Cotranslational formation of active photoprotein obelin in a cell-free translation system: direct ultrahigh sensitive measure of the translation course.

Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes.

Cell-free bioluminescent screening of translation inhibitors.

The possibility of creating new screening methods with a cell-free translation system has been demonstrated with a quantitative determination of diphtheria toxin and some antibiotics (puromycin, kanamycin and tetracycline) as examples. The approach proposed follows from the ability of various substances to inhibit protein synthesis. We used a wheat-germ cell-free translation system stabilized by freeze-drying in the presence of trehalose with the mRNA of the Ca(2+)-activated photoprotein obelin as a reporter template. This freeze-dried cell-free translation system allows prolonged storage of the detecting system before it is required, increases the reproducibility of the results and simplifies the application procedure. The obelin mRNA extends the sensitivity of the method owing to the high sensitivity of detection of the synthesized protein.

Green fluorescent protein purification by organic extraction.

Green fluorescent protein (GFP) is widely used as an excellent reporter molecule in biochemistry and cell biology. Some biochemical and immunological assays require high-purity GFP. However, the majority of current procedures for GFP purification include multiple time-consuming chromatography steps with a low yield of the desired product or require tag-containing proteins. An alternative method is described for the GFP purification without affinity extensions using organic extraction yielding a highly homogeneous protein indistinguishable in spectroscopic properties from that purified by previous methods.

Obelin mRNA--a new tool for studies of translation in cell-free systems.

Obelin mRNA obtained in vitro with the aid of SP6 RNA polymerase was translated in a wheat germ cell-free system. Only the polypeptide with a molecular mass of about 20 kDa was synthesized. The activation of apoobelin with a synthetic coelenterazine revealed a luminescence activity initiated by calcium. The specific activity was 3.6 +/- 0.4 x 10(15)photons per mg of the in vitro synthesized obelin (k=6.9s(-1)). The luminescence of the obelin was in a good correlation with the protein concentration calculated by the incorporation of [14C]Leu. The determination of the amount of de novo synthesized obelin based on measurement of its luminescence is one-thousand times more sensitive than the approach based on the incorporation of labeled amino acid. Thus, obelin mRNA has some advantages for evaluating the efficiency of cell-free translation when compared with standard methods.

Folding on the ribosome of Escherichia coli tryptophan synthase beta subunit nascent chains probed with a conformation-dependent monoclonal antibody.

Experimental analysis of protein folding during protein synthesis on the ribosome is rendered very difficult by the low concentration of nascent polypeptides and the heterogeneity of the translation mixture. In this study, an original approach is developed for analysing nascent polypeptide structures still carried by the ribosome. Folding on the ribosome of nascent chains of the beta subunit of Escherichia coli tryptophan synthase was investigated using a monoclonal antibody (mAb 19) recognizing a conformation-dependent antigenic determinant. Upon synthesis of beta subunits in an E. coli coupled transcription-translation system, it is shown that ribosome-bound nascent polypeptides can react with the monoclonal antibody provided their size is above 11.5 kDa, which is smaller than that of both the N-terminal proteolytic and crystallographic domains (29 and 21 kDa, respectively). The gene fragments coding only for the 11.5 kDa polypeptide, with and without stop codon at the end of the corresponding mRNAs, were constructed and expressed in a cell-free wheat germ translation system. It is shown that antibody 19 reacts with this polypeptide either bound to the ribosome or free in solution. That the 11.5 kDa polypeptide acquires a condensed structure is shown by gel filtration in native conditions and by urea gradient gel electrophoresis. Moreover, it is demonstrated that this condensed structure resembles that of native beta 2 in the vicinity of the epitope for antibody 19. Indeed, the affinity of antibody 19 for the 11.5 kDa fragment, either free or bound to the ribosome, was measured (6 x 10(8) M-1) and shown to be close to that for native beta 2. It is therefore proposed that the polypeptide chain may start to fold during its biosynthesis and that, even before the appearance of an entire domain, a folded intermediate is formed that already exhibits some local structural features of the native state and of an immunoreactive intermediate previously detected during the in vitro refolding of denatured complete beta chains.

Preparative in vitro synthesis of bioactive human interleukin-2 in a continuous flow translation system.

Recombinant human interleukin-2 has been synthesized in vitro in a continuous flow translation system based on the wheat germ extract. In the course of translation of mRNA the interleukin-2 becomes aggregated due to the adsorption of this protein onto the ribonucleoprotein complex. This process correlates with the cessation of translation that is usually observed in 25-30 min. This can be prevented by the use of a flow system that allows continuous removal of the synthesized protein and maintains a steady concentration of all the necessary components. This approach permitted a yield of 1,500 protein molecules per mRNA molecule. The interleukin obtained promotes the proliferation of the interleukin-2-dependent CTLL-2 cell line. The biological activity of interleukin-2 not subjected to oxidative refolding was 10(5) units per milligram of protein.

