Secretion Of Recombinant Proteins Research Articles

Synthesis of Secretory Proteins in Yarrowia lipolytica: Effect of Combined Stress Factors and Metabolic Load.

While overproduction of recombinant secretory proteins (rs-Prots) triggers multiple changes in the physiology of the producer cell, exposure to suboptimal growth conditions may further increase that biological response. The environmental conditions may modulate the efficiency of both the rs-Prot gene transcription and translation but also the polypeptide folding. Insights into responses elicited by different environmental stresses on the rs-Prots synthesis and host yeast physiology might contribute to a better understanding of fundamental biology processes, thus providing some clues to further optimise bioprocesses. Herein, a series of batch cultivations of Yarrowia lipolytica strains differentially metabolically burdened by the rs-Prots overproduction have been conducted. Combinations of different stress factors, namely pH (3/7) and oxygen availability (kLa 28/110 h−1), have been considered for their impact on cell growth and morphology, substrate consumption, metabolic activity, genes expression, and secretion of the rs-Prots. Amongst others, our data demonstrate that a highly metabolically burdened cell has a higher demand for the carbon source, although presenting a compromised cell growth. Moreover, the observed decrease in rs-Prot production under adverse environmental conditions rather results from the emergence of a less-producing cell subpopulation than from the decrease of the synthetic capacity of the whole cell population.

High Cell-Density Expression System: Yeast Cells in a Phalanx Efficiently Produce a Certain Range of "Difficult-to-Express" Secretory Recombinant Proteins.

Yeast's extracellular expression provides a cost-efficient means of producing recombinant proteins of academic or commercial interests. However, depending on the protein to be expressed, the production occasionally results in a poor yield, which is frequently accompanied with a deteriorated growth of the host. Here we describe our simple approach, high cell-density expression, to circumvent the cellular toxicity and achieve the production of a certain range of "difficult-to-express" secretory protein in preparative amount. The system features an ease of performing: (a) pre-cultivate yeast cells to the stationary phase in non-inducing condition, (b) suspend the cells to a small aliquot of inducing medium to form a high cell-density suspension or "a phalanx," then (c) give a sufficient aeration to the phalanx. Factors and pitfalls that affect the system's performance are also described.

Secretome-based screening of fusion partners and their application in recombinant protein secretion in Saccharomyces cerevisiae.

For the efficient production of heterologous proteins in the yeast Saccharomyces cerevisiae, we screened for a novel fusion partner from the yeast secretome. From twenty major proteins identified from the yeast secretome, we selected Scw4p, a cell wall protein with similarity to glucanase, and modified to develop a general fusion partner for the secretory expression of heterologous proteins in yeast. The optimal size of the SCW4 gene to act as an efficient fusion partner was determined by C-terminal truncation analysis; two of the variants, S1 (truncated at codon 115Q) and S2 (truncated at codon 142E), were further used for the secretion of heterologous proteins. When fused with S2, the secretion of three target proteins (hGH, exendin-4, and hPTH) significantly increased. Conserved O-glycosylation sites (Ser/Thr-rich domain) and hydrophilic sequences of S2 were deemed important for the function of S2 as a secretion fusion partner. Approximately 5g/L of the S2-exendin-4 fusion protein was obtained from fed-batch fermentation. Intact target proteins were easily purified by affinity chromatography after in vitro processing of the fusion partner. This system may be of general application for the secretory production of heterologous proteins in S. cerevisiae. KEY POINTS : • Target proteins were efficiently secreted with their N-terminus fused to Scw4p. • O-glycosylation and hydrophilic stretches in Scw4p were important for protein secretion. • A variant of Scw4p (S2) was successfully applied for the secretory expression of heterologous proteins.

A leader peptide of the extracellular cyanobacterial carbonic anhydrase ensures the efficient secretion of recombinant proteins in Escherichia coli

Several forms of EcaA protein, correspondent to the extracellular α-class carbonic anhydrase (CA) of cyanobacterium Crocosphaera subtropica ATCC 51142 were expressed in Escherichia coli. The recombinant proteins with no leader peptide (EcaA and its fusion with thioredoxin or glutathione S-transferase) were allocated inside cells in a full-length form; these cells did not display any extracellular CA activity. Soluble proteins (including that of periplasmic space) of E. coli cells that expressed both ЕсаА equipped with its native leader peptide (L-EcaA) as well as L-EcaA fused with thioredoxin or glutathione S-transferase at N-terminus, mainly contained the processed EcaA. The appearance of mature ЕсаА in outer layers of E. coli cells expressed leader peptide-containing forms of recombinant proteins, has been directly confirmed by immunofluorescent microscopy. Those cells also displayed high extracellular CA activity. In addition, the mature EcaA protein was detected in the culture medium. This suggests that cyanobacterial signal peptide is recognized by the secretory machinery and by the leader peptidase of E. coli even as a part of a fusion protein. The efficiency of EcaA leader peptide was comparable to that of PelB and TorA signal peptides, commonly used for biotechnological production of extracellular recombinant proteins in E. coli.

