Translational recombinant expression

When working with purified enzymes, it can be useful to perform a close examination of their phosphorylation states and molecular masses. Mass spectrometry is often useful for this purpose. Post-translational modifications or sequence truncations can potentially alter the compound binding sites available and can also change the structure of potential inhibitory sites. For example, with protein kinases, phosphorylations distal from the ATP binding site can inactivate the kinase whereas phosphorylations near the ATP binding site can activate the catalytic activity. Often, practice does not permit control of such situations because the purified systems are often mixtures and cannot be controlled in the commonly used recombinant expression technologies. 

Ribosomes needed for translation in the PURE system are isolated from E. coli using sucrose-density gradient centrifugation. The protein factors necessary for translation in E. coli are recombinantly expressed as His-tagged fusions, and purified to homogeneity. These include the factors for initiation (IFl, IF2, and IF3), elongation (EF-G, FF-Tu, FF-Ts), peptide chain release (RFl and RF3), ribosome recycling (RRF), methionyl-tRNA transformylase (MTF) for formylation of the initial Met-tRNA, and the 20 aminoacyl-tRNA synthetases (ARSs) for transfer RNA (tRNA) recy-... 

Thuku, R.N., Weber, B.W., Varsani, A., and Sewell, B.T. (2007) Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form. FEBS J., 274, 2099-2108. 

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. 

Studies have shown that plants can make biologically active recombinant proteins through both transgenic and transient expression approaches. Although the plant post-translational machinery is similar to that of mammalian cells, there are some notable differences, e.g. differences in glycosylation, particularly the absence of sia-lation, which may impact the activity of certain proteins. The absence of mammalian enzymes may prevent complex maturation processes that are critical for the biological activity of proteins such as insulin. Fortunately these shortcomings affect the activity of only a limited number of proteins. 

The desired gene/cDNA is normally amplified, sequenced and then introduced into an expression vector that facilitates its introduction and expression (transcription and translation) in an appropriate producer cell type. All recombinant therapeutic proteins approved to date are produced in E. coli, S. cerevisiae or in animal cell lines (mainly CHO or BHK cells). The general... 

Most interferons have now been produced in a variety of expression systems, including E. coli, fungi, yeast and some mammalian cell lines, such as CHO cell lines and monkey kidney cell lines. Most interferons currently in medical use are recombinant human (rh) products produced in E. coli. E. coli s inability to carry out post-translational modifications is irrelevant in most instances, as the majority of human IFN-as, as well as IFN- 3, are not normally glycosylated. Whereas IFN-y is glycosylated, the E. coli-derived unglycosylated form displays a biological activity similiar to the native human protein. 

---