A continuous cell-free translation system capable of producing polypeptides in high yield.

A cell-free translation system has been constructed that uses a continuous flow of the feeding buffer [including amino acids, adenosine triphosphate (ATP), and guanosine triphosphate (GTP)] through the reaction mixture and a continuous removal of a polypeptide product. Both prokaryotic (Escherichia coli) and eukaryotic (wheat embryos, Triticum sp.) versions of the system have been tested. In both cases the system has proven active for long times, synthesizing polypeptides at a high constant rate for tens of hours. With the use of MS2 phage RNA or brome mosaic virus RNA 4 as templates, 100 copies of viral coat proteins per RNA were synthesized for 20 hours in the prokaryotic or eukaryotic system, respectively. With synthetic calcitonin messenger RNA, 150 to 300 copies of calcitonin polypeptide were produced per messenger RNA in both types of continuous translation systems for 40 hours.

Synthesis of high-capacity immunoaffinity sorbents with oriented immobilized immunoglobulins or their Fab' fragments for isolation of proteins.

Two methods for synthesizing high-capacity immunoaffinity sorbents on Sepharose and Separon HEMA E-1000 are described. The first is the oriented immobilization of monovalent immunoglobulin Fab fragments on a maleimide derivative of Sepharose via the formation of a covalent bond between the SH group of the Fab fragment at the C-terminus of the molecule and the maleimide covalently coupled to Sepharose. The second method is based on the oxidation of the immunoglobulin carbohydrate component, located in the Fc fragment, by periodate with subsequent immobilization of the derivatives on hydrazide derivatives of Sepharose or Separon. Sorbents for the isolation of monoclonal antibodies from the culture supernatants and the elongation factor EF-G from a crude extract of Escherichia coli cells were obtained. These sorbents are characterized by a high capacity, minimal non-specific sorption and high stability.

Interaction of the N-terminal and C-terminal domains of elongation factor G on formation of complexes with guanyl nucleotides.

Polarized fluorescence studies of interaction between guanyl nucleotides (GTP and GDP) and elongation factor G and its N-terminal tryptic fragment T*/s, carrying a fluorescent group (aminorhodamine B) at the exposed cysteine residue, has shown that binding on nucleotides by an intact EF-G molecule at neutral pH essentially affects the mobility of the fluorescent group. GTP binding changes its relaxation properties to a greater extent than GDP binding. At the same time it was demonstrated that the spectrum of relaxation time of the fluorescent group practically does not change on binding of nucleotides by the N-terminal fragment T*/2 (in the absence of the C-terminal domain) or in the case when the three-dimensional structure of the intact EF-G molecule is destabilized (pH 10). Comparison of the relaxation properties of EF-G and its N-terminal fragment T*/2, carrying a fluorescent group at the exposed cysteine residue, at pH 7.5 and 10, indicates that the C-terminal domain is involved in the formation of the close environment of the exposed cysteine residue located in the N-terminal part of EF-G. A conclusion is drawn on the nucleotide-induced influence of the C-terminal domain on a change of the exposed cysteine residue environment on guanyl nucleotide binding with EF-G at neutral pH and a hypothetical model of the EF-G molecule is proposed.

Tyrosine residues in the C-terminal domain of the elongation factor G are essential for its interaction with the ribosome.

Chemical modification of the elongation factor G (EF-G) with tetranitromethane and iodine has been studied. It has been shown by spectrophotometric titration that EF-G contains two exposed tyrosine residues, one of which has an unusually low pK value for a phenol hydroxyl group at pH 8.5. Modification of one tyrosine residue with either tetranitromethane or iodine results in a 70--80% loss of EF-G activity in all ribosome-dependent reactions. Modification of three or four residues inhibits 90--100% of activity. Binding of EF-G with the 70-S ribosome and 50-S subunit is equally effective for protection of tyrosine residues against modification. The rate of EF-G modification with tetranitromethane is considerably higher in the presence of guanyl nucleotides than for free EF-G. The modified residues are located in the C-terminal domain of EF-G and are presumably contained in one of the sites of EF-G interaction with the ribosome.

Cleavage of elongation factor G into compact domains.

A limited trypsinolysis of native elongation factor G results in the formation of two large fragments resistant to further proteolysis. The fragments were isolated in homogeneous state in conditions when their native structure is retained. According to circular dichroism and scanning calorimetry data the formed fragments retain the stability and compact structure that they had in the whole protein.