Recombinant protein 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. 

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. 

Most frequently, extracts of either prokaryotic or eukaryotic origin as such from Escherichia coli, wheat germ or rabbit reticulocytes are employed for cost reasons and availability. While those based on E. coli are unable of post-translational protein modification, eukaryotic extracts do allow synthesis of glycosylated or phosphorylated proteins to some extent when additional components, such as microsomes for glycosylation are added. Care needs to be taken with cell-free systems recombinated from the individual components when a native protein is to be produced that does not fold spontaneously... 

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

Based on the vectors for the intracellular production and purification of recombinant proteins, the vector system was further expanded with vectors for the extracellular recombinant protein production. Affinity tag aided protein purification from the cell-free growth medium was made possible by the addition of the corresponding His6- and StrepII-tags [20, 35]. 

Newcomb et al. (1994) showed that B capsids also assemble in a cell-free system, using extracts prepared from Sf9 cells infected with recombinant baculoviruses encoding the HSV-1 capsid proteins. Similar to the observations by Tatman et al. (1994) and Thomson et al. (1994), the capsids formed in the cell-free system resembled native B capsids in morphology, sedimentation rate, protein compositions, and ability to react with HSV-Tspecific antibodies. Additional work by Newcomb et al. (1996) led to the identification of several assembly intermediates. In the cell-free system the first structures observed were partial capsids that consisted of an arclike segment of the external shell surrounding a region... 

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. 

An important contribution to fundamental ceU-free science has come from the laboratory of Ueda et al. at the University of Tokyo, Japan [12]. This group reconstituted a cell-free system with purified recombinant translation factors used by E. coli, producing 160 tg mL hr protein in a simple batch-mode reaction. That system is referred to as the PURE (Protein synthesis Using Recombinant Elements) system. 

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). 

Most of the patients with autosomal recessive CGD lack pAl-phox whilst the remainder lack p66-phox (details are given in 8.2). Recombinant proteins derived from these full-length cDNA clones can restore activity when added to extracts of autosomal recessive CGD neutrophils, in the cell-free oxidase activation system. 

---