US 9,428,738 B2Granted

HIV type 1 group O reverse transcriptases that are active at high temperatures

AssigneeConsejo Superior De Investigaciones Cientificas (Csic)

InventorsLuis Menéndez Arias, Tania Matamoros Grande, David Abia Holgado, Verónica Barrioluengo Fernández

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Abstract

The present invention falls within the field of biotechnology. More specifically, the invention relates to reverse transcriptases expressed and purified in bacteria and having the amino acid sequence of the reverse transcriptase of a human immunodeficiency virus type 1 (HIV-1) group O, modified at positions 358, 359 and 360; and variants of this enzyme that contain additional changes at positions 355 and 357 or at 478 or position 69 (in this case accompanied by an insertion of two amino acids). These polymerases have greater activity than the non-mutated enzyme at high temperatures (above 60° C.). In addition, they retain the capacity for DNA synthesis at temperatures greater than 70° C. Moreover, the copying fidelity of these enzymes is not significantly different from that of the non-mutated reverse transcriptase.

Claims19

Dependent claims

A polypeptide that codes for a protein with reverse transcriptase activity isolated from HIV-1 group O that has higher stability at high temperatures and has higher activity than the parent enzyme at temperatures exceeding 75° C., maintaining copying fidelity, and characterized in that its amino acid sequence has an identity of at least 80% with the parental sequence SEQ ID NO 1 and in that it comprises at least the following amino acid changes:

replacement of the original amino acid lysine (K) by the amino acid arginine (R) in the position homologous to position 358 of SEQ ID NO 1 (mutation K358R),
replacement of the original amino acid alanine (A) by the amino acid glycine (G) in the position homologous to position 359 of SEQ ID NO 1 (mutation A359G), and
replacement of the original amino acid serine (S) by the amino acid alanine (A) in the position homologous to position 360 of SEQ ID NO 1 (mutation S360A).

Dependencies are inferred from the claim text.

Facts

Application: 14/891,950Filed 8 May 2014
Priority: ES 20133070517 May 2013
Published: US 2016/0090580 A131 Mar 2016
Granted: 30 Aug 2016
Term ends: Not estimatedThe source records don’t support an estimate.

Classification8 codes

C12N 9/1276
Nucleotidyltransferases: rNA-directed DNA polymerase , i.e. reverse transcriptase or telomerase
C12P 19/34
Nucleotides: polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12Q 1/6869
Involving nucleic acids: methods for sequencing
C12Y 207/07049
Nucleotidyltransferases: rNA-directed DNA polymerase , i.e. telomerase or reverse-transcriptase