Hyperosmolarity adversely impacts recombinant protein synthesis by Yarrowia lipolytica\u2014molecular background revealed by quantitative proteomics

In this research, we were interested in answering a question whether subjecting a Yarrowia lipolytica strain overproducing a recombinant secretory protein (rs-Prot) to pre-optimized stress factors may enhance synthesis of the rs-Prot. Increased osmolarity (3 Osm kg−1) was the primary stress factor implemented alone or in combination with decreased temperature (20 °C), known to promote synthesis of rs-Prots. The treatments were executed in batch bioreactor cultures, and the cellular response was studied in terms of culture progression, gene expression and global proteomics, to get insight into molecular bases underlying an awaken reaction. Primarily, we observed that hyperosmolarity executed by high sorbitol concentration does not enhance synthesis of the rs-Prot but increases its transcription. Expectedly, hyperosmolarity induced synthesis of polyols at the expense of citric acid synthesis and growth, which was severely limited. A number of stress-related proteins were upregulated, including heat-shock proteins (HSPs) and aldo–keto reductases, as observed at transcriptomics and proteomics levels. Concerted downregulation of central carbon metabolism, including glycolysis, tricarboxylic acid cycle and fatty acid synthesis, highlighted redirection of carbon fluxes. Elevated abundance of HSPs and osmolytes did not outbalance the severe limitation of protein synthesis, marked by orchestrated downregulation of translation (elongation factors, several aa-tRNA synthetases), amino acid biosynthesis and ribosome biogenesis in response to the hyperosmolarity. Altogether we settled that increased osmolarity is not beneficial for rs-Prots synthesis in Y. lipolytica, even though some elements of the response could assist this process. Insight into global changes in the yeast proteome under the treatments is provided.Key points• Temp enhances, but Osm decreases rs-Prots synthesis by Y. lipolytica.• Enhanced abundance of HSPs and osmolytes is overweighted by limited translation.• Global proteome under Osm, Temp and Osm Temp treatments was studied.

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli.

Main reasons to produce recombinant proteins in the periplasm of E. coli rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein isolation, -iii- control the nature of the N-terminus of the mature protein, and -iv- minimize exposure to cytoplasmic proteases. However, hampered protein targeting, translocation and folding as well as protein instability can all negatively affect periplasmic protein production yields. Strategies to enhance periplasmic protein production yields have focused on harmonizing secretory recombinant protein production rates with the capacity of the secretory apparatus by transcriptional and translational tuning, signal peptide selection and engineering, increasing the targeting, translocation and periplasmic folding capacity of the production host, preventing proteolysis, and, finally, the natural and engineered adaptation of the production host to periplasmic protein production. Here, we discuss these strategies using notable examples as a thread.

Comparative performance of different antigens on the lateral flow assay (LFA) platform for the rapid serodiagnosis of paratuberculosis

