Secretion Of Recombinant Proteins Research Articles

Influence of PDI Gene Overexpression on the Production of Heterologous Proteins in Yeast Pichia pastoris

The Pichia pastoris yeast is used for recombinant secretory proteins production. The overexpression of assistant genes, the coding proteins involved in secretion, is one of the approaches used to improve the production of target proteins. PpPDI gene encodes P. pastoris yeast protein disulfide isomerase (Pdi). The aim of our study is to evaluate the effect of Pdi overproduction on the production of recombinant interferons. We obtain the yeast Pichia pastoris strains expressing PpPDI gene under the control of alcohol oxidase-1 (AOX1) promoter. The effect of the overexpression of the PpPDI gene on the production of heterogious proteins—interferon-alfa16 and chicken interferon-gamma—and on the cell growth rate is shown.

Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress.

In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Regardless of producing high protein titers, various cellular and process level bottlenecks restrict the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large-scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed-batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized.

Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags

Upon expression of a given protein in an expression host, its secretion into the culture medium or cell-surface display is frequently advantageous in both research and industrial contexts. Hence, engineering strategies targeting folding, trafficking, and secretion of the proteins gain considerable interest. Yarrowia lipolytica has emerged as an efficient protein expression platform, repeatedly proved to be a competitive secretor of proteins. Although the key role of signal peptides (SPs) in secretory overexpression of proteins and their direct effect on the final protein titers are widely known, the number of reports on manipulation with SPs in Y. lipolytica is rather scattered. In this study, we assessed the potential of ten different SPs for secretion of two heterologous proteins in Y. lipolytica. Genomic and transcriptomic data mining allowed us to select five novel, previously undescribed SPs for recombinant protein secretion in Y. lipolytica. Their secretory potential was assessed in comparison with known, widely exploited SPs. We took advantage of Golden Gate approach, for construction of expression cassettes, and micro-volume enzymatic assays, for functional screening of large libraries of recombinant strains. Based on the adopted strategy, we identified novel secretory tags, characterized their secretory capacity, indicated the most potent SPs, and suggested a consensus sequence of a potentially robust synthetic SP to expand the molecular toolbox for engineering Y. lipolytica.

Preparation of the Secretory Recombinant ALV-J gp85 Protein Using Pichia pastoris and Its Immunoprotection as Vaccine Antigen Combining with CpG-ODN Adjuvant.

This study focuses on preparing the secretory recombinant J subgroup of avian leukosis virus (ALV-J) gp85 protein using Pichia pastoris and evaluating its immunoprotection as vaccine antigen combining with CpG-ODN adjuvant. The secretory recombinant plasmid pPIC9-gp85 containing ALV-J gp85 gene was designed and was transfected into the genome of P. pastoris (GS115) cells. The recombinant plasmid was expressed under the induction of methanol. The expressed products in the medium of the cells were purified and identified with endoglycosidase digestion assay and western blot mediated with monoclonal antibody (MAb) JE9. The purified product combining with CpG-ODN adjuvant was inoculated intramuscularly into 7-day-old chickens and three booster inoculations were performed on 21 days post first inoculation (dpfi), 42, and 56 dpfi. The antibody responses and cellular immune responses were detected, and the protective effects were analyzed after challenge with ALV-J. The results showed that the secretory pPIC9-gp85 plasmid was successfully constructed and could be stably expressed in GS115 cells. The expressed products were N-acetylglucosylated and could specifically combine with MAb (JE9). The secreted gp85 protein combining with CpG-ODN adjuvant could induce higher antibody response and spleen lymphocyte proliferation response and IFN-γ-inducing response, and could protect all the inoculated chickens against the viremia and the immunosuppressive lesions caused by ALV-J challenge. The results of neutralizing test in vitro suggested that the antisera with some ALV-J antibody titers could neutralize ALV-J strain and inhibit the growth of virus in vitro. The result of IFA showed that IgG antibody in the antisera could specifically combine with ALV-J strain in cells. It can be concluded that the secretory recombinant gp85 protein, as a new acetylglucosylated gp85 protein, was successfully prepared and combining with CpG-ODN adjuvant could protect the inoculated chickens against ALV-J infection. This study first reported the methods on preparing the secretory recombinant ALV-J gp85 protein using P. pastoris and evaluated its immunoprotection.

