A comparative study of protein synthesis in <Emphasis Type="Italic">in vitro</Emphasis> systems: from the prokaryotic reconstituted to the eukaryotic extract-based

Background  

Cell-free protein synthesis is not only a rapid and high throughput technology to obtain proteins from their genes, but also provides an in vitro platform to study protein translation and folding. A detailed comparison of in vitro protein synthesis in different cell-free systems may provide insights to their biological differences and guidelines for their applications.     

High quality protein microarray using in situ protein purification

Background  

In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC). This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays.     

Encapsulated <Emphasis Type="Italic">Escherichia coli</Emphasis> in alginate beads capable of secreting a heterologous pectin lyase

Background  

Production of heterologous proteins in the E. coli periplasm, or into the extracellular fluid has many advantages; therefore naturally occurring signal peptides are selected for proteins translocation. The aim of this study was the production in high yields of a recombinant pectin lyase that is efficiently secreted and the encapsulation of transformed E. coli cells for pectin degradation in a biotechnological process.     

Transient increase of ATP as a response to temperature up-shift in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift.     

Viral promoters can initiate expression of toxin genes introduced into <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures.     

Predicting the pathway involved in post-translational modification of Elongation factor P in a subset of bacterial species

Background  

The bacterial elongation factor P (EF-P) is strictly conserved in bacteria and essential for protein synthesis. It is homologous to the eukaryotic translation initiation factor 5A (eIF5A). A highly conserved eIF5A lysine is modified into an unusual amino acid derived from spermidine, hypusine. Hypusine is absolutely required for eIF5A's role in translation in Saccharomyces cerevisiae. The homologous lysine of EF-P is also modified to a spermidine derivative in Escherichia coli. However, the biosynthesis pathway of this modification in the bacterial EF-P is yet to be elucidated.     

Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain

Background  

The expression of heterologous proteins in Escherichia coli is strongly affected by codon bias. This phenomenon occurs when the codon usage of the mRNA coding for the foreign protein differs from that of the bacterium. The ribosome pauses upon encountering a rare codon and may detach from the mRNA, thereby the yield of protein expression is reduced. Several bacterial strains have been engineered to overcome this effect. However, the increased rate of translation may lead to protein misfolding and insolubilization. In order to prove this assumption, the solubility of several recombinant proteins from plants was studied in a codon bias-adjusted E. coli strain.     

Revolutionizing membrane protein overexpression in bacteria

The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory, especially for eukaryotic membrane proteins. This has initiated a revolution of membrane protein overexpression in bacteria. Recent studies have shown that it is feasible to (i) engineer or select for E. coli strains with strongly improved membrane protein overexpression characteristics, (ii) use bacteria other than E. coli for the expression of membrane proteins, (iii) engineer or select for membrane protein variants that retain functionality but express better than the wild-type protein, and (iv) express membrane proteins using E. coli-based cell-free systems.     

Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

Escherichia coli-based cell-free protein synthesis is a powerful emerging tool for protein engineering due to the open, accessible nature of the reaction and its straightforward, economical potential for many diverse applications. One critical limitation of this system is the inability to express some complex, eukaryotic, and/or unnatural proteins at high expression yields. A potential solution is a synthetic-biology-like approach where cell-free reactions are supplemented by expressing the required supplemental components in the E. coli cells during the fermentation, which cells are used to prepare the extract for cell-free protein synthesis. Here we report adjustments to the fermentation conditions that increase yields of complex proteins upwards of 150% over standard conditions. We consider extracts containing GroEL/ES protein folding chaperones and extracts containing orthogonal tRNA/tRNA synthetase pairs for noncanonical amino acid incorporation. In contrast to standard cell-free synthesis, delaying the harvest of supplemented fermentations lead to increased and more consistent yields of proteins that required supplemental components. Protein yields enhanced by buffering the fermentation media pH lead to an average 52% decrease in yield cost, while costs for cases unchanged or negatively affected by buffering increased an average 14%. An apparent balance is required between the supplemental components and general extract protein profile.     

