An optimized yeast cell-free system: sufficient for translation of human papillomavirus 58 L1 mRNA and assembly of virus-like particles

We developed a yeast cell-free system suitable for in vitro translation of human papillomavirus 58 (HPV58) L1 mRNA. This system was systematically optimized resulting in enhanced translation efficiency. The optimal concentrations of potassium and magnesium ions observed were specific to the HPV58 L1 protein production. Supplementation with sucrose in the preparation of the yeast lysate greatly enhanced its stability. After optimization, protein production in this system was significantly superior to that produced by the rabbit reticulocyte (RRL) system. Finally, we demonstrated for the first time that virus-like particles (VLPs) were assembled from HPV58 L1 capsid protein in the yeast cell-free system. Thus, the system described here is a powerful tool for the HPV L1 protein production and will be useful for the study of VLP assembly and DNA encapsulation.     

The Saccharomyces cerevisiae poly (A) binding protein (Pab1): Master regulator of mRNA metabolism and cell physiology

Pab1, the major poly (A) binding protein of the yeast Saccharomyces cerevisiae, is involved in many intracellular functions associated with mRNA metabolism, such as mRNA nuclear export, deadenylation, translation initiation and termination. Pab1 consists of four RNA recognition motifs (RRM), a proline-rich domain (P) and a carboxy-terminal (C) domain. Due to its modular structure, Pab1 can simultaneously interact with poly (A) tails and different proteins that regulate mRNA turnover and translation. Furthermore, Pab1 also influences cell physiology under stressful conditions by affecting the formation of quinary assemblies and stress granules, as well as by stabilizing specific mRNAs to allow translation re-initiation after stress. The main goal of this review is to correlate the structural complexity of this protein with the multiplicity of its functions.     

5'-secondary structure formation, in contrast to a short string of non-preferred codons, inhibits the translation of the pyruvate kinase mRNA in yeast

The effects of poor codon bias and secondary structure formation upon the translation of the pyruvate kinase (PYK1) mRNA have been investigated in Saccharomyces cerevisiae. Following insertion mutagenesis at the 5'-end of the PYK1 coding region, the gene was transformed into yeast, and translation assessed directly in vivo by determining the distribution of the modified PYK1 mRNAs across polysomes fractionated by sucrose density gradient centrifugation. The chromosomally-encoded (wild-type) PYK1 mRNA, and the actin, ribosomal protein L3 and glyceraldehyde-3-phosphate dehydrogenase mRNAs were used to control for minor differences between polysome preparations. An insertion containing 13 non-preferred codons at the 5'-end of the coding region was found to have no significant effect upon PYK1 mRNA translation. In contrast, translation was inhibited by an insertion which increased the formation of secondary structures at the 5'-end of the mRNA (overall delta G = -36.6 kcal/mol). Control insertions were also analysed to exclude the possibility that alterations to the amino acid sequence of pyruvate kinase affect the translation of its mRNA. These insertions, which introduced preferred codons or restored wild-type levels of secondary structure formation, did not significantly influence PYK1 mRNA translation.     

Four-base codon-mediated incorporation of non-natural amino acids into proteins in a eukaryotic cell-free translation system

Various four-base codons have been shown to work for the introduction of non-natural amino acids into proteins in an Escherichia coli cell-free translation system. Here, a four-base codon-mediated non-natural mutagenesis was applied to a eukaryotic rabbit reticulocyte cell-free translation system. Mutated streptavidin mRNAs containing four-base codons were prepared and added to a rabbit reticulocyte lysate in the presence of tRNAs that were aminoacylated with a non-natural amino acid and had the corresponding four-base anticodons. A Western blot analysis of translation products indicated that the four-base codons CGGU, CGCU, CCCU, CUCU, CUAU, and GGGU were efficiently decoded by the aminoacyl-tRNAs having the corresponding four-base anticodons. In contrast, the four-base codons AGGU, AGAU, CGAU, UUGU, UCGU, and ACGU were not decoded. The stop codon-derived four-base codons UAGU, UAAU, and UGAU were found to be inefficient, whereas the amber codon UAG and opal codon UGA were efficient for the incorporation of non-natural amino acids. The application of the expanded genetic code in a eukaryotic cell-free system opens the possibility of a four-base codon-mediated incorporation of non-natural amino acids into proteins in living eukaryotic cells.     

