Overproduction Of Recombinant Proteins Research Articles

Enhanced overexpression of secreted enzymes by discrete repeat promoters in Streptomyces lividans.

Streptomyces lividans is an efficient host for extracellular overproduction of recombinant proteins. To enhance the overexpression strength of S. lividans, we designed several kinds of expression plasmids with different positioning of repeat promoters. The effect of repeat promoters was evaluated by measuring the accumulated amounts of a stable transglutaminase or an unstable carboxypeptidase that was secreted into the medium. Successive tandem positions of repeat promoters upstream of the normal promoter did not enhance the expression of transglutaminase. Discrete positions of repeat promoters both upstream and downstream of the normal promoter enhanced the expression of transglutaminase to 2-fold, and the downstream ones also enhanced the expression of carboxypeptidase to 1.7-fold. On the other hand, there were still some constructs of plasmids with discrete repeat promoters that did not promote the expression of the target enzymes, indicating the complexity of the mechanisms of repeat promoters working on gene expression.

The pre-induction temperature affects recombinant HuGM-CSF aggregation in thermoinducible Escherichia coli.

The overproduction of recombinant proteins in Escherichia coli leads to insoluble aggregates of proteins called inclusion bodies (IBs). IBs are considered dynamic entities that harbor high percentages of the recombinant protein, which can be found in different conformational states. The production conditions influence the properties of IBs and recombinant protein recovery and solubilization. The E. coli growth in thermoinduced systems is generally carried out at 30 °C and then recombinant protein production at 42 °C. Since the heat shock response in E. coli is triggered above 34 °C, the synthesis of heat shock proteins can modify the yields of the recombinant protein and the structural quality of IBs. The objective of this work was to evaluate the effect of different pre-induction temperatures (30 and 34 °C) on the growth of E. coli W3110 producing the human granulocyte–macrophage colony-stimulating factor (rHuGM-CSF) and on the IBs structure in a λpL/pR-cI857 thermoinducible system. The recombinant E. coli cultures growing at 34 °C showed a ~ 69% increase in the specific growth rate compared to cultures grown at 30 °C. The amount of rHuGM-CSF in IBs was significantly higher in cultures grown at 34 °C. Main folding chaperones (DnaK and GroEL) were associated with IBs and their co-chaperones (DnaJ and GroES) with the soluble protein fraction. Finally, IBs from cultures that grew at 34 °C had a lower content of amyloid-like structure and were more sensitive to proteolytic degradation than IBs obtained from cultures at 30 °C. Our study presents evidence that increasing the pre-induction temperature in a thermoinduced system allows obtaining higher recombinant protein and reducing amyloid contents of the IBs.Key Points• Pre-induction temperature determines inclusion bodies architecture• In pre-induction (above 34 °C), the heat shock response increases recombinant protein production• Inclusion bodies at higher pre-induction temperature show a lower amyloid contentSupplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-11908-z.

Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis

Inducer-free integrative vectors are often used to create B. subtilis strains for industrial purposes, but employing strong promoters to produce high levels of recombinant proteins in B. subtilis results in high leaky expression that can hamper cloning in Escherichia coli. To overcome the problem, we used strong IPTG-inducible Pgrac promoters harboring lac operators to construct inducer-free integrative vectors able to integrate into the B. subtilis genome at either the lacA or the amyE locus, or both and examined their ability to repress the β-galactosidase (bgaB) gene in E. coli and to overexpress BgaB in B. subtilis. The Pgrac01 vectors could repress bgaB expression about 24-fold in E. coli to low background levels. The integrated Pgrac01-bgaB constructs exhibited inducer-free expression and produced 8% of total cellular proteins, only 1.25 or 1.75 times less compared with their cognates as plasmids. The stronger promoters, Pgrac100-bgaB and Pgrac212-bgaB yielded 20.9 % and 42 % of total intracellular proteins after 12 h of incubation, respectively. Incorporation of the Pgrac212-bgaB into both amyE and lacA loci resulted in BgaB expression up to 53.4 %. In conclusion, integrative vectors containing the Pgrac promoter family have great potential for inducer-free overproduction of recombinant proteins in B. subtilis.

Redox Engineering by Ectopic Overexpression of NADH Kinase in Recombinant Pichia pastoris (Komagataella phaffii): Impact on Cell Physiology and Recombinant Production of Secreted Proteins.

