Secretory Production Of Heterologous Proteins Research Articles

Enhancing extracellular membrane vesicle productivity of Shewanella vesiculosa HM13, a prospective host for vesiculation-mediated protein secretion, by weakening outer membrane-peptidoglycan linkage

Shewanella vesiculosa HM13, a psychrotrophic gram-negative bacterium isolated from the intestinal contents of horse mackerel, produces abundant extracellular membrane vesicles (EMVs) by budding the outer membrane. The EMVs of this bacterium carry a single major cargo protein, P49, of unknown function, which may be useful as a carrier for the secretory production of heterologous proteins as cargoes of EMVs. In this study, to increase the utility of S.vesiculosa HM13 as a host for EMV-mediated protein production, we improved its EMV productivity by weakening the linkage between the outer membrane and underlying peptidoglycan layer. In gram-negative bacteria, the outer membrane is connected to peptidoglycans predominantly through Braun's lipoprotein (Lpp), and the formation of this linkage is catalyzed by an l,d-transpeptidase (Ldt). We constructed gene-disrupted mutants of Lpp and Ldt and assessed their EMV productivity. The EMVs of the lpp- and ldt-disrupted mutants grown at 18°C were evaluated using nanoparticle tracking analysis, and their morphologies were observed using transmission electron microscopy. As a result, an approximately 2.5-fold increase in EMV production was achieved, whereas the morphology of the EMVs of these mutants remained almost identical to that of the parent strain. In accordance with the increase in EMV production, the mutants secreted approximately 2-fold higher amounts of P49 than the parent strain into the culture broth as the EMV cargo. These findings will contribute to the development of an EMV-based secretory production system for heterologous proteins using S.vesiculosa HM13 as a host.

Evaluation of Different Signal Peptides for Secretory Production of Recombinant Human Interferon-gamma: Bioinformatics Approach

Background: The fusion of the secretory signal peptide to the N-terminal of polypeptide’s amino acid sequence is an attractive technique for the secretory production of heterologous proteins. On the other hand, applying computational analysis may be beneficial to overcome the barriers of trial-and-error approaches in detecting proper signal sequences. As the scope of this study, the most probable effective properties of 30 signal sequences for the extracellular production of recombinant human interferon-gamma (rhIFN-γ) were analysed. Methods: The fusion of the secretory signal peptide to the N-terminal of a polypeptide’s amino acid sequence is an attractive technique for the secretory production of heterologous proteins. On the other hand, applying computational analysis may be beneficial in overcoming the barriers of trial-anderror approaches in detecting proper signal sequences. As the scope of this study, the most probable effective properties of 30 signal sequences for the extracellular production of recombinant human interferon-gamma (rhIFN-γ) were analysed. Results: Finally, 12 high probable signal peptides, including OmpC, PhoE, AnsB, and OmpA, were theoretically detected with ideal solubility probabilities and almost balanced physicochemical properties; hopes to be helpful in future experimental studies for the secretion of rhIFN-γ. Conclusion: The experimental analysis is required to validate the in silico results and focus on in-lab affecting factors such as cultivation methods and conditions.

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.

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae.

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A polyethylene glycol (PEG)-mediated protoplast transformation method is generally used for the introduction of heterologous genes into A. oryzae. The conventional method typically requires three weeks for the screening of favorable transformants. Here, a new technique, the direct liquid-culture (DLC) screening method, is introduced which reduces the screening time to six days in a 200 mL flask format or to 10 days in a 24 well microplate format. The DLC screening method ensures the acquisition of positive transformants and evaluation of the secretory production of heterologous proteins in a single step, unlike the conventional screening method where two separate steps are required for the same. The protocol for PEG-mediated protoplast transformation of A. oryzae is described, which consists of five steps: preparation of fresh spore suspension, preculture, preparation of protoplasts, introduction of DNA, and DLC screening. For successful results in DLC screening, it is critical to use a nutrient-rich medium with optimized osmotic pressure. The protocol should further popularize the use of A. oryzae as a host of choice in the industrial production of proteins.