Four-base codon-mediated saturation mutagenesis in a cell-free translation system

Saturation mutagenesis is a useful technique for the structural and functional analyses of proteins and for protein engineering. However, the extensive mutagenesis of genes and expression of mutated proteins are tedious and time-consuming. We have developed a simple and rapid method for the expression of mutated proteins with comprehensive single amino acid substitutions from single mutated genes having a four-base codon in a cell-free translation system. Twenty types of tRNA that were aminoacylated with one of the 20 proteinogenic amino acids and that contained a four-base anticodon were prepared by chemical aminoacylation. In the presence of one of the aminoacyl-tRNAs, a streptavidin mRNA with a four-base codon at the Tyr83 position was expressed in an Escherichia coli cell-free translation system. The N-terminus of the expressed proteins was fluorescently labeled using a fluorescent-labeled initiator Met-tRNA. Fluorescence imaging of an SDS-PAGE gel showed that all the amino acids are incorporated in response to the four-base codon; however, the incorporation efficiency was dependent on the structure of the side chains. Streptavidin mutants with comprehensive amino acid substitutions at the Tyr83, Arg84, and Tyr54 positions were used for analyzing their biotin-binding activity by dot blot analysis. These results demonstrate that this method is effective for the expression and analysis of mutated proteins with comprehensive amino acid substitutions at desired positions.     

Performance of expression vector, pTD1, in insect cell-free translation system

We constructed a pTD1 vector for an insect cell-free translation system containing a 5' untranslated region (UTR) of a polyhedrin gene as a translational enhancer sequence. Its translational efficiency was about 50-fold higher than those of mRNAs without an enhancer sequence. Moreover, the pTD1 vector functioned as an effective expression vector not only in the insect cell-free translation system but also in wheat germ extract and rabbit reticulocyte lysate systems.     

Study of the precursors of ovine lactoproteins: primary structures of the 'signals' and enzymic processing of prelactoproteins by mammary microsomal membranes.

The radiolabelled primary translation products of ovine mammary mRNAs synthesized in a wheat germ cell-free system were isolated by immunoprecipitation and analysed by automated Edman degradation. The 3 'Ca-sensitive' caseins (alpha S1, alpha S2 and beta), kappa-casein, beta-lactoglobulin and alpha-lactalbumin were found to be synthesized as precursors with amino terminal extensions of 15, 21, 18 and 19 amino acid residues respectively. The extra pieces of these various lactoproteins are similar to 'signal' peptides of other secretory proteins in their length and hydrophobicity. The occurrence of an alanyl residue at the C-termini of the extra pieces of the 6 ovine prelactoproteins suggests that the mammary proteinase responsible for the cleavage of the signal peptides may have an elastase-like specificity. When mammary mRNAs were translated in a wheat germ cell-free system in the presence of mammary microsomal membranes, pre-beta-casein was converted into authentic beta-casein as demonstrated by amino terminal sequence analyses. Additionally, pre-beta-casein was post-translationally converted into authentic beta-casein by a specific proteinase(s) extracted from rough microsomes with Na deoxycholate.     

Decapping of stabilized,polyadenylated mRNA in yeast pab1 mutants

Interaction of the poly(A) binding protein, Pab1p, with mRNA plays an important role in gene expression. This work describes an analysis of pab1 mutants in Saccharomyces cerevisiae. Yeast pab1 mutants were found to be sensitive to elevated concentrations of copper (Cu) and 3‐aminotriazole (3‐AT) in the growth medium. This phenotype arises because these pab1 mutants underaccumulate mRNA, including the CUP1 and HIS3 mRNAs, the products of which are required for Cu and 3‐AT resistance, respectively. To determine the cause of the mRNA underaccumulation, mRNA turnover and production were examined in the pab1‐53 mutant. It was found that although the pattern of mRNA decay was altered, and substantial decapping of polyadenylated mRNA could be detected, mRNA was not destabilized in this strain. It was also found that the pab1 mutant was impaired in the production of mRNA. These data show that the decreased level of mRNA in the pab1‐53 mutant arises from poor production, and they suggest that yeast Pab1p is involved in an aspect of nuclear mRNA metabolism. They also indicate that deadenylation can be uncoupled from decapping without significant changes in an mRNA's stability, and that this uncoupling can be tolerated by yeast. Copyright © 1999 John Wiley & Sons, Ltd.     