Paratuberculosis is a globally prevalent disease, that adversely affects the economy of livestock farming. Control is largely based on early detection followed by ‘Test and Cull’ or ‘Test and Segregate’ Policy. Implementation of paratuberculosis control is a special challenge due to the non-availability of point of care diagnostics (PoCD). Therefore, the present study aimed to optimize and evaluate a lateral flow assay (LFA) for the rapid serodiagnosis of paratuberculosis in ruminant species, especially in the view of the resource-limited areas. Performance of three different antigenic preparations including native purified protoplasmic antigen (nPPA-LFA), commercial purified protoplasmic antigen (cPPA-LFA), and a cocktail of recombinant secretory proteins (RP-LFA) was evaluated as detection reagents for coating LFA strips. Comparative performance of the optimized LFA was also evaluated with gold standard tissue culture, fecal PCR (polymerase chain reaction), and plate ELISA. In addition, the onsite testing of animals belonging to different farms (endemic), species, and regions using optimized LFA was also done to highlight the on-farm testing approach. Findings revealed recombinant secretory proteins based LFA (RP-LFA) had a higher sensitivity of detection compared to other antigens. RP-LFA had a sensitivity of 77.7%, 75.44%, and 75.16% in comparison to gold standard tissue culture, fecal PCR, and plate ELISA, respectively. The specificity of RP-LFA was 100% with all reference tests. In comparison to plate ELISA, RP-LFA had a detection limit of 100% when the S/P ratio of the serum sample is ≥1.0 and 80% when the S/P ratio range of 0.8–1.0. Using RP-LFA, on-farm testing of 608 animals was done and 283 (46.5%) were found positive. Kappa analysis of present RP-LFA revealed ‘good strength of agreement’ with gold standard tissue culture, fecal PCR, and plate ELISA. Optimized RP-LFA had no cross-reactivity with bovine tuberculosis (bovine TB). The RP-LFA was found reproducible, user-friendly and test results can be interpreted within five minutes. In conclusion, the findings of the present study advocate the huge potential of LFA-based PoCD in the rapid diagnosis and control of paratuberculosis.

Biosensor-Based Optimization of Cutinase Secretion by Corynebacterium glutamicum.

The industrial microbe Corynebacterium glutamicum is gaining substantial importance as a platform host for recombinant protein secretion. We recently developed a fluorescence-based (eYFP) C. glutamicum reporter strain for the quantification of Sec-dependent protein secretion by monitoring the secretion-related stress response and now demonstrate its applicability in optimizing the secretion of the heterologous enzyme cutinase from Fusarium solani pisi. To drive secretion, either the poor-performing PelSP or the potent NprESP Sec signal peptide from Bacillus subtilis was used. To enable easy detection and quantification of the secreted cutinase we implemented the split green fluorescent protein (GFP) assay, which relies on the GFP11-tag fused to the C-terminus of the cutinase, which can complement a truncated GFP thereby reconstituting its fluorescence. The reporter strain was transformed with different mutant libraries created by error-prone PCR, which covered the region of the signal peptide and the N-terminus of the cutinase. Fluorescence-activated cell sorting (FACS) was performed to isolate cells that show increased fluorescence in response to increased protein secretion stress. Five PelSP variants were identified that showed a 4- to 6-fold increase in the amount and activity of the secreted cutinase (up to 4,100 U/L), whereas two improved NprESP variants were identified that showed a ∼35% increase in secretion, achieving ∼5,500 U/L. Most of the isolated variants carried mutations in the h-region of the signal peptide that increased its overall hydrophobicity. Using site-directed mutagenesis it was shown that the combined mutations F11I and P16S within the hydrophobic core of the PelSP are sufficient to boost cutinase secretion in batch cultivations to the same level as achieved by the NprESP. Screening of a PelSP mutant library in addition resulted in the identification of a cutinase variant with an increased specific activity, which was attributed to the mutation A85V located within the substrate-binding region. Taken together the biosensor-based optimization approach resulted in a substantial improvement of cutinase secretion by C. glutamicum, and therefore represents a valuable tool that can be applied to any secretory protein of interest.

The mechanism of N-acetyl-l-cysteine in improving the secretion of porcine follicle-stimulating hormone in Pichia pastoris.

Our previous study revealed that N-acetyl-l-cysteine (NAC) could enhance the secretion of recombinant proteins by Pichia pastoris, but the corresponding molecular mechanisms are still unclear. In the present study, we explored whether other thiols have a similar action on the secretion of recombinant human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHβ), to reveal the mechanism of NAC on HSA-pFSHβ secretion. Transcriptome analysis showed that genes involved in oxidoreductase activity and oxidation-reduction process were upregulated in cells supplemented with NAC. The other three thiol-reducing regents including dimercaptopropanol (DT), thioglycolic acid, and mercaptolactic acid could improve HSA-pFSHβ production in the culture supernatant. Among them, only DT had similar effect as NAC on HSA-pFSHβ secretion and the increase of GSH content. Moreover, 1-20 mM GSH, 1-10mM cysteine, or 1-20 mM N-acetyl-d-cysteine supplementation could improve the secretion of HSA-pFSHβ. Furthermore, 0.4-3.2mM ethacrynic acid, rather than 1-16 mM BSO could inhibit the effect of NAC on the production of HSA-pFSHβ. These results indicated that NAC improved the secretion of HSA-pFSHβ by increasing the intracellular GSH content through its thiol activity rather than as a precursor for GSH synthesis. In conclusion, our results demonstrate, for the first time, that the secretion of recombinant HSA-pFSHβ in Pichia pastoris could be improved through thiol-reducing agent supplementation, and the mechanism of the effect NAC has on HSA-pFSHβ secretion is associated with improving the intracellular GSH content.