A secretion biosensor for monitoring Sec-dependent protein export in Corynebacterium glutamicum

BackgroundIn recent years, the industrial workhorse Corynebacterium glutamicum has gained increasing interest as a host organism for the secretory production of heterologous proteins. Generally, the yield of a target protein in the culture supernatant depends on a multitude of interdependent biological and bioprocess parameters which have to be optimized. So far, the monitoring of such optimization processes depends on the availability of a direct assay for the respective target protein that can be handled also in high throughput approaches. Since simple assays, such as standard enzymatic activity assays, are not always at hand, the availability of a general protein secretion biosensor is highly desirable.ResultsHigh level secretion of proteins via the Sec protein export pathway leads to secretion stress, a phenomenon that is thought to be caused by the accumulation of incompletely or misfolded proteins at the membrane-cell envelope interface. We have analyzed the transcriptional responses of C. glutamicum to the secretory production of two different heterologous proteins and found that, in both cases, the expression of the gene encoding a homologue of the extracytosolic HtrA protease was highly upregulated. Based on this finding, a C. glutamicum Sec secretion biosensor strain was constructed in which the htrA gene on the chromosome was replaced by the eyfp gene. The fluorescence of the resulting reporter strain responded to the secretion of different heterologous proteins (cutinase from Fusarium solani pisi and alkaline phosphatase PhoA from Escherichia coli) in a dose-dependent manner. In addition, three differently efficient signal peptides for the secretory production of the cutinase could be differentiated by the biosensor signal. Furthermore, we have shown that an efficient signal peptide can be separated from a poor signal peptide by using the biosensor signal of the respective cells in fluorescence activated cell sorting experiments.ConclusionsWe have succeeded in the construction of a C. glutamicum biosensor strain that allows for the monitoring of Sec-dependent secretion of heterologous proteins in a dose-dependent manner, independent of a direct assay for the desired target protein.

The effects of overexpression of cytoplasmic chaperones on secretory production of hirudin-PA in E. coli

The secretory production of heterologous proteins in E. coli has revolutionized biotechnology. Efficient periplasmic production of foreign proteins in E. coli often requires a signal peptide to direct proteins to the periplasm. However, the presence of attached signal peptide does not guarantee periplasmic expression of target proteins. Overproduction of auxiliary proteins, such as chaperones can be a useful approach to enhance protein export. In the current study, three chaperone plasmid sets, including GroEL–GroES (GroELS), Dnak–Dnaj–GrpE (DnaKJE), and trigger factor (TF), were coexpressed in E. coli BL21 (DE3) in a pairwise manner with two pET22-b vectors carrying the recombinant hirudin-PA (Hir) gene and different signal sequences alkaline phosphatase (PhoA) and l-asparaginase II (l-ASP). Overexpression of cytoplasmic combinations of molecular chaperones containing GroELS and DnaKJE with PhoAHir increased the secretory production of PhoAHir by 2.6fold (p < 0.05) and 3.5fold (p < 0.01) compared with their controls, respectively. By contrast, secretory production of PhoAHir significantly reduced in the presence of overexpressed TF (p = 0.02). Further, periplasmic expression of l-ASP was significantly increased only in the presence of DnaKJE (p = 0.04). These findings suggest that using molecular chaperones can be helpful for improving periplasmic expression of Hir. However, tagged signal peptides may affect the physicochemical properties and secondary and tertiary structures of mature Hir, which may alter their interactions with chaperones. Hence, using overexpressed chaperones has various effects on secretory production of PhoAHir and l-ASPHir.

Development of a Novel Gene Expression System for Secretory Production of Heterologous Proteins via the General Secretory (Sec) Pathway in Corynebacterium Glutamicum.

