Expression of catalytic subunit of bovine enterokinase in the filamentous fungus Aspergillus niger

The cDNA encoding for catalytic subunit of bovine enterokinase (EK(L)), to which the sequence for Kex2 protease cleavage site was inserted, was expressed in the protease deficient filamentous fungus Aspergillus niger AB1.13. Fungal transformants were obtained in which expression of the glucoamylase fusion gene resulted in secretion of the protein into growth medium. Fusion polypeptide was processed to mature EK(L) by endogenous Kex-2 like protease cleavage during secretory pathway. The highest quantity of EK(L), up to 5 mg l(-1), was obtained in soya milk medium. The secreted EK(L) was easily purified from other proteins found in A. niger culture supernatant, using ion exchange and affinity chromatography. The yield of the purified and highly active EK(L) was 1.9 mg l(-1) of culture.     

Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer.

An N-terminus sequence of human interleukin 1beta (hIL-1beta) was used as a fusion expression partner for the production of two recombinant therapeutic proteins, human granulocyte-colony stimulating factor (hG-CSF) and human growth hormone (hGH), using Saccharomyces cerevisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyveromyces lactis, the N-terminus 24 amino acids (Ser5-Ala28) of mature hIL-1beta, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-CSF or hGH). The gene expression was controlled by the inducible UAS(gal)/MF-alpha1 promoter. With the expression vector above, both recombinant proteins were well secreted into culture medium with high secretion efficiencies, and especially, the recombinant hGH was accumulated up to around 1.3 g/L in the culture broth. This is due presumably to the significant role of fused hIL-1beta as secretion enhancer in the yeast secretory pathway. In our recent report, various immunoblotting analyses have shown that the presence of a core N-glycosylation resident in the hIL-1beta fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the recombinant S. cerevisiae. When the N-glycosylation was completely blocked with the addition of tunicamycin to the culture, the secretion of hG-CSF and hGH was decreased to a negligible level although the other host-derived proteins were well secreted to the culture broth regardless of the presence of tunicamycin. The N-terminal sequencing of the purified hG-CSF verified that the hIL-1beta fusion peptide was correctly removed by in vivo KEX2 protease upon the exit of fusion protein from Golgi complex. From the results presented in this article, it is strongly suggested that the N-terminus fusion of the hIL-1beta peptide could be utilized as a potent secretion enhancer in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.     

The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system

Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.     

Immunocytochemical characterization of two thyroid medullary carcinoma cell linesin vitro

Summary The immunocytochemical characterization of cell lines originating from thyroid medullary carcinoma, i.e. human TT cells and rat rMTC 6-23 cells, was undertaken. The immunocytochemical studies were supplemented by ultrastructural studies, including ultrastructural immunocytochemistry, and by radioimmunological estimation of calcitonin secretion to the medium. In rMTC 6-23 cells (subcultures 24 to 30), no hormone presence was demonstrated immunocytochemically, which corresponded to the absence of secretory granules at the ultrastructural level. Of various proteins sought, only neuron-specific enolase could be demonstrated. Nevertheless, the cells secreted calcitonin into the medium. TT cells (passages 145 to 160) produced secretory granules. The granules contained calcitonin, calcitonin gene-related peptide, somatostatin, neurotensin, met-enkephalin, leu-enkephalin, gastrin releasing peptide, parathyroid hormone-related protein, functional proteins of the chromogranin group and synaptophysin. Other functional proteins found in the cytosol of TT cells included non-specific enolase, calbindin and tyrosine hydroxylase. Receptor for calcitriol was localized in the cell nucleus. Marker proteins were localized in the cytosol (carcinoembryonic antigen) and in the cell skeleton (-tubulin, cytokeratin). Following changes in ionized calcium levels in the medium, changes in calcitonin secretion and in immunocytochemical detectability of some hormones and functional proteins were observed. TT cells demonstrated the expression of numerous hormones and functional proteins associated with calcitonin secretion. Further, the cells in their ultrastructure, immunocytochemical and secretory characteristics, resemble more closely normal parafollicular cells of the thyroid and, in our opinion, represent a more appropriate model for functional studies.     

Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2 fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9–mCherry fusion protein was detected 6 h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9–mCherry secretion, demonstrating the utility of this method in a biological experiment.     

A novel fusion partner for enhanced secretion of recombinant proteins in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.     

Role of N-terminal 28-amino-acid region of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lipase in directing proteins to secretory pathway of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

To develop a new approach for improving heterologous protein production in Aspergillus oryzae, we focused on the functional role of the N-terminal region of Rhizopus oryzae lipase (ROL). Several N-terminal deletion variants of ROL were expressed in A. oryzae. Interestingly, a segment of 28 amino acids from the C-terminal region of the propeptide (N28) was found to be critical for secretion of ROL into the culture medium. To further investigate the role of N28, the ROL secretory process was visualized in vivo using ROL-green fluorescent protein (GFP) fusion proteins. In cells producing ROL with N28, fluorescence observations showed that the fusion proteins are transported through endoplasmic reticulum (ER), Golgi, and cell wall, which is one of the typical secretory processes in a eukaryotic cell. Because the expression of the mature ROL-GFP fusion protein induced fluorescence accumulation without its translocation into the ER, N28 is considered to play a crucial role in protein transport. When N28 was inserted between the secretion signal and GFP, fluorescence observations showed that GFP, which is originally a cytoplasmic protein, was efficiently translocated into the ER of A. oryzae, resulting in an enhanced secretion of mature GFP after proteolytic cleavage of N28. These findings suggest that N28 facilitates protein translocation into ER and can be a promising candidate for improving heterologous protein production in A. oryzae.     