An overview and future prospects of recombinant protein production in Bacillus subtilis.

Bacillus subtilis is a well-characterized Gram-positive bacterium and a valuable host for recombinant protein production because of its efficient secretion ability, high yield, and non-toxicity. Here, we comprehensively review the recent studies on recombinant protein production in B. subtilis to update and supplement other previous reviews. We have focused on several aspects, including optimization of B. subtilis strains, enhancement and regulation of expression, improvement of secretion level, surface display of proteins, and fermentation optimization. Among them, optimization of B. subtilis strains mainly involves undirected chemical/physical mutagenesis and selection and genetic manipulation; enhancement and regulation of expression comprises autonomous plasmid and integrated expression, promoter regulation and engineering, and fine-tuning gene expression based on proteases and molecular chaperones; improvement of secretion level predominantly involves secretion pathway and signal peptide screening and optimization; surface display of proteins includes surface display of proteins on spores or vegetative cells; and fermentation optimization incorporates medium optimization, process condition optimization, and feeding strategy optimization. Furthermore, we propose some novel methods and future challenges for recombinant protein production in B. subtilis.Key points• A comprehensive review on recombinant protein production in Bacillus subtilis.• Novel techniques facilitate recombinant protein expression and secretion.• Surface display of proteins has significant potential for different applications.

Aberrant Membrane Structures in Hypervesiculating Escherichia coli Strain ΔmlaE ΔnlpI Visualized by Electron Microscopy.

Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing a part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE, related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of ΔmlaEΔnlpI cells using quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Plasmolysis was also observed in the single-gene knockout mutants ΔnlpI and ΔmlaE. This study has demonstrated that plasmolysis was induced in the hypervesiculating mutant E. coli cells. Furthermore, intracellular vesicles and multilamellar OMV were observed in the ΔmlaEΔnlpI cells. Meanwhile, the secretion of recombinant green fluorescent protein (GFP) expressed in the cytosol of the ΔmlaEΔnlpI cells was more than 100 times higher than that of WT and ΔnlpI, and about 50 times higher than that of ΔmlaE in the OMV fraction, suggesting that cytosolic components were incorporated into outer-inner membrane vesicles (OIMVs) and released into the extracellular space. Additionally, QFDE-EM analysis revealed that ΔmlaEΔnlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan (PG) pores in wild-type (WT) E. coli. These results suggest that in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as OIMVs.

Development of rELISA using novel markers for the diagnosis of paratuberculosis

Paratuberculosis is one of the complex livestock infections whose control has largely been hampered due to the lack of efficacious diagnostics. Present study optimized plate ELISA assay for the diagnosis and screening of paratuberculosis using recombinant secretory proteins. Five secretory antigens (2677c, 3547c, 4308c, 1693c, and 2168c) were produced in the recombinant system using the E. coli host and used for the optimization of the assay. These proteins were selected because of their prior proven specificity and antigenicity as humoral immunity markers. The assay was first optimized using traditional ELISA reader and then the performance was evaluated using a handheld ELISA reader. Findings were identical in both traditional ELISA reader as well as handheld ELISA reader. Optimized ELISA was found reproducible using different batches of the recombinant antigens as well as in terms of the inter and intra assay %CV values. The present ELISA has a sensitivity and specificity of 91.6% and 100%, respectively. Also, rELISA revealed AUCROC and Youden index J of 0.95 and 0.91, respectively. In conclusion, assay conditions of MAP-recombinant protein-based ELISA were optimized and the optimized ELISA ODs can be read using portable handheld ELISA reader. Thereby, opening a future window to develop assay for onsite testing.

