Continuous-flow cell-free system

Cell-free translation system, used for the identification of cloned genes and gene expression, has been investigated extensively as a preparative production system of commercially interesting proteins after the development of continuous-flow cell-free translation system. Many efforts have been devoted to improve the productivity of cell-free system [1], but the relatively low productivity of cell-free translation system still limits its potential as an alternative to the protein production using recombinant cells. One approach to enhance the translational efficiency is to use a condensed cell-free translation extract. However, simple addition of a condensed extract to a continuous-flow cell-free system equipped with an ultrafiltration membrane can cause fouling. Therefore, it needs to be developed a selective condensation of cell-free extract for the improvement of translational efficiency without fouling problem. 

CFCF, continuous-flow cell-free protein synthesis system PCR, polymerase chain reaction TB, transcription buffer. 

Fig. 4. Massive production of green fluorescent protein (GFP) by the continuous-flow cell-free method. Sodium dodecyl sulfide-polyacrylamide gel electrophoresis analysis of GFP produced during 14 d of reaction. mRNA produced by transcription of circular plasmid of Ehime University (pEU) was used for the translation reaction in the dialysis membrane system and was added every 48 h. A 0.1 -pL aliquot of the mixture was run on the gel and protein bands were stained with Coomassie Brilliant Blue. The arrow shows GFP and st designates an authentic GFP band (0.5 pg).

A significant leap forward was made in 1988 by Spirin et al. [4], who developed a continuous-flow apparatus for the continuous supplementation of reaction mixtures with substrates required for protein synthesis and continuous removal of reaction by-products. In this way, it was shown that the activity of a cell-free system could be sustained for many hours, compared with batch-mode reactions which became inactive after approximately 45 minutes. Since then, many reports have been made describing the use of simple dialysis systems [3, 5] that can maintain the high productivity of a reaction over many hours, without the use of a cumbersome apparatus. 

Fig. 23 (A) Schematic diagram of the analytical system for eontinuous and on-Line measurement of ascorbate in the rat brain with heat-treated SWNT-modified GC electrode in a thin-layer flow cell (B) On-line amperometric responses continuously (a) and repeatedly (b) recorded for the brain dialysate of free moving rats in a continuous-flow cell with the heat-treated SWNT-modifled GC electrode. The electrode was poised at - -30 mV. Reproduced with permission from ref. 122. Copyright 2005 American Chemical Society.

Active materials are considered to be integrated as solids in the battery electrodes. Electrodes of the second kind and insertion electrodes are treated exclusively. Sometimes, it is believed [10] that this is the only way to arrive at practical battery designs. However, nowadays a continuous hybridization of the fuel cell type of design can be observed for conventional inorganic systems, e.g., dissolved active materials in flow cells, storage outside in a separate tank (especially for gaseous reactants) and so on. These versions will not be discussed in the present review, however, for they play no role for metal-free cells. 

A cell-free extract (S30) from E. coli K12 has been developed as an efficient coupled transcription/translation system which performs protein synthesis in vitro from the genes cloned in plasmids under the T7, T3, or SP6 promoter. Capping of the mRNA for eukaryotic proteins is apparently unnecessary with the S30 system. The coupled transcription/translation system, which was originally developed as the S3 0 translation system (122), can be used in a batchwise or continuous-flow mode (123,124). Use of the ribosome fraction collected from the S30 extracts is reported to improve the yield and efficacy further and more advantageously with nonlinearized plasmids than with linearized plasmids (125). 

The biofermenter BF-F500 system consisted of a 1.5 1 culture vessel, 2 1 medium reservoir and effluent bottle (2 1 glass vessels) for fresh and expended media which were connected to the perfusion (culture) vessel by a peristaltic pump. As shown in Fig. 14, the fermenter systems have a conical shape sedimentation column in the center of the fermenter, and an impeller on the bottom of the sedimentation column. The Namalwa cells, KJM-1, were cultivated by continuous cultivation in the biofermenter. In Fig. 15, the culture has been inoculated at 1 to 2 x 10 cells/ml with an initial flow rate of approximately 10 ml/h, sufficient to support the population growth. At densities of 7 x 10 -1.5 x 10" cells/ml, we have used a nutrient flow rate of 1340 ml/h using ITPSG and ITPSG-F68 serum-free media. The flow rate of fresh media was increased step-wise from 240 to 960 ml/d in proportion to the increase in cell density. This resulted in an increase of 4 to 10 fold in cell density compared to the conventional batch culture systems. This system was then scaled up to a 45 1 SUS316L unit mounted on an auto-sterilization sequence system with a medium reservoir and an effluent vessel of 901 each. 

This procedure represents a preparative version of zone electrophoresis. The apparatus and technique has been previously elaborated [294,295] separation itself is carried out either in a stream of electrolytic solution or on a sheet of cardboard (curtain electrophoresis). In the free flow version the separation is carried out in a cell formed by two parallel glass plates (50 x 50 cm) situated 0.5-1.0 mm apart. It is necessary to ensure an equal and laminar flow of the electrolyte, which is carried out by feeding the buffer through a multichannel peristaltic pump. The sample is continuously applied in the middle near the upper edge of the cuvette (or paper sheet). The electrophoretic separation occurs transversally between vertical electrodes located on the right and left hand side of the separation cuvette (Fig. 6.33). The separated fractions are collected at the lower end of the cell by a system of small communicating vessels or by a multi-channel pump. In the version using paper the... 

Then the conditions for continuous production of L-mallc acid by a column packed with this bile extract treated immobilized cells was studied. When 1 M sodium fumarate (pH 7.0) is passed through the column at 37 C at flow rate of space velocity=0.2 hr the reaction reaches an equiliblium. From the effluent of the column, L-malic acid can be obtained by ordinary methods. Average yield of pyrogen-free pure L-malic acid from consumed fumaric acid is around 70% of the theoretical. Tanabe Seiyaku Co., Ltd. is operating this production system since 1974, and we are satisfied both with the economical efficiency and with the quality of product. 

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