High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3′,5,5′-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.     

Enhanced secretory ability for the human factor VIII light chain produced in baculovirus-infected insect cells

The light and truncated heavy chains of human factor VIII, expressed separately in baculovirus-infected insect cells, exhibited different secretory behaviour when compared with each other and with a biologically active fusion molecule of the truncated heavy and light chains.The light chain was very efficiently secreted into culture medium, as judged by high extracellular protein levels and the absence of evidence for light chain retention within cells.Alternatively, proteins containing the heavy chain sequence were poorly secreted and appeared to be sequestered within cells, suggesting that regions within the heavy chain are responsible for the low levels of secreted protein which have generally been observed for recombinant factor VIII.     

Secretome analysis uncovers an Hcp-family protein secreted via a type VI secretion system in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria.     

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates. Our observations should facilitate efforts to achieve a differentiated functional state of Sertoli and peritubular cells in culture as well as to select secretory proteins for assessing their possible biological role in testicular function.     

The CssRS two-component regulatory system controls a general secretion stress response in Bacillus subtilis

Bacillus species are valuable producers of industrial enzymes and biopharmaceuticals, because they can secrete large quantities of high-quality proteins directly into the growth medium. This requires the concerted action of quality control factors, such as folding catalysts and 'cleaning proteases'. The expression of two important cleaning proteases, HtrA and HtrB, of Bacillus subtilis is controlled by the CssRS two-component regulatory system. The induced CssRS-dependent expression of htrA and htrB has been defined as a protein secretion stress response, because it can be triggered by high-level production of secreted alpha-amylases. It was not known whether translocation of these alpha-amylases across the membrane is required to trigger a secretion stress response or whether other secretory proteins can also activate this response. These studies show for the first time that the CssRS-dependent response is a general secretion stress response which can be triggered by both homologous and heterologous secretory proteins. As demonstrated by high-level production of a nontranslocated variant of the alpha-amylase, AmyQ, membrane translocation of secretory proteins is required to elicit this general protein secretion stress response. Studies with two other secretory reporter proteins, lipase A of B. subtilis and human interleukin-3, show that the intensity of the protein secretion stress response only partly reflects the production levels of the respective proteins. Importantly, degradation of human interleukin-3 by extracellular proteases has a major impact on the production level, but only a minor effect on the intensity of the secretion stress response.     

Enhanced secretion of<Emphasis Type="Italic"> Bacillus stearothermophilus</Emphasis> L1 lipase in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> by translational fusion to cellulose-binding domain

The secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae was investigated by employing a fusion partner, a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II (THEG). The CBD was connected to the N-terminal of L1 lipase through an endogenous linker peptide from THEG. The expression cassette for the fusion protein in S. cerevisiae was constructed using the -amylase signal peptide and the galactose-inducible GAL10 promoter. Secretion of CBD-linker-L1 lipase by this fusion construct was dramatically 7-fold enhanced, compared with that of the mature L1 lipase without CBD-fusion. The fusion protein was secreted into the culture medium, reaching levels of approximately 1.3 g/l in high-cell-density fed-batch cultures. Insertion of a KEX2 cleavage site into the junction between CBD-linker and L1 lipase resulted in the same level of enhanced secretion, indicating that the CBD-linker fusion probably plays a critical role in secretion from endoplasmic reticulum to Golgi apparatus. Therefore, the CBD from THEG can be used both as an affinity tag and as a secretion enhancer for the secretory production of heterologous proteins in S. cerevisiae, since in vivo breakage at the linker was almost negligible.     

Contrasting secretory processing of simultaneously expressed heterologous proteins in Saccharomyces cerevisiae

In this study, secretory processing of cell-surface displayed Aga2p fusions to bovine pancreatic trypsin inhibitor (BPTI) and the single chain Fv (scFv) antibody fragment D1.3 are examined. BPTI is more efficiently processed than D1.3 both when secreted and surface-displayed, and D1.3 expression imparts a greater amount of secretory stress on the cell as assayed by a reporter of the unfolded protein response (UPR). Surprisingly, simultaneous expression of the two proteins in the same cell somewhat improves BPTI surface display while decreasing D1.3 surface display with minimal effect on UPR activation. Furthermore, co-expression leads to the accumulation of punctate vacuolar aggregates of D1.3 and increased secretion of the D1.3-Aga2p fusion into the supernatant. Overexpression of the folding chaperones protein disulfide isomerase (PDI) and BiP largely mitigates the D1.3 surface expression decrease, suggesting that changes in vacuolar and cell surface targeting may be due, in part, to folding inefficiency. Titration of constitutive UPR expression across a broad range progressively decreases surface display of both proteins as UPR increases. D1.3-Aga2p traffic through the late secretory pathway appears to be strongly affected by overall secretory load as well as folding conditions in the ER.     