Abundance and phosphorylation state of translation initiation factors in mammary glands of lactating and nonlactating dairy cows

To test if control of mRNA translation is involved in the increase in protein synthesis by mammary glands during lactation, cellular contents and phosphorylation states of translation factors and their upstream regulators were measured in mammary parenchyma from 12 nonpregnant dairy cows. For a 42-d period, 6 cows in late lactation continued to be milked (L) and 6 at the same stage of lactation were dried off (NL). All cows were then slaughtered and mammary glands and tissue samples obtained. Alveoli and lobules tended to be larger in L cows. Mammary parenchymal mass, cell number, cell size, and RNA, DNA, and protein contents were greater in L cows. Increases (3.1- and 1.8-fold) in the abundance of active, phosphorylated ribosomal protein S6 and its kinase, S6K1, respectively, in L vs. NL parenchyma indicated an ability to sustain greater rates of synthesis of translational machinery, which was also evident in the 102% increase in parenchymal RNA:DNA between the 2 groups. Cellular abundances of the main eukaryotic translation initiation factors (eIF), eIF2 and eIF4E, were 2.6- and 3-fold greater, respectively, in L cows. That these differences were greater than the 102% greater RNA:DNA in L mammary parenchyma suggests an elevated translational efficiency in L glands. Abundance of phosphorylated rpS6 was not different between mammary parenchyma and liver, whereas eIF2α was 50% greater in mammary tissue. In semimembranosus muscle, abundances of phosphorylated rpS6 and eIF2α were 3 to 4 times lower than in mammary parenchyma. In both L and NL mammary glands, 11% of eIF2α was in the inhibitory, phosphorylated form and 48 to 60% of eIF4E was complexed with its binding protein, 4EBP1. It is concluded that up-regulation of initiation of mRNA translation occurs in the fully differentiated milk secretory cell and that, where crucial initiation factors are not present in a maximally active form, the initiation rate might be flexible in response to external stimuli.     

Biosynthesis of proteins containing modified lysines and fluorescent labels using non-natural amino acid mutagenesis

The preparation of posttranslationally modified proteins is required to investigate the function and structure of modified proteins. However, homogeneously modified proteins are not easily isolated from natural sources or prepared using modification enzymes. Non-natural amino acid mutagenesis has enabled us to incorporate modified amino acids into specific positions of proteins in both cell-free and in-cell translation systems using tRNAs that are aminoacylated with modified amino acids. Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins. To introduce methyllysine, dimethyllysine, trimethyllysine, and acetyllysine, frameshift and amber suppressor tRNAs aminoacylated with modified lysines were synthesized by chemical aminoacylation and supplied to an Escherichia coli cell-free translation system. The immunodetection of the translation products indicated that the modified lysines were incorporated into streptavidin and histone H3 in a quantitative, position-specific manner. Calmodulin derivatives containing a fluorescent non-natural amino acid at the N-terminal region and modified lysines at the Lys115 position were also synthesized, and their binding activity to a calmodulin-binding peptide was analyzed by fluorescence correlation spectroscopy. The results obtained here demonstrate that this method is useful in preparing and analyzing naturally occurring and non-natural modified proteins.     

Analytical nutritional characteristics of seed proteins in six wild Lupinus species from Southern Spain

The nutritional characteristics of seed proteins of Spanish wild populations of Lupinus angustifolius, L. cosentinii, L. gredensis, L. hispanicus, L. luteus and L. micranthus have been studied. Protein contents in this genus ranged from 23.8% in L. gredensis to 33.6% in L. luteus. On the one hand, L. cosentinii showed the most balanced amino acid composition, being only deficient in lysine. On the other hand, L. gredensis showed the worst amino acid composition. The in vitro protein digestibility (IVPD) was high in all species examined, ranging from 82.3% in L. gredensis to 89.0% in L. cosentinii. In addition to the amino acid composition and IVPD, other nutritional parameters, such as amino acid score, calculated biological value, predicted protein efficiency ratio or protein digestibility corrected amino acid score, were studied. These data yielded L. luteus, L. hispanicus and L. cosentinii as the species with seed proteins with the best nutritional properties, similar to those observed in other legumes with recognised high quality proteins, such as soybean. Results confirm the importance of studying wild populations of cultivated and non-cultivated Lupinus species as sources of seeds with good nutritional characteristics.     