High-level expression and secretion of heterologous proteins in yeast cause an increased energy demand, which may result in altered metabolic flux distributions. Moreover, recombinant protein overproduction often results in endoplasmic reticulum (ER) stress and oxidative stress, causing deviations from the optimal NAD(P)H regeneration balance. In this context, overexpression of genes encoding enzymes catalyzing endogenous NADPH-producing reactions, such as the oxidative branch of the pentose phosphate pathway, has been previously shown to improve protein production in Pichia pastoris (syn. Komagataella spp.). In this study, we evaluate the overexpression of the Saccharomyces cerevisiaePOS5-encoded NADH kinase in a recombinant P. pastoris strain as an alternative approach to overcome such redox constraints. Specifically, POS5 was cooverexpressed in a strain secreting an antibody fragment, either by directing Pos5 to the cytosol or to the mitochondria. The physiology of the resulting strains was evaluated in continuous cultivations with glycerol or glucose as the sole carbon source, as well as under hypoxia (on glucose). Cytosolic targeting of Pos5 NADH kinase resulted in lower biomass-substrate yields but allowed for a 2-fold increase in product specific productivity. In contrast, Pos5 NADH kinase targeting to the mitochondria did not affect growth physiology and recombinant protein production significantly. Growth physiological parameters were in silico evaluated using the recent upgraded version (v3.0) of the P. pastoris consensus genome-scale metabolic model iMT1026, providing insights on the impact of POS5 overexpression on metabolic flux distributions.IMPORTANCE Recombinant protein overproduction often results in oxidative stress, causing deviations from the optimal redox cofactor regeneration balance. This becomes one of the limiting factors in obtaining high levels of heterologous protein production. Overexpression of redox-affecting enzymes has been explored in other organisms, such as Saccharomyces cerevisiae, as a means to fine tune the cofactor regeneration balance in order to obtain higher protein titers. In the present work, this strategy is explored in P. pastoris In particular, one NADH kinase enzyme from S. cerevisiae (Pos5) is used, either in the cytosol or in mitochondria of P. pastoris, and its impact on the production of a model protein (antibody fragment) is evaluated. A significant improvement in the production of the model protein is observed when the kinase is directed to the cytosol. These results are significant in the field of heterologous protein production in general and in particular in the development of improved metabolic engineering strategies for P. pastoris.

Promoter engineering for the recombinant protein production in prokaryotic systems

Recombinant proteins have an economical value with their utilization in many areas from food industry to pharmaceutical and chemical industry. Therefore, it is of great importance to establish optimum production systems for the proteins of interest. One of the critical steps in protein production is regulation of the gene expression. Promoters are among the key regulatory elements which can directly control the level of recombinant gene expression in a host cell. Thus, a suitable promoter is required for optimum gene expression. Promoter engineering is an innovative approach to find out the best promoter system for the expression of recombinant genes, which influences the overproduction of proteins of interest. In this review, some of the bacterial hosts highly used in recombinant protein production were discussed. Next, the importance of promoters in recombinant gene expression, and promoter engineering for enhanced protein production were described. Utilization of double promoter systems was highlighted as one of the successful techniques in overproduction of recombinant proteins. Increment in the variety and availability of the novel methodologies especially in the synthetic biology is expected to increase the quality and the quantity of recombinant proteins with an economical value.

Systems biology approach in the formulation of chemically defined media for recombinant protein overproduction.

The cell culture medium is an intricate mixture of components which has a tremendous effect on cell growth and recombinant protein production. Regular cell culture medium includes various components, and the decision about which component should be included in the formulation and its optimum amount is an underlying issue in biotechnology industries. Applying conventional techniques to design an optimal medium for the production of a recombinant protein requires meticulous and immense research. Moreover, since the medium formulation for the production of one protein could not be the best choice for another protein, hence, the most suitable media should be determined for each recombinant cell line. Accordingly, medium formulation becomes a laborious, time-consuming, and costly process in biomanufacturing of recombinant protein, and finding alternative strategies for medium development seems to be crucial. In silico modeling is an attractive concept to be adapted for medium formulation due to its high potential to supersede laboratory examinations. By emerging the high-throughput datasets, scientists can disclose the knowledge about the effect of medium components on cell growth and metabolism, and via applying this information through systems biology approach, medium formulation optimization could be accomplished in silico with no need of significant amount of experimentation. This review demonstrates some of the applications of systems biology as a powerful tool for medium development and illustrates the effect of medium optimization with system-level analysis on the production of recombinant proteins in different host cells.