Export of prepro-alpha-factor from Escherichia coli

Yeast prepro-alpha-factor translocates posttranslationally into yeast microsomes in vitro. This process is strongly influenced by the extreme carboxyl-terminal region of the protein. These features contrast with the properties of most eucaryotic proteins which are translocated into the endoplasmic reticulum. We have extended these studies by introducing the gene for the wild-type and several mutant forms of prepro-alpha-factor into Escherichia coli. Prepro-alpha-factor is secreted into the periplasm and processed to pro-alpha-factor. Its translocation across the plasma membrane requires the membrane potential and the secY gene product. Deletion mutant analysis showed that features of the pro-segment were essential for secretion of prepro-alpha-factor in E. coli, while the carboxyl-terminal region, which is required in yeast, is dispensible in E. coli. Neither size nor the presence of a unique topogenic sequence was sufficient to explain the requirement for the pro-segment.     

Secretion of genetically-engineered dihydrofolate reductase from Escherichia coli using an E. coli α-hemolysin membrane translocation system

Summary Secretion of fusion proteins composed of cytoplasmic protein dihydrofolate reductase (DHFR) and the Escherichia coli -haemolysin (HlyA) C-terminal sequence was examined through the haemolysin secretion machinery of E. coli. DHFR of various lengths was combined with the HlyA C-terminal region, and both secretion and DHFR activity of the fusions were measured. The secretion was found to be inversely correlated with the intracellular DHFR activity. Moreover, when one amino acid (Ile155) in a -sheet of the DHFR C-terminal region was replaced with Lys, the enzymatically active DHFR fusion protein was secreted into the medium. We discuss the possibility of a relationship between folding and secretion of HlyA-fused protein in the HlyA secretion system. Correspondence to: H. Nakano     

Comparison of proteins synthesized by polarized caprine oviductal epithelial cells and oviductal explants in vitro

Our objectives were to compare proteins secreted by caprine oviductal explants and oviductal epithelial (OE) cells in vitro. Oviducts were collected from goats on Days 1 (n=5) and 5 (n=5) of the estrous cycle. Radiolabeled secretory proteins from tissue segments and cell cultures were visualized using SDS-PAGE and fluorography. After culture, media from ampulla oviduct segments collected on Days 1 and 5 of the estrous cycle contained an acidic 97 kDa protein, which was greatly reduced in culture medium obtained from infundibulum and isthmus oviduct segments. A complex of low molecular weight proteins (14-26 kDa) could be modulated by estradiol when OE cells were cultured on plastic. This complex was constitutively expressed when OE cells were cultured on Matrigel-coated filters. Polarized OE cells were also capable of compartment-specific secretion of [L-(35)S]-methionine-labeled proteins. A 45 kDa acidic protein was predominantly secreted into the apical compartment while a 66 kDa acidic protein was preferentially localized in the basal compartment. Proteins secreted by OE cells were similar to proteins secreted by tissue segments in vitro. Therefore, under well-defined culture conditions OE cells may be useful in enhancing in vitro fertilization or early embryonic development.     

A novel strategy for enhancing extracellular secretion of recombinant proteins in Escherichia coli

Secretion of cytoplasmic expressed proteins into culture medium has significant commercial advantages in large-scale production of proteins. Our previous study demonstrated that the membrane permeability of Escherichia coli could be significantly improved when Thermobifida fusca cutinase, without a signal peptide, was expressed in cytoplasm. This study investigated the extracellular production of other recombinant proteins, including both secretory and cytosolic proteins, with co-expression of cutinase. When the secretory enzymes, xylanase and α-amylase, were co-expressed with cutinase, the culture period was shortened by half, and the productivity was 7.9 and 2.0-fold to that of their individual control without co-expression, respectively. When the normally cytosolic proteins, xylose isomerase and trehalose synthase, were co-expressed with cutinase, more than half of the target proteins were “secreted” into the culture medium. Moreover, by using β-galactosidase to detect membrane leakage, the improved secretion of the above model proteins was confirmed not to be due to cell lysis. The study provides a novel strategy for enhancing extracellular secretion of recombinant proteins in E. coli.     

Secretion and affinity purification of glutathione S-transferase fusion proteins from yeast

Summary Sequences encoding GST-fusion proteins were cloned into the Saccharomyces cerevisiae secretion vector, pYEX-S1, to direct secretion into the culture medium. GST and metallothionein fused to GST were secreted successfully and the fusion proteins purified. With several other GST-fusion proteins however, the proteins were retained inside the cell, indicating limitations to the types of proteins that can be secreted from yeast.