BackgroundCorynebacterium glutamicum (C. glutamicum) is a potential host for the secretory production of the heterologous proteins. However, to this date few secretion-type gene expression systems in C. glutamicum have been developed, which limit applications of C. glutamicum in a secretory production of the heterologous proteins.ObjectivesIn this study, a novel and efficient general secretory (Sec) pathway-dependent type gene expression system for the production of heterologous proteins was developed in C. glutamicum.Materials and MethodsThe synthesized cloning/expression cassette C was assembled into the basic E. coli-C. glutamicum shuttle vector pAU2, generating the Sec-dependent type gene expression vector pAU5. Subsequently, the applicability of the C. glutamicum/pAU5 system was tested using the α-amylase AmyE from Bacillus subtilis as a reporter protein.ResultsThe vector pAU5 was successfully constructed. The SDS-PAGE experiment showed the AmyE protein band could be observed in the original culture supernatant of the 14067/pAU5-amyE. The Western blotting experiment showed that the AmyE polypeptide could be detected in the culture supernatant of the 14067/pAU5-amyE, not in the cell lysate of 14067/pAU5-amyE. The α-amylase specific activity of the culture supernatant of 14067/pAU5-amyE was 103.24±7.14 U.mg-1 protein, while no α-amylase activity was detected in the cell homogenate supernatant of 14067/pAU5-amyE. These results demonstrate that the recombinant AmyE was efficiently expressed and completely secreted into the extracellular environmentin an active form in C. glutamicum/pAU5 system.ConclusionsA novel efficient Sec-dependent type gene expression vector pAU5 was constructed in the C. glutamicum. The vector pAU5 employs the strong promoter tac-M for controlling a constitutive transcription of the target gene, the consensus ribosome binding site (RBS) sequence of C. glutamicum to ensure protein translation, and the efficient Sec-type cgR_2070 signal sequence to mediate protein secretion in the C. glutamicum. The C. glutamicum/pAU5 system is an efficient expression system for the secretory production of the heterologous proteins.

Enhanced cell-surface display and secretory production of cellulolytic enzymes with Saccharomyces cerevisiae Sed1 signal peptide.

Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived from S. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae α-mating pheromone (MFα1SP) were constructed for cell-surface display of Aspergillus aculeatus β-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MFα1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MFα1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. Biotechnol. Bioeng. 2016;113: 2358-2366. © 2016 Wiley Periodicals, Inc.

Construction of a novel twin-arginine translocation (Tat)-dependent type expression vector for secretory production of heterologous proteins in Corynebacterium glutamicum

Corynebacterium glutamicum is recognized as a favorable host for the secretory production of heterologous proteins. However, there are few secretion-type expression vectors available for protein production in C. glutamicum. In this study, we constructed a shuttle expression vector pAU3, which harbors the strong promoter tac-M for constitutive gene transcription, the consensus RBS sequence for protein translation, and the strong cgR_0949 signal sequence for protein secretion via the Tat pathway in C. glutamicum. The applicability of pAU3 was confirmed by the highly efficient expression and secretion of the CAT protein in C. glutamicum. The vector pAU3 is highly useful for secretory production of heterologous proteins in C. glutamicum.

Signal peptides from filamentous fungi efficiently mediate the secretion of recombinant proteins in Kluyveromyces lactis

Kluyveromyces lactis is an established host for the secretory production of heterologous proteins. The secretion of enhanced green fluorescent protein (EGFP) in K. lactis mediated by nine secretion signals originating from different filamentous fungi – Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus awamori and Trichoderma reesei were investigated. EGFP was fused to the carboxyl terminus of signals and was expressed under the control of the lac4 promoter. In all the cases, EGFP was secreted into the extracellular fluid. SDS-polyacrylamide gel electrophoresis, western blot analysis of the culture supernatant, and fluorescence measurements confirmed the efficient secretion of EGFP mediated by the novel secretion sequences. In addition, we confirmed that codon optimization and addition of kex2 protease cleavage site in filamentous fungal signal peptide elevated the expression level of recombinant proteins. Efficiency of fungal signal peptide in secretion of other heterologous proteins was demonstrated through secretion of recombinant human interferon β.

