Improve Protein Secretion Research Articles

Optimization of heterologous protein production in Chinese hamster ovary cells under overexpression of spliced form of human X-box binding protein.

BackgroundThe optimization of protein production is a complex and challenging problem in biotechnology. Different techniques for transcription, translation engineering and the optimization of cell culture conditions have been used to improve protein secretion, but there remain many open problems involving post-translational modifications of the secreted protein and cell line stability.ResultsIn this work, we focus on the regulation of secreted protein specific productivity (using a recombinant human immunoglobulin G (IgG)) by controlling the expression of the spliced form of human X-box binding protein (XBP-(s)) in Chinese hamster ovary cells (CHO-K1) under doxycycline (DOX) induction at different temperatures. We observed a four-fold increase in specific IgG productivity by CHO cells under elevated concentrations of DOX at 30°C compared to 37°C, without detectable differences in binding activity in vitro or changes in the structural integrity of IgG. In addition, we found a correlation between the overexpression of human XBP-1(s) (and, as a consequence, endoplasmic reticulum (ER) size expansion) and the specific IgG productivity under DOX induction.ConclusionsOur data suggest the T-REx system overexpressing human XBP-1(s) can be successfully used in CHO-K1 cells for human immunoglobulin production.

Degradation of Extracytoplasmic Catalysts for Protein Folding in Bacillus subtilis

The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold into their active and protease-resistant conformations once membrane passage is completed. The lipoprotein PrsA and the membrane proteins HtrA and HtrB facilitate the extracytoplasmic folding and quality control of exported proteins. Among the native exported proteins of B. subtilis are at least 10 proteases that have previously been implicated in the degradation of heterologous secreted proteins. Recently, we have shown that these proteases also degrade many native membrane proteins, lipoproteins, and secreted proteins. The present studies were therefore aimed at assessing to what extent these proteases also degrade extracytoplasmic catalysts for protein folding. To this end, we employed a collection of markerless protease mutant strains that lack up to 10 different extracytoplasmic proteases. The results show that PrsA, HtrA, and HtrB are indeed substrates of multiple extracytoplasmic proteases. Thus, improved protein secretion by multiple-protease-mutant strains may be related to both reduced proteolysis and improved posttranslocational protein folding and quality control.

Highly robust protein production by co-culture of CHO spheroids layered on feeder cells in serum-free medium

Recombinant Chinese hamster ovary (rCHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. Although recent advances in 3D culture of rCHO cells is preferred to 2D monolayer culture for highly productive and robust expression of therapeutic proteins, there exists still limitation for efficient protein production. Therefore, a new cell culture system is essentially required for an efficient protein production. Here, we report on a new 3D cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein by CHO cells. Particularly, cocultivation of CHO spheroids with bovine aortic endothelial cells (BAEC) as a feeder layer cells was essential to stably increase a protein production. We investigated the co-culture mechanism of functional enhancement with respect to the cell–cell interactions. Functional comparison between 2D and 3D co-cultures suggested the preferred configuration as spheroid for higher protein production. Specifically, to estimate the effect of respective cell constitution in co-cultured spheroids on the protein production per CHO cell, the number of viable cells in cell proliferation was determined with culture periods. These studies demonstrated the significant role of micropatterned BAEC as a feeder layer for the retained formation of CHO spheroids, resulting in predominantly enhanced production of proteins, although the functional enhancement of CHO cells was obtained by co-culture with BAECs in both 2D and 3D configurations. Thus, heterotypic cell communications that play indispensable roles in increasing CHO functions should be properly obtained in 3D cell configurations. Significantly, these spheroids in the serum-free medium drastically enhanced protein expression level up to sevenfold compared with CHO monospheroids, suggesting that a suitable culture conditions for heterotypic cell–cell interactions would allow improved protein secretion to occur unimpeded.

Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe – A quantitative approach using 13C-based metabolic flux analysis

Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying 13C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.

Artificial signal peptide prediction by a hidden markov model to improve protein secretion via Lactococcus lactis bacteria.

A hidden Markov model (HMM) has been utilized to predict and generate artificial secretory signal peptide sequences. The strength of signal peptides of proteins from different subcellular locations via Lactococcus lactis bacteria correlated with their HMM bit scores in the model. The results show that the HMM bit score +12 are determined as the threshold for discriminating secreteory signal sequences from the others. The model is used to generate artificial signal peptides with different bit scores for secretory proteins. The signal peptide with the maximum bit score strongly directs proteins secretion.

