Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Utility Patent
1999-03-17
2001-01-02
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
Utility Patent
active
06168931
ABSTRACT:
BACKGROUND OF THE INVENTION
The directed synthesis of proteins and other biological macromolecules is one of the great achievements of biochemistry. The development of recombinant DNA techniques has allowed the characterization and synthesis of highly purified coding sequences, which in turn can be used to produce highly purified proteins, even though in native cells the protein may be available only in trace amounts. Polypeptide chains can be synthesized by chemical or biological processes. The biological synthesis may be performed within the environment of a cell, or using cellular extracts and coding sequences to synthesize proteins in vitro.
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 systems have 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, deamination or oxidation. In addition, many products inhibit metabolic processes and their synthesis must compete with other cellular process 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. 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). However, despite all its promising aspects, the in vitro system has not been widely accepted as a practical alternative, mainly due to the short reaction period, which causes a poor yield of protein synthesis.
The development of a continuous flow in vitro protein synthesis system by Spirin et al (1988)
Science
242:1162-1164 proved that the reaction could be extended up to several hours. Since then, numerous groups have reproduced and improved this system (Kigawa et al. (1991)
J. Biochem.
110:166-168; Endo et al. (1992)
J. Biotechnol.
25:221-230. Recently, Kim and Choi (1996) Biotechnol. Prog. 12: 645-649, reported that the merits of batch and continuous flow systems could be combined by adopting a ‘semicontinuous operation’ using a simple dialysis membrane reactor. They were able to reproduce the extended reaction period of the continuous flow system while maintaining the initial rate of a conventional batch system. However, both the continuous and semi-continuous approaches require quantities of expensive reagents, which must be increased by a significantly greater factor than the increase in product yield.
Several improvements have been made in the conventional batch system (Kim et al. (1996)
Eur. J. Biochem.
239: 881-886; Kuldlicki et al. (1992)
Anal. Biochem.
206:389-393; Kawarasaki et al (1995)
Anal. Biochem.
226: 320-324). Although the semicontinuous system maintains the initial rate of protein synthesis over extended periods, the conventional batch system still offers several advantages, e.g. convenience of operation, easy scale-up, lower reagent costs and excellent reproducibility. Also, the batch system can be readily conducted in multiplexed formats to express various genetic materials simultaneously.
Most recently, Patnaik and Swartz (1998)
Biotechniques
24:862-868 reported that the initial specific rate of protein synthesis could be enhanced to a level similar to that of in vivo expression through extensive optimization of reaction conditions. It is notable that they achieved such a high rate of protein synthesis using the conventional cell extract prepared without any condensation steps (Nakano et al. (1996)
J. Biotechnol.
46:275-282; Kim et al. (1996)
Eur. J. Biochem.
239:881-886). Their result implies that further improvement of the batch system, especially in terms of the longevity of the protein synthesis reaction, would substantially increase the productivity for batch in vitro protein synthesis. However, the reason for the early halt of protein synthesis in the conventional batch system has remained unclear.
As shown from the above, both protein productivity and production amount are still low, which is an obstacle in implementing the industrialization of cell-free protein synthesis. Therefore, improvements are greatly required in terms of the total productivity of the protein by increasing the specific production rate and the length of system operation, and in terms of a cost-reduction by regenerating an expensive energy source in order to recycle the regenerated energy source. It has been suggested that accumulation of small molecular weight by-products could inhibit protein synthesis. The continuous removal of the inhibitory by-product(s) as well as the continuous supply of substrates for protein synthesis may enable the continuous or semicontinuous reaction system to support protein synthesis over long reaction periods. Elucidation of inhibitory products, and prevention of their synthesis is of great interest for development of in vitro synthetic systems.
SUMMARY OF THE INVENTION
Compositions and methods are provided for the enhanced in vitro synthesis of biological molecules where ATP is required for synthesis. Of particular interest is the synthesis of polymers, e.g. nucleic acids, polypeptides, and complex carbohydrates. A homeostatic system is used for production of ATP, where use of this energy system prevents a net increase in free phosphate as a result of ATP hydrolysis. The phosphate that is released from ATP hydrolysis is recycled into high energy phosphate bonds, thereby preventing its accumulation. Energy homeostasis is accomplished by addition of a regenerative energy source, e.g. pyruvate in combination with the enzyme pyruvate oxidase to generate acetyl phosphate. In a preferred embodiment, energy sources such as phosphoenol pyruvate, which increase the net free phosphate in the reaction mix, are omitted.
Relevant literature
Muller et al. (1993)
Science
259:965-967 describes the structure of the thiamine- and flavin-dependent enzyme, pyruvate oxidase. Ryabova et al. (1995)
Anal. Biochem.
226:184-186 describe the use of acetyl phosphate as an energy source for bacterial cell-free translation systems. Pyruvate oxidase mutants are described in U.S. Pat. No. 5,153,138, and pyruvate oxidase is further described in U.S. Pat. Nos. 4,666,832 and 4,246,342.
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Kawarasaki, Yasuaki, et al., “A Long-Lived Batch Reaction System Of Cell-Free Protein Synthesis1”Analytical Biochemistry(1995) vol. 226:320-324.
Kigawa, Takanori, et al., “A Continuous Cell-Free Protein Synthesis System For Coupled Transcription-Translation1”J. Biochem.(1991) vol. 110:166-168.
Kim, Dong-Myung, et al., “A Semicontinuous Prokaryotic Coupled
Kim Dong-Myung
Swartz James R.
Achutamurthy Ponnathapu
Bozicevic, Field & Francis
Kerr Kathleen
Sherwood Pamela J.
The Board of Trustees of the Leland Stanford Junior University
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