Entropic bristle domain sequences and their use in recombinant protein production

a bristle domain and entropic technology, applied in the field of compositions and methods can solve the problems of large percentage of proteins identified via different genome sequencing efforts that are difficult to express and/or purify as recombinant proteins using standard methods, reduce the success level between cloned and crystallized, and reduce the cost of recombinant protein production

Inactive Publication Date: 2009-09-03
MOLECULAR KINETICS
View PDF9 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

Fusion polypeptides with EBD sequences exhibit increased solubility and reduced aggregation, leading to improved protein production yields and stability, effectively addressing the limitations of existing methods.

Problems solved by technology

A large percentage of the proteins identified via the different genome sequencing effort have been difficult to express and / or purify as recombinant proteins using standard methods.
For example, a trial study using Methanobacterium thermoautotrophicum as a model system identified a number of problems associated with high throughput structure determination (Christendat et al.
The study concluded that considerable effort must be invested in improving the attrition rate due to proteins with poor expression levels and unfavorable biophysical properties.
A significant component of the decrease between the cloned and crystallized success levels was due to poor protein solubility and stability (Kuhn et al.
Similarly low success rates have been reported for eukaryotic proteomes.
Interactions between individual recombinant proteins are responsible for a significant number of the previously mentioned failures.
These strategies have advantages for some proteins but they generally do not succeed when used, for example, with membrane proteins or proteins capable of strong protein-protein interactions.
However, these fusion approaches used to date have not been amendable to all classes of proteins, and have thus met with only limited success.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Entropic bristle domain sequences and their use in recombinant protein production

Examples

Experimental program
Comparison scheme
Effect test

example 1

Use of Neurofilament Triplet M Protein (NF-M) in an Entropic Bristle Domain Vector

[0182]The heterogeneity in the charge distribution of the human NF-M protein sequence was determined (shown below). The observed heterogeneity of the sequence suggests that EBDs with different characteristics may result for different regions of the sequence. For example, a 422-600 fragment is predominantly negatively charged. This fragment could be used as a basis to design EBDs for negatively charged proteins. The charge distribution in the 601-916 fragment is very heterogeneous. It can be used as a basis to design EBDs both for positively- and negatively-charged proteins.

[0183]Cloning of EBD sequence: We obtained the full-length cDNA for human NF-M from Origene Technologies (Rockville, Md.) and cloned the coding region for a 494-residue EBD sequence (residues 422 to 916 of the NF-M protein) into a pMALc2E vector from which the maltose-binding protein coding region had been deleted. (See FIG. 1.) Rest...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

Compositions and methods for recombinant protein production and, more particularly, fusion polypeptides, polynucleotides encoding fusion polypeptides, expression vectors, kits, and related methods for recombinant protein production, are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 11 / 485,613, filed Jul. 11, 2006, now U.S. Pat. No. 7,494,788, which application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60 / 698,456, filed Jul. 11, 2005, where these applications are incorporated herein by reference in their entireties.STATEMENT REGARDING SEQUENCE LISTING[0002]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 670098—402Cl_SEQUENCE_LISTING.txt. The text file is 151 KB, was created on Jan. 22, 2009, and is being submitted electronically via EFS-Web.FIELD OF THE INVENTION[0003]The present invention relates generally to compositions and methods for recombinant protein production and, more particularly, to fusion polypeptides, polynuc...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12P21/06C12N5/00C12N15/63C07K14/00C07K7/08C07K7/06C07H21/04
CPCC07K14/47C12N15/62C07K2319/35
InventorDUNKER, A. KEITHUVERSKY, VLADIMIR N.CORTESE, MARC S.MUELLER, JAMES
OwnerMOLECULAR KINETICS