Engineering a highly efficient expression system to produce BcaPRO protease in Bacillus subtilis by an optimized promoter and signal peptide

Bacillus subtilis has been extensively utilized as a host to express heterologous protein used in various industrial processes. Hence, the secretory overproduction of recombinant proteins is highly dependent on the strong promoters and signal peptides with high efficiency in B. subtilis. To enlarge the limited information of signal peptides and promoters suitable for specific proteins expression at a high-level, a series of plasmids carrying various signal peptides and single, dual, or triple promoters were engineered using the coding sequence of reporter protein, alkaline serine protease (BcaPRO) from B. clausii. Finally, the signal peptide DacB was selected from a library composed of 73 Sec-type signal peptides, and the dual promoter PBsamy-PBaamy demonstrated the best performance. Furthermore, BcaPRO activity was as high as 27,860 U/mL in the supernatant of the recombinant B. subtilis WB600 containing the plasmid with the dual promoter PBsamy-PBaamy and signal peptide DacB with batch fermentation in a 5-L fermenter after 56 h. It indicated that this new engineered expression system would be potential for high-level BcaPRO expression in B. subtilis.

Construction and characterization of broad-host-range reporter plasmid suitable for on-line analysis of bacterial host responses related to recombinant protein production

BackgroundBacteria are widely used as hosts for recombinant protein production due to their rapid growth, simple media requirement and ability to produce high yields of correctly folded proteins. Overproduction of recombinant proteins may impose metabolic burden to host cells, triggering various stress responses, and the ability of the cells to cope with such stresses is an important factor affecting both cell growth and product yield.ResultsHere, we present a versatile plasmid-based reporter system for efficient analysis of metabolic responses associated with availability of cellular resources utilized for recombinant protein production and host capacity to synthesize correctly folded proteins. The reporter plasmid is based on the broad-host range RK2 minimal replicon and harbors the strong and inducible XylS/Pm regulator/promoter system, the ppGpp-regulated ribosomal protein promoter PrpsJ, and the σ32-dependent synthetic tandem promoter Pibpfxs, each controlling expression of one distinguishable fluorescent protein. We characterized the responsiveness of all three reporters in Escherichia coli by quantitative fluorescence measurements in cell cultures cultivated under different growth and stress conditions. We also validated the broad-host range application potential of the reporter plasmid by using Pseudomonas putida and Azotobacter vinelandii as hosts.ConclusionsThe plasmid-based reporter system can be used for analysis of the total inducible recombinant protein production, the translational capacity measured as transcription level of ribosomal protein genes and the heat shock-like response revealing aberrant protein folding in all studied Gram-negative bacterial strains.

Exploitation of Bacillus subtilis as a robust workhorse for production of heterologous proteins and beyond.

Bacillus subtilis, belonging to the type species of Bacillus, is a type of soil-derived, low %G+C, endospore-forming Gram-positive bacterium. After the discovery of B. subtilis 168 that displayed natural competence, this bacterium has been intensively considered to be an ideal model organism and a robust host to study several basic mechanisms, such as metabolism, gene regulation, bacterial differentiation, and application for industrial purposes, such as heterologous protein expression and the overproduction of an array of bioactive molecules. Since the first report of heterologous overproduction of recombinant proteins in this strain, the bulk production of a multitude of valuable enzymes, especially industrial enzymes, has been performed on a relatively large scale. Since B. subtilis can non-specifically secrete recombinant proteins using various signal peptides, it has tremendous advantages over Gram-negative bacterial hosts. Along with the report of the complete genome sequence of B. subtilis, a number of genetic tools, including diverse types of plasmids, bacterial promoters, regulatory elements, and signal peptides, have been developed and characterized. These novel genetic elements tremendously accelerated genetic engineering in B. subtilis recombinant systems. In addition, with the development of several complex gene expression systems, B. subtilis has performed a number of more complex functions. This ability enables it to be a substantial chassis in synthetic biology rather than just a workhorse for the overproduction of recombinant proteins. In this review, we review the progress in the development of B. subtilis as a universal platform to overproduce heterologous diverse high-value enzymes. This progress has occurred from the development of biological parts, including the characterization and utilization of native promoters, the fabrication of synthetic promoters and regulatory elements, and the assembly and optimization of genetic systems. Some important industrial enzymes that have been produced in large quantities in this host are also summarized in this review. Furthermore, the ability of B. subtilis to serve as a cellular tool was also briefly recapitulated and reviewed.

