Method for synthesis of polypeptides in cell-free systems

Details Method for synthesis of polypeptides in cell-free systems Method for synthesis of polypeptides in cell-free systems

2003-01-21

2004-08-31

Wax, Robert A. (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C536S026260

Reexamination Certificate

active

06783957

ABSTRACT:

FIELD OF THE INVENTION
The invention pertains to the field of molecular biology, in particular to synthesis of proteins and polypeptides in cell-free systems prepared from prokaryotic and eukaryotic cells.
BACKGROUND
Synthesis of polypeptides and proteins in cell-free translation systems of the first generation (U.S. Pat. No. 4,668,624, Roberts, 1978) was performed in a static (batch) mode where the reaction mixture is in static conditions with constant Mg
2+
, K
+
and NTP concentrations, constant pH and temperature. To this end, extracts and lysates of prokaryotic (Zubay, 1973) and eukaryotic cells (Roberts and Paterson, 1973; Pelham and Jackson, 1976) were prepared, and natural and synthesized mRNAs were used (U.S. Pat. No. 4,937,190, Palmberg, 1990).
Rapid development of biotechnology has called for alternative methods that would increase the yield of synthesized proteins. The design of more productive translation systems in which the concentration of basic components is maintained constant during the synthesis is one direction of efforts aimed at improvement of the existing methods. In the second generation systems (Spirin et al., 1988), a continuous flow of low weight substrates included in the feeding solution (CFCF mode) into the reactor volume and removal of target polypetides and low molecular weight products inhibiting the cell-free system increases the time of its operation and raises the yield of the desired protein as compared to the classic system of synthesis in static (batch) conditions. Numerous studies have been focused on optimization of the conditions for CFCF protein synthesis (Baranov, 1989; Ryabova et al., 1989; Takanori et al., 1991; Spirin, 1992, Baranov and Spirin, 1993; Volyanik et al., 1993; Erdman et al., 1994; Kim and Choi, 1996; Yamamoto, 1996; Ryabova et al., 1998, EP Patent 0312617; Alakhov et al., 1993, EP Patent 0401369, Baranov et al., 1995, U.S. Pat. No. 5,434,079; Mozayeni, 1995; JP Patent 7075592, Shimizu, 1995; JP Patent 7031494, Sakurai, 1995; JP Patent 5076381, Sato, 1995; EP Patent 0593757, Baranov et al., 1997; U.S. Pat. No. 5,593,856, Choi et al., 1997).
U.S. Pat. No. 5,478,730 (Alakhov et al., 1995) describes a method in which the synthesis in cell-free translation systems is based on continuous exchange (CECF mode) of the feeding solution components with the component of the reaction mixture via a semipermeable barrier by a diffusion process. The results obtained by many authors (Davis et al., 1996; Kim and Choi, 1996; U.S. Pat. No. 5,593,856, Choi, 1997; JP Patent 10080295, Yamane, 1998) demonstrate a significant increase in the yield of the target polypeptide upon continuous exchange, as compared to the static (batch) mode of operation.
In addition to improvement of the components of the translation system, efforts were made to improve methods for preparation of mRNA in transcription systems including RNA polymerase and DNA. In these systems, preparation of mRNA depends on the concentration of RNA polymerase and DNA, as well as on the concentration of Mg
2+
, K
+
and NTP and other ionic conditions (Kern and Davis, 1997). The cost of components of the in vitro transcription including RNA polymerase, DNA and NTP is very high. Therefore it is necessary to analyze conditions of transcription and optimize the process of mRNA preparation (Gurevich et al., 1991).
There are methods for synthesis of polypeptides in a CFCF mode in prokaryotic cell-free systems in conditions of a coupled transcription-translation (Baranov et al., 1989; EP Patent 0401369, Baranov et al., 1995; Ryabova et al., 1998) and the process was patented where transcription and translation occur in eukaryotic cell-free systems in the same reaction volume (Spirin, 1992; Baranov and Spirin, 1993; EP Patent 0593757, Baranov et al., 1997).
It is known (Craig et al., 1993) that translation and transcription conditions in eukaryotic cell-free systems differ and are determined largely by the concentrations of Mg
2+
and K
+
