Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
2001-09-05
2003-04-15
Bugaisky, Gabrielle (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C435S068100, C435S025000, C435S007900, C435S189000, C435S215000, C435S320100, C530S350000, C514S012200, C536S023100
Reexamination Certificate
active
06548276
ABSTRACT:
BACKGROUND OF THE INVENTION
Escherichia coli
is a widely used organism for the expression of heterologous proteins. It easily grows to a high cell density on inexpensive substrates to provide excellent volumetric and economic productivities. Well established genetic techniques and various expression vectors further justify the use of Escherichia coli as a production host. However, a high rate of protein synthesis is necessary, but by no means sufficient, for the efficient production of active biomolecules. In order to be biologically active, the polypeptide chain has to fold into the correct native three-dimensional structure, including the appropriate formation of disulfide bonds.
In many cases, the recombinant polypeptides have been found to be sequestered within large refractile aggregates known as inclusion bodies. Active proteins can be recovered from inclusion bodies through a cycle of denaturant-induced solubilization of the aggregates followed by removal of the denaturant under conditions that favor refolding. But although the formation of inclusion bodies can sometimes ease the purification of expressed proteins; in most occasions, refolding of the aggregated proteins remains a challenge.
Various attempts have been made to improve the folding of heterologous proteins in the bacterial cytoplasm. In addition to the traditional methods, including lowering the temperature of the culture, increasing knowledge of the mechanism and effectors of protein folding has enabled new approaches to solve the problem of aggregation.
Studies in vitro have demonstrated that, for the vast majority of polypeptides, folding is a spontaneous process directed by the amino acid sequence and the solvent conditions. Yet, even though the native state is thermodynamically favored, the time-scale for folding can vary from milliseconds to days. Kinetic barriers are introduced, for example, by the need for alignment of subunits and sub-domains. And particularly with eukaryotic proteins, covalent reactions must take place for the correctly folded protein to form. The latter types of reaction include disulfide bond formation. cis/trans isomerization of the polypeptide chain around proline peptide bonds, preprotein processing and the ligation of prosthetic groups. These kinetic limitations result in the accumulation of partially folded intermediates, that contain exposed hydrophobic ‘sticky’ surfaces which promote self-association and formation of aggregates.
Expression of mammalian proteins is more complicated than bacterial proteins because most of them require intramolecular disulfide bonds for their activity. Thus additional effectors such as foldases and proper redox potential are required to achieve their native structures. Even though the periplasmic space of Escherichia coli provides an oxidizing environment as well as folding proteins such as DsbA, B, C, and D; in many cases, simple secretion of complex proteins into the periplasmic space is not sufficient to form correct disulfide bonds.
Accessory proteins known as foldases and chaperones have been found to assist in the proper folding of proteins in vivo. Foldases have a catalytic activity that serves to accelerate rate-limiting covalent steps in folding. Chaperones, on the other hand, perform many functions, the most important of which is to provide an environment for nascent proteins to fold without the competing process of self-association. In addition to the well-characterized molecular chaperones, such as GroEL and DnaK proteins, a number of additional cytoplasmic proteins have been identified to affect the folding of heterologous proteins.
Following the discovery of numerous bacterial or eukaryotic foldases and their specific roles in the oxidation and isomerization of disulfide bonds, many attempts have been made to use those proteins in the periplasmnic space or even in the cytoplasm of
Escherichia coli
(see, for example, Bessette et al. (1999)). The co-expression of molecular chaperones has been shown to partially solve the problem of inclusion body formation in the expression of certain recombinant proteins (see, for example, Richardson et al. (1998)
Trends Biochem. Sci.
23:138-143; and Bukau et al. (1998)
Cell
92:351-366).
However, the effect of molecular chaperones is rather product-specific and the co-expression of each molecular chaperone with the target proteins is often cumbersome. Moreover, in some cases, the expression of a molecular chaperone is harmful or even detrimental to cell growth. Despite the recent advances, the expression of properly folded mammalian proteins in
Escherichia coli
still remains as a great challenge. This is mainly due to the difficulties in the control of the key parameters for disulfide bond formation including the redox potential inside the cells.
For several decades, in vitro protein synthesis has served as an effective tool for lab-scale expression of cloned or synthesized genetic materials. In recent years, in vitro protein synthesis has been considered as an alternative to conventional recombinant DNA technology, because of disadvantages associated with cellular expression. In vivo, proteins can be degraded or modified by several enzymes synthesized with the growth of the cell, and, after synthesis, may be modified by post-translational processing, such as glycosylation, deamidation or oxidation. In addition, many products inhibit metabolic processes and their synthesis must compete with other cellular processes required to reproduce the cell and to protect its genetic information.
Because it is essentially free from cellular regulation of gene expression, in vitro protein synthesis has advantages in the production of cytotoxic, unstable, or insoluble proteins. The over-production of protein beyond a predetermined concentration can be difficult to obtain in vivo, because the expression levels are regulated by the concentration of product. The concentration of protein accumulated in the cell generally affects the viability of the cell, so that over-production of the desired protein is difficult to obtain. In an isolation and purification process, many kinds of protein are insoluble or unstable, and are either degraded by intracellular proteases or aggregate in inclusion bodies, so that the loss rate is high.
In vitro synthesis circumvents many of these problems (see Kim and Swartz (1999)
Biotechnol. Bioeng.
66:180-188; and Kim and Swartz (2000)
Biotechnol. Prog.
16:385-390). Also, through simultaneous and rapid expression of various proteins in a multiplexed configuration, this technology can provide a valuable tool for development of combinatorial arrays for research, and for screening of proteins. In addition, various kinds of unnatural amino acids can be efficiently incorporated into proteins for specific purposes (Noren et al. (1989)
Science
244:182-188).
Unlike in vivo gene expression, cell-free protein synthesis uses isolated translational machinery instead of entire cells. As a result, this method eliminates the requirement to maintain cell physiology and allows direct control of various parameters to optimize the synthesis/folding of target proteins. Of particular interest is the problem of cell-free synthesis of biologically active mammalian proteins having multiple disulfide bonds. The present invention addresses the coupled synthesis and folding of mammalian proteins through the control of redox potential during protein synthesis.
SUMMARY OF THE INVENTION
Compositions and methods are provided for the enhanced in vitro synthesis of protein molecules, by optimizing the redox conditions in the reaction mix. In one embodiment of the invention, a redox buffer is included in the reaction mix to maintain the appropriate oxidizing environment for the formation of proper disulfide bonds, for example by the inclusion of glutathione in an appropriate ratio of oxidized to reduced forms.
The reaction mix is preferably further modified to decrease the activity of molecules in the extract, e.g. endogenous enzymes that have reducing activity. Preferably such molecules are chemically
Kim Dong-Myung
Swartz James Robert
Bozicevic Field & Francis LLP
Bugaisky Gabrielle
Kam Chih-Min
Sherwood Pamela J.
The Board of Trustees of the Leland Stanford Junior University
LandOfFree
Enhanced in vitro synthesis of active proteins containing... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Enhanced in vitro synthesis of active proteins containing..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Enhanced in vitro synthesis of active proteins containing... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3081738