Global transcriptome profiling reveals genes responding to overproduction of a small secretory, a high cysteine- and a high glycosylation-bearing protein in Yarrowia lipolytica

Investigation of the yeast cell’s response to recombinant secretory protein (rs-Prot) overproduction is relevant for both basic and applied research. Imbalance, overloading or stress within this process impacts the whole cell. In the present study, by using steady-state cultures and transcriptomics, we investigated the cellular response of Yarrowia lipolytica challenged with high-level expression of genes encoding proteins with significantly different biochemical characteristics: a small protein retained within the cell i) or secreted ii), a medium size secretory protein with a high number of disulfide bonds iii), or glycosylation sites iv). Extensive analysis of omics data, supported by careful manual curation, led to some anticipated observations on oxidative and unfolded protein stress (CTT1, PXMP2/4, HAC1), glycosylation (ALGs, KTRs, MNTs, MNNs), folding and translocation (SSAs, SSEs) but also generated new exciting knowledge on non-conventional protein secretion (NCE102), transcriptional regulators (FLO11, MHY1, D01353 g, RSFA, E23925g or MAF1), vacuolar proteolysis targets in Y. lipolytica (ATGs, VPSs, HSE1, PRB1, PRC1, PEP4) or growth arrest (CLN1) upon rs-Prots overproduction.

Modulating Oral Delivery and Gastrointestinal Kinetics of Recombinant Proteins via Engineered Fungi.

A new modality in microbe-mediated drug delivery has recently emerged wherein genetically engineered microbes are used to locally deliver recombinant therapeutic proteins to the gastrointestinal tract. These engineered microbes are often referred to as live biotherapeutic products (LBPs). Despite advanced genetic engineering and recombinant protein expression approaches, little is known on how to control the spatiotemporal dynamics of LBPs and their secreted therapeutics within the gastrointestinal tract. To date, the fundamental pharmacokinetic analyses for microbe-mediated drug delivery systems have not been described. Here, we explore the pharmacokinetics of an engineered, model protein-secreting Saccharomyces cerevisiae, which serves as an ideal organism for the oral delivery of complex, post-translationally modified proteins. We establish three methods to modulate the pharmacokinetics of an engineered, recombinant protein-secreting fungi system: (i) altering oral dose of engineered fungi, (ii) co-administering antibiotics, and (iii) altering recombinant protein secretion titer. Our findings establish the fundamental pharmacokinetics which will be essential in controlling downstream therapeutic response for this new delivery modality.

Constitutively active Rheb mutants [T23M] and [E40K] drive increased production and secretion of recombinant protein in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are high value agents used for disease therapy ("biologic drugs") or as diagnostic tools which are widely used in the healthcare sector. They are generally manufactured in mammalian cells, in particular Chinese hamster ovary (CHO) cells cultured in defined media, and are harvested from the medium. Rheb is a small GTPase which, when bound to GTP, activates mechanistic target of rapamycin complex 1, a protein kinase that drives anabolic processes including protein synthesis and ribosome biogenesis. Here, we show that certain constitutively active mutants of Rheb drive faster protein synthesis in CHO cells and increase the expression of proteins involved in the processing of secreted proteins in the endoplasmic reticulum, which expands in response to expression of Rheb mutants. Active Rheb mutants, in particular Rheb[T23M], drive increased cell number under serum-free conditions similar to those used in the biotechnology industry. Rheb[T23M] also enhances the expression of the reporter protein luciferase and, especially strongly, the secreted Gaussia luciferase. Moreover, Rheb[T23M] markedly (2-3 fold) enhances the amount of this luciferase and of a model immunoglobulin secreted into the medium. Our data clearly demonstrate that expressing Rheb[T23M] in CHO cells provides a simple approach to promoting their growth in defined medium and the production of secreted proteins of high commercial value.

Non-classical secretion of a type I L-asparaginase in Bacillus subtilis

L-asparaginase (EC 3.5.1.1) showed great commercial value owing to its effective treatment of acute lymphoblastic leukemia (ALL), lymphoid system malignancies and Hodgkin disease, and also to its use in the prevention of acrylamide formation in fried and baked foods. In this study, a type I L-asparaginase gene from Bacillus licheniformis Z-1 (BlAase) was cloned and expressed in Bacillus subtilis RIK 1285. Results showed that even without the mediation of any N-terminal signal peptides, BlAase can efficiently secrete into the medium. Further investigation indicated that the secretion of the BlAase was via neither Sec- nor Tat-dependent secretion pathway, and both the N- and C-terminal regions of the BlAase were essential for its expression and secretion, implying that BlAase might be secreted via a non-classical secretion pathway. To explore its secretion ability, BlAase was used as a signal peptide to direct the secretion of various heterologous proteins, where two of five proteins were successfully secreted with the mediation of BlAase. To the best of our knowledge, this is the first time to achieve extracellular expression of L-asparaginase via non-classical protein secretion pathway in B. subtilis, and provide a potential tool for secretion of recombinant proteins expressed in B. subtilis using BlAase as a signal peptide.