Insights into the Balance between Folding and Aggregation during a Protein's Life

Despite its fundamental importance for life, the delicate balance between protein folding and aggregation in the cellular environment is poorly understood. This lack of knowledge poses serious challenges in basic science, biotechnology and medicine, as inclusion body formation limits the efficient overproduction of recombinant proteins and protein-based therapeutics. In this work, we combine experimental and computational approaches to better understand how proteins undertake and discriminate folding and aggregation pathways in the cell. A wide variety of proteins studied, including apomyoglobin and the soluble E. coli proteome, are able to populate stable aggregate states under physiologically-relevant conditions. The aggregate and native states of these proteins are found to be kinetically trapped relative to each other over a wide range of low concentrations. Kinetic simulations modeling apomyoglobin's folding and aggregation landscape recapitulate these findings. Surprisingly, our simulations also show that the addition of stable pre-formed aggregated seeds, especially if small in size, facilitate the conversion of native proteins into aggregates on biologically-relevant timescales. Further studies probing the earlier stages of a protein's life, i.e. translation and ribosome release, unveiled that these two events are critical for directing the relative flux between competing protein folding and aggregation pathways. Overall, we found that the ribosome serves as a solubility tag for nascent proteins that are not yet fully synthesized. In addition, cotranslationally-active molecular chaperones help preventing longer nascent proteins from forming insoluble aggregates during the later stages of translation, prior to ribosome release. Ongoing studies are aimed at elucidating the structural features and chaperone-dependence of cotranslationally-aggregated proteins. In all, these findings shed light on the cellular mechanisms underlying the fidelity of protein folding upon ribosome release in healthy cells.

New high-cloning-efficiency vectors for complementation studies and recombinant protein overproduction in Escherichia coli and Salmonella enterica

Galloway et al. recently described a method to alter vectors to include Type IIS restriction enzymes for high efficiency cloning. Utilizing this method, the multiple cloning sites of complementation and overexpression vectors commonly used in our laboratory were altered to contain recognition sequences of the Type IIS restriction enzyme, BspQI. Use of this enzyme increased the rate of cloning success to >97% efficiency. L(+)-Arabinose-inducible complementation vectors and overexpression vectors encoding N-terminal recombinant tobacco etch virus protease (rTEV)-cleavable H6-tags were altered to contain BspQI sites that allowed for cloning into all vectors using identical primer overhangs. Additionally, a vector used for directing the synthesis of proteins with a C-terminal, rTEV-cleavable H6-tag was engineered to contain BspQI sites, albeit with different overhangs from that of the previously mentioned vectors. Here we apply a method used to engineer cloning vectors to contain BspQI sites and the use of each vector in either in vivo complementation studies or in vitro protein purifications.

Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis

A strong promoter and highly efficient signal peptides are essential for the secretory overproduction of recombinant proteins in Bacillus subtilis. To enhance the limited overexpression capability of natural promoters, various strategies for promoter engineering have been developed and used to construct gene expression systems in B. subtilis and other hosts. By applying a semi-rational approach for promoter engineering, a series of expression plasmids containing single and dual promoters were constructed using aminopeptidase (AP) with an intrinsic signal peptide as the reporter protein. Of the single and dual promoters investigated, the dual promoter PgsiB-PHpaII gave the best performance. To optimize secretion efficiency, the signal peptide YncM was selected after screening a library containing 19 different Sec-type signal peptides. The AP activity detected in the supernatants of a recombinant strain containing the plasmid pBSG24-YncM was as high as 88.86U/mL. The capacity of the expression plasmid pBSG24-YncM was also evaluated with batch fermentation in a 5-L fermentor. Increased production of AP (205U/mL, equal to 1.7g/L) was achieved after 45h of fermentation. These results suggest that this expression system can be used for high-level protein expression in B. subtilis.

Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum.

BackgroundLeuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc.ResultsUsing an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α–amylase.ConclusionsThe high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.

Prokaryotic expression systems

For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities.

Overproduction of anti-Tn antibody MLS128 single-chain Fv fragment in Escherichia coli cytoplasm using a novel pCold-PDI vector

Overproduction of recombinant proteins in Escherichia coli is often hampered by their failure to fold correctly, leading to their accumulation within inclusion bodies. To overcome the problem, a variety of techniques aimed at soluble expression have been developed including low temperature expression and/or fusion of soluble tags and chaperones. However, a general protocol for bacterial expression of disulfide bond-containing proteins has hitherto not been established. Single chain Fv fragments (scFvs) are disulfide bond-containing proteins often difficult to express in soluble forms in E. coli. We here examine in detail the E. coli expression of a scFv originating from an anti-carbohydrate MLS128 antibody as a model system. We combine three techniques: (1) tagging scFv with thioredoxin, DsbC and protein disulfide isomerase (PDI), (2) expressing the proteins at low temperature using the pCold vector system, and (3) using Origami E. coli strains with mutations in the thioredoxin reductase and glutathione reductase genes. We observed a high expression level of soluble MLS128-scFv in the Origami strain only when PDI is used as a tag. The recombinant protein retains full binding activity towards synthetic carbohydrate antigens. The developed “pCold-PDI” vector has potential for overproduction of other scFvs and disulfide-containing proteins in the Origami strains.