Secretion of recombinant proteins from E. coli.

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E.coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one- or two-step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often-overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

The impact of ERAD on recombinant protein secretion in Pichia pastoris (syn Komagataella spp.).

The yeast Pichia pastoris (syn. Komagataella spp.) is a popular cell factory for recombinant protein production. Yeasts in general provide a good starting point for cell factory engineering. They are intrinsically robust and easy to manipulate and cultivate. However, their secretory pathway is not evolutionarily adapted to high loads of secretory protein. In particular, more complex proteins, like the antibody fragment (Fab) used in this study, overwhelm the folding and secretion capacity. This triggers cellular stress responses, which may cause excessive intracellular degradation. Previous results have shown that, in fact, about 60 % of the newly synthesized Fab is intracellularly degraded. Endoplasmic reticulum-associated protein degradation (ERAD) is one possible intracellular degradation pathway for proteins aimed for secretion. We therefore targeted ERAD for cell factory engineering and investigated the impact on recombinant protein secretion in P. pastoris. Three components of the ERAD-L complex, which is involved in the degradation of luminal proteins, and a protein involved in proteasomal degradation, were successfully disrupted in Fab-secreting P. pastoris. Contrary to expectation, the effect on secretion was marginal. In the course of more detailed investigation of the impact of ERAD, we took a closer look at the intracellular variants of the recombinant protein. This enabled us to further zero in on the issue of intracellular Fab degradation and exclude an overshooting ER quality control. We propose that a major fraction of the Fab is actually degraded before entering the secretory pathway.

Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast

The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein anterograde trafficking from the endoplasmic reticulum to the Golgi apparatus by the moderate overexpression of SEC16 could increase recombinant protein secretion in S. cerevisiae. In this study, the retrograde trafficking in a strain with moderate overexpression of SEC16 was engineered by overexpression of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of α-amylase but did not induce production of reactive oxygen species. An expanded ER membrane was detected in both the GCS1 and GLO3 overexpression strains. Physiological characterizations during batch fermentation showed that GLO3 overexpression had better effect on recombinant protein secretion than GCS1 overexpression. Additionally, the GLO3 overexpression strain had higher secretion of two other recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-α-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast.

Co-Expression of ORFCma with PHB Depolymerase (PhaZCma ) in Escherichia coli Induces Efficient Whole-Cell Biodegradation of Polyesters.

Whole-cell degradation of polyesters not only avoids the tedious process of enzyme separation, but also allows the degraded product to be reused as a carbon source. In this study, Escherichia coli BL21(DE3) harboring phaZCma , a gene encoding poly(3-hydroxybutyrate) (PHB) depolymerase from Caldimonas manganoxidans, is constructed. The extra-cellular fraction of E. coli/pPHAZ exhibits a fast PHB degradation rate where it only took 35 h to completely degrade PHB films, while C. manganoxidans takes 81 h to do the same. The co-expression of ORFCma (a putative periplasmic substrate binding protein that is within the same operon of phaZCma ) further improves the PHB degradation. While 28 h is needed for E. coli/pPHAZ to cause an 80% weight loss in PHB films, E. coli/pORFPHAZ needs only 21 h. Furthermore, it is able to degrade at-least four different polyesters, PHB, poly(lactic acid) (PLA), polycaprolactone (PCL), and poly(butylene succinate-co-adipate) (PBSA). Testing of the time course of 3-hydroxybutyrate concentration and the turbidity of the degradation solutions over time shows that PhaZCma has both exo- and endo-enzymatic activity. The whole-cell E. coli/pORFPHAZ can be used for recycling various polyesters while ORFCma can potentially be a universal element for enhancing the secretion of recombinant protein.

