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Home / Equine Research Bank / Biochem J / Molecular cloning and expression of an intracellular serpin:...
The Biochemical journal1993; 293 ( Pt 1)(Pt 1); 187-193; doi: 10.1042/bj2930187

Molecular cloning and expression of an intracellular serpin: an elastase inhibitor from horse leucocytes.

Abstract: Horse blood leucocytes contain an elastase inhibitor (HLEI) belonging to the serpin family. Poly(A)+RNA isolated from these cells was used to construct a cDNA library in lambda gt10, which was first screened with a synthetic degenerate oligonucleotide probe corresponding to the amino acid sequence of the reactive centre of the inhibitor. Three clones were obtained covering the entire coding region of the protein. Sequencing of these clones showed identity with the amino acid sequence obtained from Edman degradation of the elastase inhibitor. The coding sequence of the HLEI cDNA was cloned into the bacterial expression vector pKK233-2 and expressed in Escherichia coli cells. Transformed bacteria expressed significant amounts of the protein, which was immunoprecipitated with a specific anti-HLEI antiserum. Furthermore, HLEI expressed in bacteria inhibited the activity of elastase but not trypsin.
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Publication Date: 1993-07-01 PubMed ID: 7687128PubMed Central: PMC1134338DOI: 10.1042/bj2930187Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research focuses on the successful molecular cloning and expression of a horse blood cell-based elastase inhibitor known as HLEI, a member of the serpin family.

Process of HLEI Cloning

  • Researchers first isolated poly(A)+RNA from horse blood leucocytes. Polyadenylated RNA, as it is also known, is a type of RNA molecule that has been modified with a poly-A-tail, which typically promotes its stability and translation.
  • This poly(A)+RNA served to construct a cDNA library in a bacteriophage cDNA library system known as lambda gt10. A cDNA library is a combination of cloned cDNA fragments that forms a stable representation of the total complement of mRNA present in a particular tissue.
  • The cDNA library was screened using a synthetic degenerate oligonucleotide probe. The probe corresponds to the amino acid sequence of the reactive centre of the elastase inhibitor HLEI. A synthetic degenerate oligonucleotide is a short strand of artificially generated DNA or RNA that has the ability to bind to matching sequences and is often used in gene targeting and detection.

Identification of the HLEI Clone

  • The above process led to the identification of three clones that cover the entire coding region of the elastase inhibiting protein.
  • When sequenced, these clones were found to align with the amino acid sequence obtained from a process called Edman degradation of the elastase inhibitor. Edman degradation is an iterative method used to understand the amino acid sequence of a peptide, in this case, the elastase inhibitor HLEI.

Bacterial Expression of the HLEI Clone

  • The coding sequence of the HLEI cDNA was then cloned into a bacterial expression vector, referred to as pKK233-2.
  • This cloned material was then expressed in Escherichia coli (E. coli) cells.
  • Post transformation, it was observed that the E. coli expressed significant quantities of the HLEI protein.

Efficacy of the HLEI

  • Researchers then verified the expression of the HLEI protein using a specific anti-HLEI antiserum. An antiserum is a blood serum that contains antibodies against the protein, and is used for testing whether the protein indeed has been expressed.
  • It was also found that the HLEI expressed in E. coli was effective in inhibiting the activity of elastase, a type of enzyme that degrades elastin – an essential protein in connective tissues.
  • However, it did not inhibit the activity of trypsin, another type of enzyme that contributes to the degradation of proteins.

Cite This Article

APA
Kordula T, Dubin A, Schooltink H, Koj A, Heinrich PC, Rose-John S. (1993). Molecular cloning and expression of an intracellular serpin: an elastase inhibitor from horse leucocytes. Biochem J, 293 ( Pt 1)(Pt 1), 187-193. https://doi.org/10.1042/bj2930187

Publication

The Biochemical journal
ISSN: 0264-6021
NlmUniqueID: 2984726R
Country: England
Language: English
Volume: 293 ( Pt 1)
Issue: Pt 1
Pages: 187-193

Researcher Affiliations

Kordula, T
  • Institute of Molecular Biology, Jagiellionian University, Krakow, Poland.
Dubin, A
    Schooltink, H
      Koj, A
        Heinrich, P C
          Rose-John, S

            MeSH Terms

            • Amino Acid Sequence
            • Animals
            • Base Sequence
            • Blotting, Northern
            • Cloning, Molecular
            • Horses
            • Leukocyte Elastase
            • Leukocytes / metabolism
            • Molecular Sequence Data
            • Pancreatic Elastase / antagonists & inhibitors
            • Poly A / chemistry
            • RNA / chemistry
            • RNA, Messenger / chemistry
            • RNA, Messenger / isolation & purification
            • Serpins / biosynthesis
            • Serpins / blood

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            Citations

            This article has been cited 4 times.
            1. Hayashi KG, Ushizawa K, Hosoe M, Takahashi T. Differential gene expression of serine protease inhibitors in bovine ovarian follicle: possible involvement in follicular growth and atresia.. Reprod Biol Endocrinol 2011 May 27;9:72.
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