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Molecular cloning and expression of cDNA for human antileukoprotease from cervix uterus
Abstract:
We have isolated cDNA clones for the human antileukoprotease HUSI-I, an elastase inhibitor, from a library, containing cDNA inserts made from human cervix uterus. A library of 10 000 recombinants was screened using a mixture of 16 different oligodeoxyribonucleotides which correspond to amino acids 79-84 and one 20mer oligodeoxyribonucleotide corresponding to amino acids 19-26. Two overlapping cDNA clones, containing the entire coding sequence and part of the 5'- and 3'-untranslated region, were isolated. DNA sequence data showed that our clone corresponds with the available protein sequence data. For expression, the cDNA fragment was inserted in a derivative of plasmid pPLc236 and expressed under the control of lambda PL promoter. Expression of antileukoprotease was proven by Western blot analysis and inhibition of chymotrypsin.
Insights
Researchers isolated human antileukoprotease (HUSI-I) cDNA clones from human cervix uterus tissue. These clones encode an elastase inhibitor, successfully expressed and validated through Western blot and functional assays.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Human antileukoprotease (HUSI-I) is an elastase inhibitor.
- Understanding its genetic basis is crucial for therapeutic development.
Purpose of the Study:
- To isolate and characterize cDNA clones encoding human antileukoprotease (HUSI-I).
- To express the HUSI-I protein for functional analysis.
Main Methods:
- Screening of a human cervix uterus cDNA library using degenerate oligodeoxyribonucleotides.
- DNA sequencing of isolated cDNA clones.
- Recombinant expression of HUSI-I using a plasmid vector under lambda PL promoter control.
- Validation of expression via Western blot analysis and enzyme inhibition assays.
Main Results:
- Isolation of two overlapping cDNA clones containing the full coding sequence and untranslated regions of HUSI-I.
- Confirmation that the isolated cDNA sequence aligns with existing protein data.
- Successful expression of functional HUSI-I, demonstrated by Western blot and chymotrypsin inhibition.
Conclusions:
- The study successfully isolated and characterized cDNA clones for human antileukoprotease (HUSI-I).
- The expressed HUSI-I protein retains its elastase inhibitory function, paving the way for further research and potential applications.