Anaerobic α-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae

In this study, we focus on production of heterologous α-amylase in the yeast Saccharomyces cerevisiae under anaerobic conditions. We compare the metabolic fluxes and transcriptional regulation under aerobic and anaerobic conditions, with the objective of identifying the final electron acceptor for protein folding under anaerobic conditions. We find that yeast produces more amylase under anaerobic conditions than under aerobic conditions, and we propose a model for electron transfer under anaerobic conditions. According to our model, during protein folding the electrons from the endoplasmic reticulum are transferred to fumarate as the final electron acceptor. This model is supported by findings that the addition of fumarate under anaerobic (but not aerobic) conditions improves cell growth, specifically in the α-amylase-producing strain, in which it is not used as a carbon source. Our results provide a model for the molecular mechanism of anaerobic protein secretion using fumarate as the final electron acceptor, which may allow for further engineering of yeast for improved protein secretion under anaerobic growth conditions.

Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased heterologous α-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously.

Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A

Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.

Improving protein secretion of a transglutaminase-secreting Corynebacterium glutamicum recombinant strain on the basis of 13C metabolic flux analysis

Corynebacterium glutamicum is known as a host species for amino acid production. This microorganism was recently noticed as a host that produces secreted proteins. In this study, we performed (13)C metabolic flux analysis ((13)C-MFA) on a recombinant C. glutamicum strain that secretes a heterologous transglutaminase (TGase) to improve TGase secretion. For the (13)C-MFA of a TGase-secreting C. glutamicum strain in batch cultivation, a (13)C-labeling experiment and measurement of mass isotopomer distributions of proteinogenic amino acids were performed, and metabolic fluxes were determined considering the changes in fractional (13)C-labeling of proteinogenic amino acids with respect to culture time. The TGase yield increased at the stationary phase but decreased toward its end. The results of (13)C-MFA revealed that the flux from glycolysis to the TCA cycle gradually increased during TGase secretion. We speculate that the NADH/NAD(+) ratio in the cells increases and that as a result, the specific glucose uptake rate decreases in the stationary phase because of the increased flux of the TCA cycle. Since it is expected that a decrease in the NADH/NAD(+) ratio would improve the TGase yield, we tried to enhance lactate production in a TGase-secreting C. glutamicum strain to decrease cellular NADH levels by increasing the pH level in the culture. The TGase yield increased in 1.4-fold by increasing the pH from 6.7 to 7.2, indicating that the TGase yield was successfully improved on the basis of the (13)C-MFA.

Collection of in vivo-like liver cell secretome with alternative sample enrichment method using a hollow fiber bioreactor culture system combined with tangential flow filtration for secretomics analysis

A hollow fiber bioreactor (HFB) culture system coupled with a tangential flow filtration (TFF) device was used for HepG2 cell secretome analysis. In order to reduce the loss of low-molecular-weight proteins, two new features, the hollow fiber with 0.1 μm pore size and a TFF device with a membrane of 1 kDa molecular weight cutoff, were added to the system described previously. The HFB culture system and the conventional dish culture method for secretome collection were compared side by side. It was observed that only a small fraction of cells (<0.01%) were lysed in the HFB culture system, in contrast to the 2.73% in the conventional dish culture. A total of 111 proteins were identified in the collected conditioned medium (CM) by liquid chromatography–tandem mass spectrometry (LC–MS/MS) with this improved collection procedure. Many of these proteins reported to be biomarkers for liver-related diseases. About 16% of the identified proteins were smaller than 20 kDa, demonstrating that the modified collection system had the ability to reduce the loss of low-molecular-weight proteins, in contrast to our previous collection system. The percentage increase of proteins classified as extracellular space or plasma membrane between the conventional dish culture and the HFB culture system was 40–60%. We believed that in vivo-like culture environments could support liver cells to improve protein secretion than conventional dish cultures. We suggest that the combination of the HFB culture system, TFF device, and LC–MS/MS analysis, would be an efficient procedure for the collection and characterization of in vivo-like cell secretome.

