New England Biolabs, Inc.

Francine B Perler

Donald G Comb

William E Jack

Rebecca B Kucera

Eric E Adam

Ming-Qun Xu

Maurice W Southworth

Robert A Hodges

XiaQiang Hong

Jhon Santiago Mejia

Clotilde K Carlow

Christopher J Noren

Clotilde K S Carlow

Shaorong S Chong

In accordance with the present invention, there are provided selection systems and methods for screening for agents that control splicing of inteins in their native host protein (extein) or in homologous exteins. Specifically, there are provided positive genetic selection systems for the screening of agents which inhibit or activate protein splicing which comprise: a host cell containing a chromosomal gene encoding either a drug-resistant form of a target enzyme or a wild-type target enzyme, and a plasmid-borne gene encoding either a drug-sensitive form of the target enzyme, which is dominantly cytotoxic upon interaction with the drug, or a dominantly cytotoxic form of the target enzyme. In these systems the plasmid-borne gene contains an intein, and the inhibition or activation of splicing of the dominant cytotoxic form of the target enzyme by a given reagent results in the survival or death of the host cell. More specifically, positive genetic selection systems which utilize theGyrA intein orDnaB helicase intein are provided. Similar reporter systems utilizing native or homologous exteins and systems utilizing controllable inteins are provided, as are methods of controlling in vivo expression of proteins by modulating protein splicing with inhibiting or activating agents, and methods of controlling the delivery of proteinaceous drugs in vivo by modulating protein splicing.

Screening and use of agents which block or activate intein splicing utilizing natural or homologous exteins

In accordance with the present invention, a 31-33 kDa glycoprotein of (DiT33) is provided which represents another member of the family of putative pepsin inhibitors. Other known members include Ov33 (a.k.a. Ov33.3, Oc3.6, OvD 5B), Bm33 and Av33. These filarial molecules possess significant homology to the known pepsin inhibitor (Aspi3) of Using DiT33 or Ov33, in the form of a recombinant fusion or non-fusion protein, antibody responses to DiT33 may be monitored and used in immunodiagnosis of heartworm infection in mammals. Antibodies reactive with the DiT33 or Ov33 may also be used to detect DiT33 antigen as a means of immunodiagnosis of heartworm infection in mammals.

DNA and protein from Dirofilaria immitis

In accordance with the present invention, there are provided selection systems and methods for screening for agents that control splicing of inteins in their native host protein (extein) or in homologous exteins. Specifically, there are provided positive genetic selection systems for the screening of agents which inhibit or activate protein splicing which comprise: a host cell containing a chromosomal gene encoding either a drug-resistant form of a target enzyme or a wild-type target enzyme, and a plasmid-borne gene encoding either a drug-sensitive form of the target enzyme, which is dominantly cytotoxic upon interaction with the drug, or a dominantly cytotoxic form of the target enzyme. In these systems the plasmid-borne gene contains an intein, and the inhibition or activation of splicing of the dominant cytotoxic form of the target enzyme by a given reagent results in the survival or death of the host cell. More specifically, positive genetic selection systems which utilize the GyrA intein or DnaB helicase intein are provided. Similar reporter systems utilizing native or homologous exteins and systems utilizing controllable inteins are provided, as are methods of controlling in vivo expression of proteins by modulating protein splicing with inhibiting or activating agents, and methods of controlling the delivery of proteinaceous drugs in vivo by modulating protein splicing.

Screening and use of reagents which block or activate intein splicing utilizing natural or homologous exteins

Method for immunodiagnosis of heartworm in mammals

The present invention is directed to modified proteins and methods of their production. The modified proteins comprise a controllable intervening protein sequence (CIVPS) inserted into or adjacent a target protein, the CIVPS being capable of excision from or cleavage of the modified protein under predetermined conditions in cis or in trans, i.e., increase in temperature, exposure to light, unblocking of amino acid residues by dephosphorylation, treatment with chemical reagents or deglycosylation. If desired, the modified protein can be subjected to these conditions. The CIVPS may also be inserted into a region that substantially inactivates target protein activity. The CIVPS may be used in a number of applications including purification of the target protein in a one-step protocol.

Modified proteins comprising controllable intervening protein sequences

Recombinant DNA polymerases from archaebacteria as well as isolated DNA coding for such polymerases are provided. The isolated DNA is obtained by use or DNA or antibody probes prepared from the DNA encoding T. litoralis DNA polymerase and the T. litoralis DNA polymerase respectively. Also provided are methods for producing recombinant archaebacteria thermostable DNA polymerase and methods for enhancing the expression of such polymerases by identifying, locating and removing introns from within the DNA coding for such DNA polymerases.

Recombinant thermostable DNA polymerase from archaebacteria

The present invention is directed to an extremely thermostable enzyme. More specifically, the invention is directed to a thermostable DNA polymerase 9.degree.N-7, as well as the recombinant form of 9.degree.N-7. In another embodiment, there is provided a method for mutagenizing the exo motif I of the 3'-5' exonuclease DNA polymerases from the native conserved DXE in the 3'-5' exo motif I DNA segment to DXD or AXA.

Thermostable DNA polymerase from 9.degree.N-7 and methods for producing

Recombinant DNA polymerases from archaebacteria as well as isolated DNA coding for such polymerases are provided. The isolated DNA is obtained by use of DNA or antibody probes prepared from the DNA encoding T. litoralis DNA polymerase and the T. litoralis DNA polymerase respectively. Also provided are methods for producing recombinant archaebacteria thermostable DNA polymerase and methods for enhancing the expression of such polymerases by identifying, locating and removing introns from within the DNA coding for such DNA polymerases.

The present invention is directed to modified proteins and methods of their production. The modified proteins comprise a controllable intervening protein sequence (CIVPS) inserted into a target protein, the CIVPS being capable of excision from the modified protein under predetermined conditions, i.e., increase in temperature, exposure to light, unblocking of amino acid residues by dephosphorylation or deglycosylation. If desired, the modified protein can be subjected to these conditions. The CIVPS may also be inserted into a region that substantially inactivates target protein activity.

Modification of protein by use of a controllable interveining protein

Recombinant DNA polymeraset from archaebacteria as well as isolated DNA coding for such polymeraset are provided. The isolated DNA is obtained by use of DNA or antibody probet prepared from the DNA encoding T. litoralis DNA polymerate and the T. litoralis DNA polymerate respectively. Also provided are meshocs for producing recombinant archaebacteria thermostable DNA polymerase and methods for enhancing the expression of such polymeraset by identifying, locating and removing introns from within the DNA coding for such DNA polymerases.

There is provided an extremely thermostable enzyme obtainable from Thermococcus litoralis. The thermostable enzyme has a molecular weight of about 90,000-95,000 daltons, a half-life of about 60 minutes at 100.degree. C. in the absence of stabilizer, and a half-life of about 95 minutes at 100.degree. C. in the presence of stabilizer, such as octoxynol (TRITON X-100) or bovine serum albumin. The thermostable enzyme possesses a 3'-5' proofreading exonuclease activity. The thermostable enzyme may be native or recombinant and may be used for second-strand cDNA synthesis in cDNA cloning, DNA sequencing, and DNA amplification.

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Brookline, MA, United States of America

Francine B Perler