Formation Of Proteins In Cells Research Articles

High-throughput expression, purification, and characterization of recombinant Caenorhabditis elegans proteins

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more difficult as functional proteomics efforts focus on the proteins from higher organisms, since issues of correctly identifying intron–exon boundaries and efficiently expressing and solubilizing the (often) multi-domain proteins from higher eukaryotes are challenging. Recently, 12,000 open-reading-frame (ORF) sequences from Caenorhabditis elegans have become available for functional proteomics studies [Nat. Gen. 34 (2003) 35]. We have implemented a high-throughput screening procedure to express, purify, and analyze by mass spectrometry hexa-histidine-tagged C. elegans ORFs in Escherichia coli using metal affinity ZipTips. We find that over 65% of the expressed proteins are of the correct mass as analyzed by matrix-assisted laser desorption MS. Many of the remaining proteins indicated to be “incorrect” can be explained by high-throughput cloning or genome database annotation errors. This provides a general understanding of the expected error rates in such high-throughput cloning projects. The ZipTip purified proteins can be further analyzed under both native and denaturing conditions for functional proteomics efforts.

Growth of lithotrophic ammonia-oxidizing bacteria on hydroxylamine

Nitrosomonas europaea, Nitrosomonas nitrosa and Nitrosococcus oceanus were successfully grown on hydroxylamine. Significant cell yields were obtained in media containing ammonia supplemented with successive small additions of hydroxylamine. The molar growth yield on hydroxylamine, measured as formation of cell protein per unit of substrate oxidized, was found to be approximately twice that on ammonia. In respiration experiments, the oxygen consumption was 1.5 mol O2 per mol ammonia and 1.0 mol O2 per mol hydroxylamine oxidized to nitrite.

Control of the formation of bacteriochlorophyll, and B875- and B850-bacteriochlorophyll complexes in Rhodopseudomonas sphaeroides mutant strain H5.

Rhodopseudomonas sphaeroides mutant H5 lacking 5-aminolevulinic acid synthase was employed to study the control of the formation of total bacteriochlorophyll as well as of the B875- and B850-bacteriochlorophyll protein complexes. The organisms were grown phototrophically in a chemostat where cell protein formation was limited by iron ions and bacteriochlorophyll by 5-aminolevulinic acid. 0.07 mol of bacteriochlorophyll was formed per mol of 5-amino-levulinic acid consumed. This stoichiometric relationship was not influenced by a twelve-fold variation in light energy flux. However, cell protein levels increased and, consequently, cellular specific bacteriochlorophyll contents decreased with increases in light energy flux. The ratio of B875- to B850-pigment protein complexes was inversely proportional to the velocity of 5-aminolevulinic acid supply (mol per cell protein and time) which in this system equals the velocity of 5-aminolevulinic acid consumption and the velocity of bacteriochlorophyll formation. Light had no direct effect on the ratio of B875- per B850-pigment complexes but an indirect effect via its control of protein formation. Changes in the ratio of the two pigment complexes resulted from the fact that significantly lower amounts of 5-aminolevulinic acid supplied per protein and time were required to saturate the system assembling the B875-complexes than that assembling the B850-complexes. The data suggest lack of light-dependent control in the formation of bacteriochlorophyll and its complexes subsequent to the 5-aminolevulinic acid pool.

Differences in the control of bacteriochlorophyll formation by light and oxygen

Control of bacteriochlorophyll formation was studied with continuous cultures of Rhodospirillum rubrum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas capsulata. Oxygen controlled specific bacteriochlorophyll contents of the three species in a hyperbolical fashion irrespective of the presence of light. In Rps. sphaeroides, this applied to oxygen concentrations above 16% air saturation of the medium while at lower oxygen concentrations control followed a kinetics with negative cooperativity. Cell protein formation of R. rubrum and Rsp. sphaeroides was independent of oxygen concentrations while protein formation of Rps. capsulata increased at lower concentrations. Light controlled bacteriochlorphyll contents of R. rubrum and Rps. sphaeroides in a sigmoidal fashion. When growing at a constant low oxygen concentration cell protein formation increased with light energy flux in Rps. sphaeroides but remained unaffected in R. rubrum. Protein formation of R. rubrum increased with light energy flux only under anaerobic conditions. Two factor analyses were performed with R. rubrum and Rps. sphaeroides to study the combined effects of light and oxygen on bacteriochlorophyll formation. The results showed that both factors act independent of each other.

Cellulase and protein production by Chaetomium cellulolyticum strains grown on cellulosic substrates

Fermentation with Chaetomium cellulolyticum was carried out on media containing either Avicel cellulose or newspaper. Production of free cellulolytic enzymes, cellulose degradation and the formation of cell protein were studied with the original strain and a mutant strain.

Iron deficiency and the growth of pea roots.

Pea roots have been cultured in sterile media in the presence and in the absence of added iron, and the effects of deficiency have been studied in three series of experiments. In the first, roots grown in deficient and full nutrient media were taken at intervals and on each sample growth and metabolic measurements were made. In the second, roots in which the earliest growth effects of deficiency had been observed were dissected into successive centimetre segments and metabolic and growth measurements were made on the separate fragments. In the third series of experiments the effects have been analyzed of transferring a root from a deficient medium in which growth had ceased to a full medium. It has been shown that after culture for 7 days in the deficient medium, increases in length and number of cells virtually cease; after this stage also the increase in respiration is relatively small. On the other hand protein continues to increase throughout the whole cultured period of 11 days. Oxygen absorption has been analyzed into a cyanide insensitive and a cyanide sensitive fraction, and it has been found that in the absence of iron, whereas the cyanide insensitive fraction increases continuously from the third to the eleventh day, the sensitive fraction ceases to increase after the seventh day. When roots in which growth has ceased were transferred to a full medium growth was resumed. The deficient roots were therefore not moribund. The arrest in growth is due to an abrupt cessation in division that occurs at about 7 days, and all the evidence indicates that this is the result of a disturbance confined to the meristem. The arrest in division cannot be attributed to an inhibition in the synthesis of protein. Evidence is presented which shows that in normal circumstances amino acids are probably synthesized in expanding or mature zones of the root, that they are carried forward in a polar translocation stream, and that they condense with the formation of proteins in cells that are being formed by meristematic activity. In the absence of iron division ceases, but the synthesis of amino acids continues. The sumps into which these acids are normally discharged are no longer provided, and they therefore tend to condense to proteins in the more mature cells adjacent to the apex. The cyanide-sensitive fraction of respiration is attributed to the activity of a cytochrome oxidase system. There is little or no further increase in this system after the time at which division ceases, and the coincidence may indicate a causal relation between the two events. But at the time when division ceases the cytochrome system is apparently normal in the apex and adjacent regions of the root. Therefore, if the cessation of division is due to arrest in the synthesis of cytochrome the position would be that a minimal quantity of cytochrome is required in the formation of a cell, and that when the supply of labile iron has been reduced to the level at which this minimal quantity cannot be provided then division ceases. This interpretation is consistent with the observation that the cessation of division is abrupt. The evidence, however, is not sufficiently extensive to warrant this conclusion, and the data do not exclude the alternative hypothesis that the depression in cytochrome synthesis is a consequence of the arrest in division.