Recombinant protein expression cell-free

Nunoi and co-workers (1988) fractionated neutrophil cytoplasm by Mono Q anion-exchange chromatography and obtained three fractions (NCF-1, -2 and -3) that were active in the assembly of the oxidase. Independently, Volpp and colleagues (Volpp, Nauseef Clark, 1988) prepared antiserum from cytosolic factors that eluted from a GTP-affinity column, and this antiserum (Bl) recognised cytoplasmic factors of relative molecular masses 47 kDa and 66 kDa. It was later shown by this group that these cytosolic factors translocated to the plasma membrane during activation. NCF-1 was shown to contain the 47-kDa protein and NCF-2 the 66-kDa protein. Analysis of the defect in the cytosol of autosomal recessive CGD patients revealed that most of these (88%) lacked the 47-kDa protein (p41 -phox), whereas the remainder lacked the 66-kDa protein (p66-phox). Both of these components have now been cloned and recombinant proteins expressed. Interestingly, in the cell-free system, recombinant p47-phox and p66-phox can restore oxidase activity of the cytosol from autosomal recessive CGD patients who lack these components. 

An Insect Cell-Free System for Recombinant Protein Expression Using cDNA Resources... 

In the broadest terms, cell-free synthesis offers advantages over traditional in vivo methods of recombinant protein expression, as it enables the experimentalist strictly to control conditions under which expression reactions are performed. Benefits of such an open expression system are exemplified by the fact that cell-free reaction conditions can be sufficiently modified to support co-translational folding of active disulfide-bonded proteins. 

The previous ELP fusions all are examples of protein purification in which the ELP is covalently connected to the protein of choice. This approach is suitable for the purification of recombinant proteins that are expressed to high levels, but at very low concentrations of ELP the recovery becomes limited. Therefore this approach is not applicable for proteins expressed at micrograms per liter of bacterial culture, such as toxic proteins and complex multidomain proteins. An adjusted variant of ITC was designed to solve this problem. This variant makes use of coaggregation of free ELPs with ELP fusion proteins. In this coaggregation process, an excess of free ELP is added to a cell lysate to induce the phase transition at low concentrations of... 

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. 

Andersen et al. (1996) and Andersen (1995) have studied the effect of temperature on the recombinant protein production using a baulovinis/insect cell expression system. In Tables 17.15, 17.16, 17.17, 17.18 and 17.19 we reproduce the growth data obtained in spinner flasks (batch cultures) using Bombyx mori (Bm5) cells adapted to serum-free media (Ex-Cell 400). The working volume was 125 ml and samples were taken twice daily. The cultures were carried out at six different incubation temperatures (22, 26,28, 30 and 32 TT). 

The third time constraint depends on whether the product can be extracted from seeds or fruits. This uncouples protein expression and purification. Large batches of seeds containing the recombinant protein can be produced and stored at low costs. Provided the protein remains stable in the stored seeds, purification can be carried out on demand or shifted according to free capacities. The advantage of one large harvest, with seeds mixed to uniformity, is that this allows production on demand. In contrast, mammalian cell culture is prone to minor batch-to-batch variations in... 

As a result, protein molecules can be constructed in quantities well beyond those accessible in cell-free recombinant expression systems, which incorporate residues or linkages that cannot be specified by the genetic code. Very large chimeric fusion proteins can be made, and since the reaction conditions are generally quite mild, this can be done under circumstances where the individual protein modules retain their native conformation at all... 

In a batch configuration, host cells that contain an expression vector for the recombinant product are added to a predetermined volume of growth medium (Figure 4.12A). The cells are allowed to grow until the nutrients in the medium are depleted or the excreted by-products reach inhibitory levels. At that time, the cells are harvested, and recombinant protein, found in inclusion bodies, cell-membrane fractions, or cytoplasm, are isolated after disruption of the harvested cells. Because the host cells are destroyed at the end of each run, they must be replaced every three to seven days for fermentation or every two weeks for roller-bottle or microcarrier-support production of adherent cells. To ensure uniformity and reproducibility, the FDA requires manufacturers of recombinant proteins to establish and validate a seed stock of recombinant host cells that are validated to contain the characterized expression vector and to be free of contaminants. 

Any plasmid designed to express recombinant proteins in E. coli under the control of a bacteriophage RNA polymerase promoter can be used in an E. coli cell-free system. As a mle of thumb, constructs that express weU in vivo tend to express well in vitro. T7 RNA polymerase is often considered intrinsically more efficient than other RNA polymerases (see Part IV, Chapter 12). However, this differential efficiency can often be attributed to differential sensitivity to salt concentration. Because of the relative robustness and efficiency of the T7 polymerase, plasmids under the control of a T7 promoter are almost exclusively used in E. coli cell-free systems. 

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