Conformational determinants necessary for secretion of Paecilomyces thermophila \u03b2-1,4-xylosidase that lacks a signal peptide

In this study, we investigated the secretion mechanism of the hyper-secretion signal peptide-lacking β-xylosidase PtXyl43, a non-classically secreted protein, from the fungus Paecilomyces thermophila in Escherichia coli BL21(DE3). PtXyl43 secretion is a two-step process, and the second step is accompanied by cell periplasmic leakage, indicating that PtXyl43 secretion is the result of semi-specific secretion. Homology modeling of PtXyl43 suggested that PtXyl43 had a canonical GH43 family β-xylosidase structure containing five blades. Seventeen blade deletions or circular mutants were designed to identify the conformational motif(s) involved in secretion. These mutants were expressed as recombinant, codon-optimized proteins in E. coli. Notably, only mutants containing blades 2–4 were effectively secreted. Blades 2–4 are necessary for secretion, but it appears that blade 1 or 5 must be present to maintain the structure of blades 2–4. Simultaneous deletion of blades 1 and 5 dramatically reduces excretion. The covalent and sequential linking of blades of 2, 3 and 4 are important for the excretion of mutants, as separate blades of 2 and 3 or 3 and 4 abolishes excretion. Fusion with PtXyl43 promotes the excretion of GFP from the periplasm to the extracellular milieu, which suggested that PtXyl43 had the potential to carry proteins. This study provides new insights into secretory mechanism of secretable signal peptide-lacking proteins in E. coli. To our knowledge, this is the first to definitively identify the conformational determinants for secretion of a signal peptide-lacking GH43 family β-xylosidase. This finding also has application potential for the secretion of recombinant proteins.

Protein profile in Aspergillus nidulans recombinant strains overproducing heterologous enzymes.

SummaryFilamentous fungi are robust cell factories and have been used for the production of large quantities of industrially relevant enzymes. However, the production levels of heterologous proteins still need to be improved. Therefore, this article aimed to investigate the global proteome profiling of Aspergillus nidulans recombinant strains in order to understand the bottlenecks of heterologous enzymes production. About 250, 441 and 424 intracellular proteins were identified in the control strain Anid_pEXPYR and in the recombinant strains Anid_AbfA and Anid_Cbhl respectively. In this context, the most enriched processes in recombinant strains were energy pathway, amino acid metabolism, ribosome biogenesis, translation, endoplasmic reticulum and oxidative stress, and repression under secretion stress (RESS). The global protein profile of the recombinant strains Anid_AbfA and Anid_Cbhl was similar, although the latter strain secreted more recombinant enzyme than the former. These findings provide insights into the bottlenecks involved in the secretion of recombinant proteins in A. nidulans, as well as in regard to the rational manipulation of target genes for engineering fungal strains as microbial cell factories.

Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger

Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers of heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. This strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.

The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells

The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.

A lower isoelectric point increases signal sequence–mediated secretion of recombinant proteins through a bacterial ABC transporter

Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions.

A propeptide toolbox for secretion optimization of Flavobacterium meningosepticum endopeptidase in Lactococcus lactis

