID:Demonstration of relaxin precursors in pregnant rat ovaries with antisera against bacterially expressed rat prorelaxin. 154 14

Relaxin is a peptide hormone produced by the corpora lutea of ovaries during pregnancy, softening and lengthening the ligaments of the pelvis and softening the cervix in order to make childbirth easier. In attempts to determine the nucleotide sequence coding for relaxin, recombinant DNA techniques were used to obtain a cDNA clone bank from total mRNA isolated from the ovaries of pigs in late pregnancy. Clones were screened using cDNA initiated by synthetic oligonucleotide primers coding for the Trp Val Glu Ile sequence of the porcine relaxin B chain. The synthetic undecamer [5'-ATCTCCACCCA-3'] was found to prime a specific 32P-labeled cDNA of approximately 300 nucleotides containing B chain and signal peptide coding sequences, as verified by nucleic acid sequence analysis. This cDNA was used to probe the ovarian clone bank. Several clones containing large inserts which hybridized to this probe were subjected to sequence analysis and some of these were found to contain the preprorelaxin coding region, comprising a signal peptide of 24 amino acids, a B chain of 32 amino acids, a large C peptide of 104 amino acids, and an A chain of 22 amino acids. From the amino acid sequence of prorelaxin derived in this way, it appears that the processing of prorelaxin involves two enzymes with chymotrypsin-like and trypsin-like specificity, respectively. In comparisons of porcine and rat preprorelaxins, the C region had as much amino acid sequence homology as the B and A chains. The C region is also rich in charged amino acids, suggesting a role for it beyond simply ensuring proper disulfide bond formation.
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PMID:Porcine relaxin: molecular cloning and cDNA structure. 689 21