Intellectual Property Office
Non-Confidential Disclosures
Novel Cold-Active and Heat-Labile Phosphatase Activities From a Psychrophilic Isolate
PSU Invention Disclosure No. 1436
Field of the Invention:
alkaline phosphatase, enzymes, molecular biology tool
Inventors:
J. Brenchley, J. Loveland-Curtze, P. DePrada
Background:
A group of Penn State researchers has discovered a natural psychrophilic microorganism, which produces a proprietary alkaline phosphatase that could replace those presently used in cloning procedures. First, the enzyme can be easily obtained in a highly purified preparation by following a simple and inexpensive procedure. Second, this proprietary alkaline phosphatase does not require any special co-factor for activity and is active in buffers commonly used in molecular biology. Most importantly, the heat lability of this enzyme provides an easy alternative to the time consuming phenol extraction required with commonly used enzymes
Invention description:
This enzyme utilizes many different phosphorylated substrates, including nucleotides and sugars. A highly purified preparation can be obtained using a simple chromatographic step. Treatment of the enzyme preparation yields a sample that is free of nuclease activities and suitable for use as a molecular biology reagent. The enzyme possesses an unusually high activity at low temperatures; activity can be detected at temperatures as low as 0oC, with a peak of optimal activity at 45oC. Incubation of the proprietary alkaline phosphatase at temperatures above 65oC causes an irreversible loss of enzymatic activity.
The inventors tested this enzyme in several ways to determine its usefulness as a molecular biology tool: This enzyme’s ability to dephosphorylate linearized plasmid DNA and prevent recircularization upon treatment with DNA-ligase is comparable to commercially available alkaline phosphatases. Electrophoretic analysis demonstrated that the proprietary enzyme prevented religation of plasmids treated with three restriction enzymes which generate different DNA ends: EcoRI (protruding), SmaI (blunt) and PstI (recessive). In addition, treatment of digested plasmid DNA with this isolate’s alkaline phosphatase significantly reduces the number of transformants containing plasmids that religated without containing an insert. The proprietary enzyme-treated pUC18 could also be used to clone DNA fragments as effectively as the commercial control.
The results show that this proprietary phosphatase effectively dephosphorylates linearized plasmid DNA and can be readily heat inactivated prior to the addition of DNA fragments and incubation with ligase. Our results suggest that this alkaline phosphatase could be readily produced, easily purified, packaged and marketed as a convenient, useful enzyme for recombinant DNA experiments. Its rapid inactivation at relatively low temperatures provides an excellent alternative to the more time consuming phenol/chloroform extraction.
Advantages:
- can be readily heat inactivated
- rapid inactivation at relatively low temperatures
- could be readily produced, easily purified, packaged and marketed as a convenient, useful enzyme for recombinant DNA experiments
Contact:
Dr. James F. Kolonay
Technology Licensing Officer
Intellectual Property Office
The Pennsylvania State University
113 Technology Center
University Park, PA 16802-7000
Phone: (814) 863-7070
Fax: (814) 865-3591
E-mail: jfk11@psu.edu
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