Cloning of the Zygosaccharomyces bailii GAS 1 homologue and effect of cell wall engineering on protein secretory phenotype

BackgroundZygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a β-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion.ResultsThe GAS1 homologue of Z. bailii was cloned by PCR, and when expressed in a S. cerevisiae GAS1 null mutant was able to restore the parental phenotype. The respective Z. bailii Δgas1 deleted strain was obtained by targeted deletion of both alleles of the ZbGAS1 gene with deletion cassettes having flanking regions of ~400 bp. The morphological and physiological characterization of the Z. bailii null mutant resulted very similar to that of the corresponding S. cerevisiae mutant. As for S. cerevisiae, in the Z. bailii Δgas1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the Candida rugosa lipase CRL1 and of the Yarrowia lipolytica protease XPR2, while for human IL-1β secretion disruption had no relevant effect.ConclusionsThe data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that Z. bailii is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as already widely reported in literature, our data confirmed that an "always working" solution to the problems related to recombinant protein production can be hardly, if never, found; instead, manipulations have to be finely tuned for each specific product and/or combination of host cell and product.

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Protein misfolding in the endoplasmic reticulum (ER) contributes to the pathogenesis of many diseases. Although oxidative stress can disrupt protein folding, how protein misfolding and oxidative stress impact each other has not been explored. We have analyzed expression of coagulation factor VIII (FVIII), the protein deficient in hemophilia A, to elucidate the relationship between protein misfolding and oxidative stress. Newly synthesized FVIII misfolds in the ER lumen, activates the unfolded protein response (UPR), causes oxidative stress, and induces apoptosis in vitro and in vivo in mice. Strikingly, antioxidant treatment reduces UPR activation, oxidative stress, and apoptosis, and increases FVIII secretion in vitro and in vivo. The findings indicate that reactive oxygen species are a signal generated by misfolded protein in the ER that cause UPR activation and cell death. Genetic or chemical intervention to reduce reactive oxygen species improves protein folding and cell survival and may provide an avenue to treat and/or prevent diseases of protein misfolding.

Improvement of the secretion of extracellular proteins and isolation and characterization of the amylase I ( amy1) gene from Ophiostoma floccosum

UV mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein production after repeated rounds of mutagenic treatment. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the increase in total secreted protein was about six times greater than the parental strain. Secreted proteinase and lipase activities of the parental strain and four key mutants were also investigated. N-terminal sequencing of the five dominant protein bands separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an α-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the α-amylase amino acid sequence. The amplified fragment contained the entire gene encoding α-amylase ( amy1) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single α-amylase gene locus.

Ero1-Lα plays a key role in a HIF-1-mediated pathway to improve disulfide bond formation and VEGF secretion under hypoxia: implication for cancer

Oxygen is the ultimate source of oxidizing power for disulfide bond formation, suggesting that under limiting oxygen proper protein folding might be compromised. We show that secretion of vascular endothelial growth factor (VEGF), a protein with multiple disulfide bonds, was indeed impeded under hypoxia and was partially restored by artificial increase of oxidizing equivalents with diamide. Physiologically, the oxireductase endoplasmic reticulum oxidoreductin-1 (Ero1)-L alpha, but not other proteins in the relay of disulfide formation, was strongly upregulated by hypoxia and independently by hypoglycemia, two known accompaniments of tumors. Further, we provide genetic evidence that induction of Ero1-L alpha by hypoxia and hypoglycemia is mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1) but is independent of p53. In natural human tumors, Ero1-L alpha mRNA was specifically induced in hypoxic microenvironments coinciding with that of upregulated VEGF expression. To establish a physiological relevance to modulations in Ero1-L alpha levels, we showed that even a modest, two- to three-fold reduction in Ero1-L alpha production via siRNA leads to significant inhibition of VEGF secretion, a compromised proliferation capacity and enhanced apoptosis. Together, these findings demonstrate that hypoxic induction of Ero1-L alpha is the key adaptive response in a previously unrecognized HIF-1-mediated pathway that operates to improve protein secretion under hypoxia and might be harnessed for inhibiting tumor growth via inhibiting VEGF-driven angiogenesis.

Improving protein secretion by engineering components of the bacterial translocation machinery.

The increased insight into the mechanism of bacterial protein translocation has resulted in new concepts for the production of heterologous proteins. The periplasm of gram-negative bacteria is revealed to have a role as a 'protein construction compartment', which can be used to fold complex proteins. Passage across the outer membrane, however, remains a challenge due to the high selectivity of the outer membrane translocase. In gram-positive bacteria, slow folding at the membrane-cell-wall interface can make heterologous proteins vulnerable to degradation by wall-associated proteases. The recent identification of thiol-disulfide oxidoreductases in Bacillus subtilis might open the possibility of secreting proteins containing multiple disulfide bonds from this host.

Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure.

Production of extracellular proteins plays an important role in the physiology of Trichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene of Saccharomyces cerevisiae, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated DPM1, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of cbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of the DPM1-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in Trichoderma can result in improved protein secretion.