Structure and expression of yeast DPR1, a gene essential for the processing and intracellular localization of ras proteins

The ras proteins represent a unique example of membrane proteins which apparently do not utilize the secretory pathway for their membrane localization. Instead, it is believed that palmitic acid, covalently attached to the protein, acts as an anchor to the membranes. Recent identification of yeast mutants defective in the processing of the ras proteins has provided a novel approach for defining these biosynthetic processes. We report here the characterization of yeast DPR1, a gene essential for the processing of the ras proteins. The sequence of the gene indicates that it encodes a protein of 431 amino acids which contains no significant homology with any known proteins. It is a relatively hydrophilic protein with no apparent hydrophobic stretches. The C-terminal half of the encoded protein has an unusually high content of cysteine. The DPR1 gene product has been identified in a cell-free translation system as a protein having an apparent molecular weight of 43 kd. This represents the first step in the investigation of a novel protein-processing pathway, one that is distinct from the secretory pathway.     

Regulation of translation initiation by the yeast eIF4E binding proteins is required for the pseudohyphal response

The eukaryotic translation initiation factor eIF4E is responsible for the recognition of the mRNA cap structure and, as such, plays a key role in the selection of mRNAs for translation. The interaction of eIF4E with the 'multi-adaptor' eIF4G (and thus recruitment of ribosomes to mRNA) can be regulated via competitive binding of 4E-binding proteins (4E-BPs). 4E-BPs have broad functions in cell growth, proliferation and development. We have found that disruption of the genes for either of the yeast 4E-BPs (Eap1p or Caf20p) leads to an inhibition of pseudohyphal growth in the resulting diploid yeast strain following nitrogen limitation. Specific 4E-binding domain mutations destroy the capacity of each 4E-BP gene to complement the non-pseudohyphal phenotype, suggesting that a translational function for the 4E-BPs is important for pseudohyphal growth. In addition, neither of the 4E-BP deletion strains is deficient in global or stress-regulated protein synthesis. However, our evidence reveals that the two 4E-BPs are functionally distinct with regard to pseudohyphal growth. Therefore, this work supports a model where the yeast 4E-BPs are acting on specific mRNAs to facilitate a defined proliferative response to environmental stress in yeast.     

Comparing cytosolic expression to peroxisomal targeting of the chimeric L1/L2 (ChiΔH-L2) gene from human papillomavirus type 16 in the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha

The chimeric ChiΔH‐L2 gene from human papillomavirus type 16, consisting of structural proteins L1 and L2, was successfully expressed in the cytosol of both Pichia pastoris and Hansenula polymorpha during methanol induction. In addition, a novel approach was employed whereby ChiΔH‐L2 was targeted to the peroxisome using peroxisomal targeting sequence 1 (PTS1) to compare ChiΔH‐L2 yields in the peroxisome vs the cytosol. The ChiΔH‐L2 gene was yeast‐optimized and cloned into plasmids aimed at genomic integration. Levels of intracellular ChiΔH‐L2 accumulation in the cytosol were highest in P. pastoris KM71 strain KMChiΔH‐L2 (1.43 mg/l), compared to the maximum production level of 0.72 mg/l obtained with H. polymorpha. ChiΔH‐L2 targeting to the peroxisome was successful; however, it appeared to negatively affect ChiΔH‐L2 production in both P. pastoris and H. polymorpha. Copyright © 2012 John Wiley & Sons, Ltd.     