A Glucose-Insensitive T7 Expression System for Fully-Induced Expression of Proteins at a Subsaturating Level ofl-Arabinose

The L-arabinose (Ara)-controlled T7 expression system was previously constructed by creation of an Escherichia coli BL21(BAD) strain. The production of recombinant proteins in this strain was stringently regulated and reached a high level upon induction with Ara. Nevertheless, this system is still associated with inherent problems of interference with glucose and of the all-or-nothing induction profile at a subsaturating level of Ara. In this study, these problems were circumvented by modifying the physiological traits of BL21(BAD) strain. This was followed by deletion of ptsG gene and the araFGH and araBAD operon. The former encodes the glucose transporter while the latter two gene operons produce proteins responsible for Ara uptake and catabolism. In addition, the expression of genomic araE (encodes the Ara transporter) was constitutively enhanced. The resulting strain was designated BAD-5. By expression of the faster degrader GFP(LAA) at a subsaturating level of Ara, 80% of BAD-5 strain was found visually bright in the presence or absence of glucose. A further analysis by flow cytometry showed a uniform distribution of GFP expression for BAD-5 strain. In marked contrast, BL21(BAD) strain exhibiting visual brightness was less than 10% of the cell population and remained dark in the presence of glucose. Moreover, a saturated level of luciferase from Renilla reniformis (Rluc) could be readily obtained in BAD-5 strain at 20 μM Ara regardless of glucose. Rluc in BL21(BAD) strain was produced in an Ara dose-dependent manner, and the protein production became arrested when glucose was present. Overall, it illustrates the usefulness of the improved system for overproduction of recombinant proteins in an efficient, homogeneous, and glucose-insensitive way.

Molecular responses of E. coli caused by heat stress and recombinant protein production during temperature induction

In a recent review, we discussed the extensively used temperature-inducible expression system, based on the pL and/or pR phage lambda promoters that are finely regulated by the thermo-labile cI857 repressor. In this system, an increase in temperature induces the heterologous protein production and activates the heat shock response, as well as the stringent and SOS responses. The same responses are activated just by the overproduction of recombinant protein. All such responses result in a metabolic burden to the cells, a decrease in the specific growth rate, and alterations in the central carbon metabolism. Altogether, these effects can alter the quantity and quality of the produced foreign protein. Here, we compare and discuss the transcription of selected genes, and the concomitant synthesis of heat-shock proteins (hsp) soon after thermal induction, in relation to the responses that occur in other expression systems that also trigger the heat-shock response.

Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins

BackgroundOverexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R).ResultsOverexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction.ConclusionsDifferential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of overexpressed recombinant proteins could be enhanced by maintaining the expression of σ32, a bacterial heat shock transcription factor, at higher levels during overproduction.

Changes in the quality of antibodies produced by Chinese hamster ovary cells during the death phase of cell culture

Although overproduction of recombinant proteins by mammalian cells is well established, little attention has been paid to analysis of the quality of the products. We focused on the quality of antibodies produced during the death phase of a recombinant Chinese hamster ovary (CHO) cell line. The quality of the monoclonal antibody against HM1.24 antigen and the post-translational characteristics of the subunits during CHO cell culture in a 160-L bioreactor were investigated. The culture supernatant of a stable cell line was collected and purified by affinity chromatography and then analyzed. There were no significant changes in gel-permeation chromatography variables, carbohydrate structure, or antibody-dependent cellular cytotoxicity activity during the death phase of cell culture. However, ion-exchange chromatography analysis revealed that antibody heterogeneity changed, as indicated by a decrease in cell viability. The results presented here provide useful information that will help in determining the time to end each batch culture.

Demonstration That CobG, the Monooxygenase Associated with the Ring Contraction Process of the Aerobic Cobalamin (Vitamin B12) Biosynthetic Pathway, Contains an Fe-S Center and a Mononuclear Non-heme Iron Center

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.