A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.     

Biotinylated-sortase self-cleavage purification (BISOP) method for cell-free produced proteins

Background  

Technology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins. In general, affinity tags, such as glutathione-S-transferase or six-histidines, are used to purify recombinant proteins. Since such affinity tags often interfere negatively with the structural and functional analyses of proteins, they are usually removed by treatment with proteases. Previously, Dr. H. Mao reported self-cleavage purification of a target protein by fusing the sortase protein to its N-terminal end, and subsequently obtained tag-free recombinant protein following expression in Escherichia coli. This method, however, is yet to be applied to the cell-free based protein production.     

Genomic transcriptional response to loss of chromosomal supercoiling in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure.     

Slowing Bacterial Translation Speed Enhances Eukaryotic Protein Folding Efficiency

The mechanisms for de novo protein folding differ significantly between bacteria and eukaryotes, as evidenced by the often observed poor yields of native eukaryotic proteins upon recombinant production in bacterial systems. Polypeptide synthesis rates are faster in bacteria than in eukaryotes, but the effects of general variations in translation rates on protein folding efficiency have remained largely unexplored. By employing Escherichia coli cells with mutant ribosomes whose translation speed can be modulated, we show here that reducing polypeptide elongation rates leads to enhanced folding of diverse proteins of eukaryotic origin. These results suggest that in eukaryotes, protein folding necessitates slow translation rates. In contrast, folding in bacteria appears to be uncoupled from protein synthesis, explaining our findings that a generalized reduction in translation speed does not adversely impact the folding of the endogenous bacterial proteome. Utilization of this strategy has allowed the production of a native eukaryotic multidomain protein that has been previously unattainable in bacterial systems and may constitute a general alternative to the production of aggregation-prone recombinant proteins.     

Identifying the mechanism of Escherichia coli O157:H7 survival by the addition of salt in the treatment with organic acids

Aim

In this study, the effects of the addition of salt to treatment with acids (one of several organic acids and salt in various solutions including rich or minimal broth, buffer, or distilled water) on the reduction of Escherichia coli O157:H7 were investigated. The protein expression profiles corresponding to acid stress (acetic acid) with or without salt addition were studied using a comparative proteomic analysis of E. coli O157:H7.

Methods and Results

When acetic, lactic, or propionic acid was combined with 3% NaCl, mutually antagonistic effects of acid and salt on viability of E. coli O157:H7 were observed only in tryptone and yeast extract broth. After exposure to acetic acid alone or in combination with salt, approximately 851 and 916 protein spots were detected, respectively. Analysis of 10 statistically significant differentially expressed proteins revealed that these proteins are mainly related to energy metabolism.

Conclusions

When we compared protein expression of E. coli O157:H7 treated with acetic acid and the combination of the acid and salt, the differentially expressed proteins were not related to acid stress‐ and salt stress‐inducible proteins such as stress shock proteins.

Significance and Impact of the Study

According to these results, the increased resistance of E. coli O157:H7 to acetic acid after the addition of salt may not be the result of synthesis of proteins related to these phenomena; therefore, further research needs to be conducted to identify the mechanism of the mutually antagonistic effect of some organic acids and salt.     

RecA Proteins from <Emphasis Type="Italic">Deinococcus geothermalis</Emphasis> and <Emphasis Type="Italic">Deinococcus murrayi</Emphasis> - Cloning,Purification and Biochemical Characterisation

Background  

Escherichia coli RecA plays a crucial role in recombinational processes, the induction of SOS responses and mutagenic lesion bypasses. It has also been demonstrated that RecA protein is indispensable when it comes to the reassembly of shattered chromosomes in γ-irradiated Deinococcus radiodurans, one of the most radiation-resistant organisms known. Moreover, some functional differences between E. coli and D. radiodurans RecA proteins have also been shown.