Stabilization effect of zeolite on DHFR mRNA in a wheat germ cell-free protein synthesis system

The effects of zeolites and monocations on the protein synthesis in a cell-free system derived from wheat germ were investigated. M type of synthetic zeolite markedly enhanced the translation efficiency. Whereas this kind of stimulatory effect of zeolite in an Escherichia coli cell-free system resulted from a change in the salt compositions of the reaction solution with the addition of zeolite, the enhancement of protein synthesis in a wheat germ cell-free system was not due to the ion exchange reaction of zeolites. From the results of mRNA stability analysis, it was found that zeolite could stabilize the mRNA in a wheat germ cell-free protein synthesis system. The stabilization of mRNA by the simple addition of zeolites is useful for the enhancement of protein synthesis in a wheat germ cell-free system, since conventional methods to improve mRNA stability, such as the addition of nuclease inhibitor, have not been effective for a wheat germ cell-free system.     

A quantitative model for mRNA translation in Saccharomyces cerevisiae

Messenger RNA (mRNA) translation is an essential step in eukaryotic gene expression that contributes to the regulation of this process. We describe a deterministic model based on ordinary differential equations that describe mRNA translation in Saccharomyces cerevisiae. This model, which was parameterized using published data, was developed to examine the kinetic behaviour of translation initiation factors in response to amino acid availability. The model predicts that the abundance of the eIF1–eIF3–eIF5 complex increases under amino acid starvation conditions, suggesting a possible auxiliary role for these factors in modulating translation initiation in addition to the known mechanisms involving eIF2. Our analyses of the robustness of the mRNA translation model suggest that individual cells within a randomly generated population are sensitive to external perturbations (such as changes in amino acid availability) through Gcn2 signalling. However, the model predicts that individual cells exhibit robustness against internal perturbations (such as changes in the abundance of translation initiation factors and kinetic parameters). Gcn2 appears to enhance this robustness within the system. These findings suggest a trade‐off between the robustness and performance of this biological network. The model also predicts that individual cells exhibit considerable heterogeneity with respect to their absolute translation rates, due to random internal perturbations. Therefore, averaging the kinetic behaviour of cell populations probably obscures the dynamic robustness of individual cells. This highlights the importance of single‐cell measurements for evaluating network properties. Copyright © 2010 John Wiley & Sons, Ltd.     

A deviation from the universal genetic code in Candida maltosa and consequences for heterologous expression of cytochromes P450 52A4 and 52A5 in Saccharomyces cerevisiae

We demonstrate that serine instead of leucine is specified by the CUG codon in the yeast Candida maltosa. Evidence for this deviation from the universal genetic code was obtained by means of in vitro translation experiments. Depending on the cell-free system used, either serine, in the C. maltosa system, or leucine, in the control with the conventional wheat germ system, was found to be incorporated into the translation products of artificial CUG-containing mRNAs. Moreover, we were able to transfer the non-universal decoding of CUG to the wheat germ system by adding a tRNA fraction isolated from C. maltosa. This finding indicates the presence in C. maltosa of an unusual serine tRNA that recognizes CUG. As a consequence of the altered genetic code, expression in Saccharomyces cerevisiae of C. maltosa cytochrome P450 genes required an exchange of their CTG triplets by TCT encoding serine in order to produce the authentic proteins. In contrast, heterologous expression of the original C. maltosa genes resulted in the formation of still active but unstable enzymes probably subject to selective proteolysis in the host cells.     

Effects of gamma irradiation on edible seed protein,amino acids and genomic DNA during sterilization

This work describes radiation-induced effects on edible seed protein profiles, carbohydrates, amino acids and genomic DNA during gamma sterilization. The total protein and carbohydrate was decreased with increasing dose compared to control samples. Oryzasativa L. Cv-2233 exhibited a minimum effect in terms of its loss in total soluble protein content, compared to other seeds at 6 kGy, and the soluble protein fraction, containing 14–16 kDa albumins and 22 kDa globulin, was unchanged up to 6 kGy. In Cicer arietinum, the effect of gamma rays was more pronounced on albumin and prolamin with respect to glutelin and globulin. The easy-to-digest and difficult-to-digest proteins were not significantly affected up to 4 kGy. However, the soluble free amino acids of all the seeds increased with increasing dose. The total DNA content and band intensities both decreased with increasing absorbed dose; however, the band positions were unchanged for all seed types.