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Compositions and methods for monitoring and altering protein folding and solubility

a technology of applied in the field of microorganisms, molecular biology and protein biochemistry, can solve the problems of inability to develop a robust assay for in vivo protein folding and solubility, and inability to detect the tendency of protein misfolding and aggrega

Inactive Publication Date: 2007-02-01
CORNELL RES FOUNDATION INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013] The present invention also provides a method for screening for mutations in a host cell that alters solubility and / or folding of a target protein, comprising: providing a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises a Tat leader signal, the target protein, and a marker protein; expressing the fusion protein in a host cell; wherein the host cell has undergone mutagenesis; and correlating the ability of the host cells comprising the fusion protein to grow under selective pressure with the solubility and / or folding of the target protein. In some embodiments, an increase in growth correlates with a host cell environment that improves target protein solubility.

Problems solved by technology

As if this weren't enough, existing biochemical means for assessing the tendency of proteins to misfold and aggregate are tedious.
As a result, screening for constructs and / or conditions that favor solubility is inefficient and genetic selection of folded structures has not been forthcoming.
Development of a robust assay for in vivo protein folding and solubility has been challenging for researchers because of limitations on detecting and reporting the solubility of a protein.
This fusion approach is often problematic as certain reporter proteins can remain active even when the target protein to which they are fused aggregates or forms inclusion bodies (See, e.g., Tsumoto et al., Biochem Biophys Res Commun 312, 1383-1386 (2003)) while the gene expression response is limited by its indirect connection to the folding process.
Additionally, existing assays for protein expression in soluble form are tedious, usually requiring lysis and fractionation of cells followed by protein analysis by SDS-polyacrylamide gel electrophoresis.
Using these traditional approaches, screening for protein constructs and / or physiological conditions yielding improved solubility is inefficient, and genetic selection nearly impossible.

Method used

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  • Compositions and methods for monitoring and altering protein folding and solubility
  • Compositions and methods for monitoring and altering protein folding and solubility
  • Compositions and methods for monitoring and altering protein folding and solubility

Examples

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example 1

Materials and Methods

[0251] Bacterial strains and plasmids. Wildtype E. coli strain MC4100 and a ΔtatC derivative of MC4100, strain B1LK0 (See, e.g., Bogsch et al., J Biol Chem 273, 18003-18006 (1998)), were used for all experiments. Plasmids for cytoplasmic expression of MBP (wt) and its folding mutant derivatives (See, e.g., Betton and Hofnung, J Biol Chem 271, 8046-8052 (1996)) were generated by inserting the gene encoding each MBP sequence into the NcoI / HindIII position of pTrc99A (Amersham Pharmacia). Plasmids for expressing MBP and its derivatives via the Tat pathway were created by excising the phoA gene from pTorA-AP (See, e.g., DeLisa et al., Proc Natl Acad Sci U S A 100, 6115-6120 (2003)) with XbaI and HindIII and inserting the gene encoding mature MBP (wt) or a folding mutant into the resulting XbaI / HindIII sites. Similarly, plasmids for localizing DsRed and its derivatives to the Tat pathway were generated by inserting either the DsRed gene sequence or its derivatives, ...

example 2

Folding Quality Control of the Tat Pathway

[0257] Tat transport of E. coli maltose binding protein (MBP) and three well-characterized MBP mutants prone to varying levels of off-pathway folding intermediates: MBP-G32D, MBP-I33P, and MalE31 (G32D / I33P) (See, e.g., Betton and Hoffnung, J Biol Chem 271, 8046-8052 (1996)) was evaluated. These proteins display a >100-fold difference in in vivo solubility with unfolding / refolding stability ranging from −5.5 kcal / mol to −9.5 kcal / mol ((See, e.g., Betton and Hofnung, J Biol Chem 271, 8046-8052 (1996)). The coding region for the well-characterized E. coli TMAO reductase twin-arginine signal peptide plus the first 4 residues of mature TorA (ssTorA, amino acids 1-46) (DeLisa et al., J Biol Chem 277, 29825-29831 (2002)) was fused upstream of the gene encoding the mature form of each MBP (residues 26-396), thus creating four ssTorA-MBP chimeras. Cell fractionation of wildtype MC4100 E. coli cells was performed to track subcellular localization an...

example 3

Tat-Based Solubility Reporter

[0259] To exploit the quality control feature of the Tat pathway for monitoring protein solubility, a genetic assay that employs a tripartite fusion of the TorA signal peptide, a ‘target’ protein, and mature TEM1 β-lactamase (Bla) (FIG. 1A) was developed. The premise for this assay is as follows: a soluble target protein is exported to the periplasm via the Tat pathway and, by virtue of the Bla fusion, confers ampicillin resistance to E. coli cells expressing the ssTorA-target-Bla chimera. To verify that Bla is indeed capable of reporting Tat dependent transport in the assay, a vector (pTMB, FIG. 1B) was first constructed with no gene in the target position that expresses ssTorA-Bla. Upon expression of ssTorA-Bla in MC4100 and B1LK0, only periplasmic Bla localization was observed with a corresponding ampicillin resistance phenotype in MC4100 cells that possess a functional Tat pathway (FIG. 2C). Thus, Bla can be specifically transported by the Tat pathw...

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Abstract

The present invention relates to the fields of microbiology, molecular biology and protein biochemistry. More particularly, it relates to compositions and methods for analyzing and altering (e.g., enhancing or inhibiting) protein folding and solubility.

Description

[0001] This invention was funded, in part, under NSF Grant BES-0449080. The government may have certain rights in the invention.FIELD OF THE INVENTION [0002] The present invention relates to the fields of microbiology, molecular biology and protein biochemistry. More particularly, it relates to compositions and methods for analyzing and altering (e.g., enhancing or inhibiting) protein folding and solubility. BACKGROUND OF THE INVENTION [0003] The expression of heterologous proteins represents a cornerstone of the biotechnology enterprise. Unfortunately, many commercially important proteins misfold and aggregate when expressed in a heterologous host (See, e.g., Makrides, Microbiol Rev 60, 512-538 (1996); Baneyx and Mujacic, Nat Biotechnol 22, 1399-1408 (2004); Georgiou and Valax, Curr Opin Biotechnol 7, 190-197 (1996)). Similarly, protein misfolding and aggregation is the pathological hallmark of more than a dozen diseases including Alzheimer's (See, e.g., Radford et al., Cell 97, 29...

Claims

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Application Information

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IPC IPC(8): A61K39/02C07K14/245G01N33/53C12N1/21C12N15/74C07H21/04
CPCC07K2319/034C12N15/1086C12N15/625C12N15/1034C12N15/62
Inventor DELISA, MATTHEW P.FISHER, ADAM CHARLES
Owner CORNELL RES FOUNDATION INC
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