. Therefore, two-stage (U.S. Pat. No. 5,665,563, Beckler, 1997; Operating Guide, Single Tube Protein™, Novagen Inc., 1998) or three-stage synthesis (Roberts and Paterson, 1973) is widely used in a static (batch) mode. At the first stage optimal conditions are achieved for mRNA transcription, then the mRNA is purified or immediately added to a new reaction mixture with conditions for translation. A one-stage synthesis of polypeptides in a transcription-translation eukaryotic cell-free systems is known (U.S. Pat. No. 5,34,637, Thompson et al., 1994; Operation Guide, Linked in vitro SP6/T7 Transcription/Translation Kit, Roche Diagnostics GmbH, 1998). The authors of the patent (U.S. Pat. No. 5,324,637, Thompson et al., 1994) used a known principle of optimization of Mg
2+
concentration in the reaction mixture. By adding Mg
2+
to the reaction mixture prior to the synthesis, they achieved such a concentration of Mg
2+
in the reaction system which is intermediate between the transcription optimum and the translation optimum. Further studies showed that such optimization has no advantages over the two-stage or three-stage procedures. The study of Laios et al. (1998) demonstrates that optimization of separate stages of transcription and translation is from 2 to 6 times more efficient than that of a coupled process. On the other hand, optimization of the selection of Mg
2+
concentrations is based on a preliminary measurement of the magnesium concentration in the lysate or in the reaction volume which devalues the principle of the one-stage procedure.
European Patent 0593 757 (Baranov et al., 1997) describes the possibility to perform continuous CFCF synthesis of polypeptides in eukaryotic cell-free transcription-translation systems for 20 hours. During the synthesis, the Mg
2+
concentration in the reaction mixture is maintained at the required level due to the constant concentration of Mg
2+
in the feeding solution. Since ribonuclease activity in the reaction volume is low and the mRNA templates retain their activity for a prolonged time, the reaction system works with both the earlier and newly synthesized mRNA templates and synthesizes a target product due to the constant Mg
2+
concentration. For a more productive synthesis, the transcription system should synthesize an adequate amount of mRNA. Therefore a large quantity of expensive polymerase SP6 or T7 (30,000 units) is required. It is mentioned in the text of the patent that optimal conditions of synthesis should be chosen in each individual case. To make an appropriate choice, it is necessary to perform a series of syntheses in a batch volume at different Mg
2+
concentrations and determine its optimal value for the given polypeptide. Optimization of the process is time consuming and rather expensive.
There are many devices in which the continuous exchange mode (CECF) is maintained due to a diffusion process. The device, in the form of a dialysis container for synthesis of polypeptides in a cell-free system, was first described in U.S. Pat. No. 5,478,730 (Alakhov et al., 1995). Promega Corp. (Davis et al., 1996) made a comparative analysis of syntheses (in a static (batch) mode and at a continuous exchange mode) during coupled transcription-translation in a
E. coli
prokaryotic cell-free system. To this end, the authors used “DispoDialyser” instruments manufactured by Spectrum Medical Industr. (U.S. Pat. No. 5,324,428, Flaherty, 1994) and “Slidealyzer” dialysers manufactured by Pierce Chemical Comp. (U.S. Pat. No. 5,503,741, Clark. 1996).
For the synthesis of polypeptides upon coupled transcription-translation in a preliminarily concentrated prokaryotic cell-free system of
E. coli
, Kim and Choi (1996) used a dialysis membrane fixed at the bottom of a cylinder.
Yamamoto (1996) constructed a dialyser in which the membrane is made from hollow fibers. The feeding solution passes through the hollow fibers. Due to diffusion, the components of the reaction mixture exchange with those of the feeding solution.
In the device designed by

Affiliated with

Also associated with

Rating

Method for synthesis of polypeptides in cell-free systems does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method for synthesis of polypeptides in cell-free systems, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for synthesis of polypeptides in cell-free systems will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3335490