BackgroundLactic acid bacteria are a family of “generally regarded as safe” organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases for the treatment of celiac disease. To facilitate these applications, an efficient recombinant protein expression toolbox is required, especially for recombinant protein secretion. While current tools for enhancing protein secretion consist mainly of signal peptides, secretion propeptides have also been observed to play a crucial role for protein secretion and improved yields.ResultsTo expand the propeptide library for secretion optimization, we have mined and characterized three naturally occurring propeptides from the sequenced genomes of 109 Lactococcus species. These newly-mined propeptides were introduced after the N-terminal USP45 secretion signal to characterize and compare their effects on the secretion of Escherichia coli thioredoxin (TRX) and Flavobacterium meningosepticum prolyl endopeptidase (Fm PEP) in Lactococcus lactis NZ9000. All three propeptides, along with the positive control LEISSTCDA, improved volumetric secretion yields by 1.4–2.3-folds. However, enhancement of secretion yield is dependent on protein of interest. For TRX, the optimal combination of USP45 signal peptide and LEISSTCDA produced a 2.3-fold increase in secretion yields. Whilst for Fm PEP, propeptide 1 with USP45 signal peptide improved volumetric secretion yields by 2.2-fold compared to a 1.4-fold increase by LEISSTCDA. Similar trends in Fm PEP activity and protein yield also demonstrated minimal effect of the negative charged propeptides on PEP activity and thus folding.ConclusionsOverall, we have characterized three new propeptides for use in L. lactis secretion optimization. From success of these propeptides for improvement of secretion yields, we anticipate this collection to be valuable to heterologous protein secretion optimisation in lactic acid bacteria. We have also demonstrated for the first time, secretion of Fm PEP in L. lactis for potential use as a therapy agent in celiac disease.

In silico Analysis of Several Signal Peptides for the Excretory Production of Reteplase in Escherichia coli

Background: Solubility of recombinant therapeutic enzymes is one of the major purpose their production in bacterial host The factors such as metabolic system, a cellular stress response of the bacterial host and formation of inclusion bodies cause undesirable expression results. One method to overcome this problem is secretion of recombinant protein into the extracellular growth medium by selection of proper signal peptides. Reteplase (Retavase) is a serine protease simulating endogenous tissue plasminogen activator (t-PA), fibrin-specific plasminogen activator, playing role in thrombolysis and fibrinolysis (clot lysis) by converting plasminogen to plasmin. Objective: The aim of this study was to evaluate several signal peptides in order to selection the proper one which suitable for excretory production of the reteplase. Method: The presence and cleavage site location in signal peptides was predicted by SignalP 4.1. The prediction of secretory pathway of signal peptides was performed using high accurate database. Several critical features of the signal peptide were evaluated applying various online web-servers such as ProtParam and Solpro with high sensitivity and specificity. Finally, the selected signal peptides were compared and the one which was appropriate candidate for high yields production of secretory reteplase in the E. coli host was defined. Results: Among total 30 signal peptides, 6 of them, CHIS_BACSU, MPT53_MYCTU, PET_ECOLX, PIC_ECOL6, PIC_ECLOX, TSH_ECOLX were omitted. CPLR_DESHA, AGAR_ALTAT, CDGT_BACS2, PAG_BACAN could be considered as proper candidate for efficient extracellular production of reteplase in E. coli. Conclusion: Theoretical analysis and evaluation of signal peptides by using computational tools can be acceptably carried out, but these results must be established via practical effort. Keywords: E. coli host, bioinformatics, secretion, physicochemical properties, solubility, signal peptides.

Non-peptide guided auto-secretion of recombinant proteins by super-folder green fluorescent protein in Escherichia coli

Protein secretion in Escherichia coli is usually led by a signal peptide that targets the protein to specific secretory pathways. In this study, we demonstrated that the superfolder green fluorescent protein (sfGFP) could be served as a non-signal peptide to guide protein auto-secretion in E. coli. This auto-secretion was characterized as a three-step process through the sub-cellular localization analysis: inner membrane trans-location followed by anchoring at outer membrane, and then being released into culture media. We further determined that the beta-barrel structure and net negative charges of sfGFP played important roles in its auto-extracellular secretion property. Using sfGFP as a carrier, heterologous proteins ranging from peptide to complex protein, including antibacterial peptide PG4, endo-beta-N-acethylglucosamindase H (Endo H), human arginase-1 (ARG1), and glutamate decarboxylase (GAD) were all successfully expressed and secreted extracellularly when fused to the carboxyl end of sfGFP. Besides facilitating the extracellular secretion, sfGFP fusion proteins can also be correctly folded and formed the active complex protein structure, including the trimetric human ARG1 and homo-hexametric GAD. This is the first report that sfGFP can guide the secretion of recombinant proteins out of the cells from cytoplasm in E. coli without affecting their conformation and function.

Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used to produce biopharmaceutical proteins. However, the limited capacity of the secretory pathway may reduce its productivity. Here, we increased the secretion of a heterologous α-amylase, a model protein used for studying the protein secretory pathway in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased α-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species resulting from the heterologous α-amylase production was reduced. A genome-wide expression analysis indicated decreased endoplasmic reticulum stress in the strain that moderately overexpressed SEC16, which was consistent with a decreased volume of the endoplasmic reticulum. Additionally, fewer mitochondria were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-α-glucosidase, indicating that this mechanism is of general relevance.IMPORTANCE There is an increasing demand for recombinant proteins to be used as enzymes and pharmaceuticals. The yeast Saccharomyces cerevisiae is a cell factory that is widely used to produce recombinant proteins. Our study revealed that moderate overexpression of SEC16 increased recombinant protein secretion in S. cerevisiae This new strategy can be combined with other targets to engineer cell factories to efficiently produce protein in the future.

Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in Escherichia coli

Secretion of recombinant proteins in heterologous host has drawn attention for its simpler purification and downstream processes. Searching for secretion aid molecules to improve protein secretion can be done through synthetic biology, screening of genome data and proteome-based approach. In the present study, the extracellular proteome on starch-containing medium of Bacillus lehensis G1 was analyzed to identify naturally secreted proteins with signal peptide. A total of 87 protein spots were identified by mass spectrometry, which were categorized mostly in the metabolism of carbohydrates and related molecules (20%). Over-expression and secretion studies were performed for all the 14 selected signal peptides fused to a reporter protein, cyclomaltodextrin glucanotransferase (CGTase). All clones were found to allow CGTase to be excreted into the medium, as observed and measured from the iodine plate assay and enzyme activity assay. Compared to native signal peptide (G1) of CGTase, signal peptide of GlcNAc-binding protein A (GAP) significantly improved CGTase activities by 735% and 205% in extracellular and periplasmic compartment, respectively, with an increase of only ∼1.7 fold the amount of β-galactosidase (cell lysis) in the medium. GAP has the highest secretion rate of 45.6 U/ml/h among all clones, where physicochemical characteristics of signal peptide play significant role.

Influence of PDI gene overexpression on heterological proteins production in yeast Pichia pastoris

Summary: Background. The yeast Pichia pastoris is used for synthesis of recombinant secretory proteins. Overexpression of assistant genes, coding proteins involved in secretion, is one of approaches to improve the production of target protein. PpPDI gene encodes P. pastoris yeast protein disulfide isomerase (Pdi). The aim of our study was to evaluate the effect of Pdi overproduction on recombinant interferons (human interferon-alfa16 and chicken interferon-gamma) production.
 Materials and Methods. PpPDI gene was cloned under the control of the AOX1 gene promoter in plasmid pPICZA. Primers for AJ302014.1 nucleotide sequence of NCBI data base were used for PpPDI gene cloning. The chromosomal DNA of the GS115 strain was used as a template. To generate strains with PpPDI gene overexpression we used a previously obtained strains producing human interferon-alfa16 and chicken interferon-gamma. Yeast transformation was performed by electroporation. Cultivation was performed using single and two-stage strategies in standard media containing methanol as the sole carbon source to induce the AOX1 gene promoter.
 Results. We obtained interferon-producing strains with PpPDI gene overexpression. Over-expression of the PpPDI gene in yeast P. pastoris increases the production of interferon-alfa16, a protein containing disulfide bonds, regardless of the mode of cultivation. Effect of PpPDI gene over-expression on the production of interferon-gamma the protein without disulfide bonds, depends on cultivation mode.
 Conclusion. PpPDI gene overexpression can be used to enhance the production of interferons and other proteins that contain disulfide bonds. Effect of PpPDI gene overexpression on recombinant proteins without disulfide bonds may depend on cultivation procedure.