An optimized growth medium for increased recombinant protein secretion titer via the type III secretion system

BackgroundProtein secretion in bacteria is an attractive strategy for heterologous protein production because it retains the high titers and tractability of bacterial hosts while simplifying downstream processing. Traditional intracellular production strategies require cell lysis and separation of the protein product from the chemically similar cellular contents, often a multi-step process that can include an expensive refolding step. The type III secretion system of Salmonella enterica Typhimurium transports proteins from the cytoplasm to the extracellular environment in a single step and is thus a promising solution for protein secretion in bacteria. Product titer is sensitive to extracellular environmental conditions, however, and T3SS regulation is integrated with essential cellular functions. Instead of attempting to untangle a complex web of regulatory input, we took an “outside-in” approach to elucidate the effect of growth medium components on secretion titer.ResultsWe dissected the individual and combined effects of carbon sources, buffers, and salts in a rich nutrient base on secretion titer. Carbon sources alone decreased secretion titer, secretion titer increased with salt concentration, and the combination of a carbon source, buffer, and high salt concentration had a synergistic effect on secretion titer. Transcriptional activity measured by flow cytometry showed that medium composition affected secretion system activity, and prolonged secretion system activation correlated strongly with increased secretion titer. We found that an optimal combination of glycerol, phosphate, and sodium chloride provided at least a fourfold increase in secretion titer for a variety of proteins. Further, the increase in secretion titer provided by the optimized medium was additive with strain enhancements.ConclusionsWe leveraged the sensitivity of the type III secretion system to the extracellular environment to increase heterologous protein secretion titer. Our results suggest that maximizing secretion titer via the type III secretion system is not as simple as maximizing secreted protein expression—one must also optimize secretion system activity. This work advances the type III secretion system as a platform for heterologous protein secretion in bacteria and will form a basis for future engineering efforts.

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris

BackgroundPichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains.ResultsPotential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter.DiscussionThe microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.

A method for the transient inhibition of toxicity of secretory recombinant proteins, exemplified by bacterial alkaline phosphatase. Novel protocol for problematic DNA termini dephosphorylation

Genes encoding proteins ‘toxic' to recombinant host are difficult for cloning/expression and recombinant clones are unstable. Even tightly controlled inducible T7-lac, PBAD, PL, PR promoters are not totally silent in an uninduced state and thus not adequate for highly toxic proteins. An innovative approach to engineering and expression of the gene, encoding bacterial alkaline phosphatase (BAP) is proposed. The native precursor enzyme contains a signal peptide at the N-terminus and is secreted to the Escherichia coli (E. coli) periplasm. The signal peptide is then removed that allows oxidation and formation of active dimers. To decrease toxicity of the bap gene, its secretion leader coding section was replaced with a N-terminal His6-tag. The gene was expressed in E. coli in a PBAD vector, resulting in the accumulation of soluble His6-BAP in the cytoplasm. The His6-BAP was neutral to the cells, as no maturation was possible in the reducing cytoplasm. The purified homogenous protein was further reactivated in a redox buffer containing the protein structure stabilizing cofactors. The His6-BAP exhibited high activity. A dephosphorylation protocol for all types of DNA termini was developed.The method appears well suited for the industrial production of BAP and can be applied to other problematic proteins.•Efficient toxic gene expression•Novel approach to toxic gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme•Scaled-up production of ultrapure BAP•Improved protocol for all types of DNA termini dephosphorylation

Improving the Recombinant Protein Expression of Human Galectin-3 in BL21 Bacterial Host System

Background: Regardless of the broad explore in the territory of glycobiology concerning structure and capacity of glycans, lectins and glycosylation forms, numerous viewpoints are still left unexplored.
 Aim: In this study, we analyzed the effect of shuttle vector on the secretion of human galectin recombinant protein.
 Methods: The galectin was expressed in E. coli BL21 by growing the bacterial culture in SOC medium and purified by nickel-based affinity chromatography due to its His-tag.
 Results: After cell lysis the protein was identified as a single 29 KDa band by 12% SDS-PAGE. Conclusion: Characterization studies clearly revealed that the purified protein was indeed galectin 3.