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Cloning, functional expression, and characterization of recombinant pig liver esterase
S Lange1, A Musidlowska, C Schmidt-Dannert
1Institute for Technical Biochemistry, Stuttgart University, Allmandring 31, 70569 Stuttgart, Germany.
Chembiochem : a European Journal of Chemical Biology
|February 6, 2002
Summary
Researchers successfully cloned and expressed pig liver esterase (PLE) in yeast by removing a retention signal. The recombinant PLE (rPLE) showed high activity and specific properties, confirming the expression of a single isoenzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Pig liver esterase (PLE) is an enzyme with esterolytic activity.
- Previous studies have characterized commercial PLE preparations, but heterologous expression has been challenging.
Purpose of the Study:
- To clone and express pig liver esterase (PLE) in a heterologous system.
- To characterize the recombinant enzyme and compare its properties to commercial preparations.
Main Methods:
- Determined the N-terminal amino acid sequence of commercial PLE.
- Cloned the PLE cDNA using reverse transcription and PCR.
- Expressed the cDNA in Pichia pastoris after deleting a putative endoplasmic reticulum retention signal.
- Purified and characterized the recombinant PLE (rPLE) using biochemical assays and gel electrophoresis.
Main Results:
- Successfully expressed and secreted active rPLE in P. pastoris.
- rPLE exhibited high specific activity (approx. 600 U/mg) and a Vmax/Km of 139 µmol/min/mM with p-nitrophenyl acetate.
- rPLE showed a high preference for proline-beta-naphthylamide, characteristic of the gamma subunit of PLE.
- Optimal activity was observed at pH 8.0 and 60°C.
- rPLE molecular weight was ~61-62 kDa, and it functions as a trimer.
Conclusions:
- Deletion of the C-terminal retention signal (His-Ala-Glu-Leu) was crucial for successful heterologous expression and secretion of active PLE.
- The recombinant enzyme represents a single PLE isoenzyme with properties consistent with published data.
- This study provides a method for producing functional PLE for further research.