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tRNA synthetase fragments

a synthetase and fragment technology, applied in the direction of ligases, peptide/protein ingredients, enzymology, etc., can solve the problems of not being able to provide a polypeptide preparation substantially free of endotoxins, not being able to provide a polypeptide preparation, and not being able to reduce the amount of endotoxins

Inactive Publication Date: 2013-05-02
GLIDDEN PAUL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
However, clarification, by itself, is not designed to provide a polypeptide preparation that is substantially free of endotoxins.
In general, however, such surfactants and glycols are not used in compositions for intraocular administration except in very low doses because of their potential to cause certain harmful side effects, such as retinal detachment.
In ocular diseases, neovascularization can lead to catastrophic loss of vision.
However, the 6-His tag was not used in the final system chosen for the expression and purification of material for pre-clinical development.
The acetonitrile is less hydrophobic than water and therefore interferes with lipid interactions of proteins and the column resin surface.

Method used

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Examples

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example 1

Preparation of Endotoxin-Free Recombinant TrpRS Purified by Laboratory Techniques (Nickel Affinity Column and Triton X-114)

[0485]Endotoxin-free recombinant human TrpRS (GD and SY variants) were prepared as follows: Plasmids encoding full-length TrpRS (amino acid residues 1-471 of SEQ ID NO: 1 and the SY variant thereof), or truncated TrpRS, hereinafter referred to as T2 (SEQ ID NO: 12 (GD variant) or SEQ ID NO: 24 (SY variant)), consisting essentially of residues 94-471 of full length TrpRS and a second truncated TrpRS fragment, hereinafter referred to as T1 (SEQ ID NO: 13 (GD variant) or SEQ ID NO: 25 (SY variant)), consisting essentially of residues 71-471 of full length TrpRS were prepared.

[0486]Each plasmid also encoded a C-terminal tag consisting of six histidine residues (e.g. amino acid residues 472-484 of SEQ ID NO: 1), and an initial methionine residue. The His6-tagged T1 (SEQ ID NOS: 13 and 25) had the amino acid sequence of SEQ ID NO: 5 (or SY variant thereof), whereas th...

example 2

Cleavage of Human TrpRS by PMN Elastase

[0489]Cleavage of human full-length TrpRS by PMN elastase was examined. TrpRS was treated with PMN elastase in PBS (pH 7.4) at a protease:protein ratio of 1:3000 for 0, 15, 30, or 60 minutes. Following cleavage, samples were analyzed on 12.5% SDS-polyacrylamide gels. PMN elastase cleavage of a full-length TrpRS of about 53 kDa generated a major fragment of about 46 kDa (SEQ ID NO: 5, T1, having the C-terminal histidine tag, or an SY variant thereof) and a minor fragment of about 43.5 kDa (SEQ ID NO: 7, T2 having the C-terminal histidine tag or the SY variant thereof). In particular, cleavage of full-length TrpRS (SY variant) by PMN elastase generated a major fragment of about 46 kDa (SEQ ID NO: 25) and a minor fragment of about 43.5 kDa (SEQ ID NO: 24).

[0490]Western blot analysis with antibodies directed against the carboxyl-terminal His6-tag of the recombinant TrpRS proteins revealed that both fragments, which were apparent at approximately 46...

example 3

Truncated Fragments of Trp-RS Show Potent Angiostatic Effect for Retinal Angiogenesis

[0492]Angiostatic activity of truncated forms derived from full length tryptophanyl-tRNA synthetase was examined, in a post-natal mouse retinal angiogenesis model. Friedlander et al. (Abstracts 709-B84 and 714-B89, IOVS 41(4): 138-139 (Mar. 15, 2000)) reported that postnatal retinal angiogenesis proceeds in stages in the mouse. The present invention provides a method of assaying angiogenesis inhibition by exploiting this staged retinal vascularization.

[0493]Endotoxin-free recombinant mini-TrpRS and T2 (e.g., SEQ ID NOS: 12 and 24) were prepared as recombinant proteins. These proteins were injected intravitreally into neonatal Balb / C mice on postnatal (P) day 7 or 8 and the retinas harvested on P12 or P13. Collagen IV antibody and fluorescein-conjugated secondary antibody were used to visualize the vessels in retinal whole mount preparations. Anti-angiogenic activity was evaluated by confocal microsc...

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Abstract

The present invention relates to compositions and methods for treating conditions associated with angiogenesis. In particular the present invention relates to multi-unit complexes of tRNA synthetase fragments and uses thereof; diverse multi-unit complexes including a tRNA synthetase fragment; compositions and methods for modulating angiogenesis; polynucleotides encoding tRNA synthetase fragments and uses thereof; antibodies and epitopes specific to tRNA synthetase fragments; variants of tRNA synthetase fragments and uses thereof; methods for treating angiogenesis; methods for screening for anti-angiogenic agents; methods of modulating angiogenesis; kits for modulating angiogenesis; and business methods for modulating angiogenesis. Preferably the tRNA synthetase fragments are tryptophanyl tRNA synthetase fragments, and more preferably human tryptophanyl tRNA synthetase fragments.

Description

CROSS REFERENCES[0001]This application is a continuation-in-part of U.S. application Ser. No. 12 / 399,468, filed on Mar. 6, 2009, which is a continuation-in-part of U.S. application Ser. No. 11 / 196,019 filed on Aug. 2, 2005 (abandoned), which is: a continuation-in-part of U.S. application Ser. Nos. 10 / 962,171, 10 / 962,217, 10 / 962,058, 10 / 961,528, 10 / 962,375, 10 / 962,062, 10 / 962,218, 10 / 961,529, 10 / 961,526, and 10 / 961,486, all of which were filed on Oct. 7, 2004 (all abandoned), and a continuation-in-part of U.S. application Ser. No. 11 / 019,969 filed on Dec. 20, 2004 (abandoned), and a continuation-in-part of U.S. application Ser. No. 10 / 980,866 filed on Nov. 2, 2004 (abandoned), which is a continuation-in-part of U.S. application Ser. No. 10 / 962,218 filed on Oct. 7, 2004 (abandoned); and U.S. application Ser. No. 11 / 196,019 claims the benefit of U.S. Provisional Application No. 60 / 598,019 filed on Aug. 2, 2004 and U.S. Provisional Application No. 60 / 624,656 filed on Nov. 2, 2004; all o...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/00A61K38/53
CPCC12N9/93A61K38/53
Inventor GLIDDEN, PAUL
Owner GLIDDEN PAUL
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