In Silico Evaluation of Different Signal Peptides for the Secretory Production of Human Growth Hormone in E. coli

Various advantages of protein secretion have prompted scientists to search for secretory production of heterologous proteins. Signal peptides are one of the most important factors for prosperous secretion of the recombinant proteins. The aim of this study was to evaluate 23 different signal peptides and theoretically determine suitable ones for secretory production of the human growth hormone (hGH) in the E. coli host. The signal peptide sequences and the precise location of their cleavage sites were predicted using SignalP 4.0 server. Accordingly, six of the signal peptides, including hGH, major outer membrane lipoprotein, protease VII, protein TolB, periplasmic protein TorT and beta-lactamase TEM were excluded from the further study, due to their inappropriate cleavage scores. Different physico-chemical properties, which are essential for selecting a proper signal peptide, were evaluated using ProtParam and Solpro as the most accurate and reliable servers. Computational analysis of the above-mentioned factors, indicates that outer membrane protein C, fimbrial chaperone SfmC, outer membrane protein F and disulfide interchange protein DsbA can theoretically be suitable signal peptides for hGH secretion.

Improving the secretory production of the heterologous protein in Pichia pastoris by focusing on protein folding.

Pichia pastoris has currently been developed as an effective host system for the expression of heterologous genes owing to its potential use for the production of soluble and high-yield proteins. However, the secretory production of the different heterologous proteins in P. pastoris varies widely. Some factors restrict the effective secretory production of heterologous proteins in P. pastoris, among which the folding and processing of proteins is a major one. Besides optimizing the fermentative process, current strategies focus on investigating protein folding process. Thus, this paper is the first time to review the improvement of the secretory production of the heterologous protein in P. pastoris by focusing on its folding process.

Development of a secretion system for the production of heterologous proteins in Corynebacterium glutamicum using the Porin B signal peptide

Corynebacterium glutamicum is one of the useful hosts for the secretory production of heterologous proteins because of intrinsic attributes such as the presence of few endogenous proteins and proteases in culture medium. Here, we report the development of a new secretory system for the production of heterologous proteins by using the porin B (PorB) signal peptide in C. glutamicum. We examined two different endoxylanases and an antibody fragment (scFv) as model proteins for secretory production. In the flask cultivations, all the examined proteins were successfully produced as active forms into the culture medium with high efficiency. For the high-level production of endoxylanase, fed-batch cultivation was also performed in a lab-scale (5L) bioreactor, and the endoxylanases were efficiently secreted in the culture medium at levels as high as 615mg/L. From the culture supernatant, the secreted endoxylanases could be purified with high purity via one-step affinity column chromatography.

Authentic human basic fibroblast growth factor produced by secretion in Bacillus subtilis

Bacillus subtilis is generally accepted as an inborn host candidate employed for secretory production of heterologous proteins. However, this ideal host system has never been employed for commercial production of medically useful proteins. In this communication, we report for the first time the employment of an engineered B. subtilis system, in conjunction with a facile cell-wall destabilization protocol, to successfully obtain an alluring yield of 40 mg l(-1) of secreted human basic fibroblast growth factor (hbFGF) in the culture supernatant. The product was not only shown to exhibit potent bioactivity but also revealed to possess a protein sequence identical to that of mature native hbFGF (Mat-hbFGF). Our findings may pave way for the development of a cost-effective process for producing Mat-hbFGF, which is currently sold at an unusually expensive price of over US $1 million g(-1), for medical and skin care applications.

The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses

BackgroundThe adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications.ResultsThe impact of a step-wise restricted supply of phosphate on the physiological state of S. cerevisiae cells producing human Insulin was studied. The focus was to determine the changes within the global gene expression of cells being cultured to an industrially relevant high cell density of 33 g/l cell dry weight and under six distinct phosphate concentrations, ranging from 33 mM (unlimited) to 2.6 mM (limited). An increased flux through the secretory pathway, being induced by the PHO circuit during low Pi supplementation, proved to enhance the secretory production of the heterologous protein. The re-distribution of the carbon flux from biomass formation towards increased glycerol production under low phosphate led to increased transcript levels of the insulin gene, which was under the regulation of the TPI1 promoter.ConclusionsOur study underlines the dynamic character of adaptive responses of cells towards a change in their nutrient access. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. Our work emphasizes the importance of reduced phosphate supply for improved secretory production of heterologous proteins.

Functional Implementation of the Posttranslational SecB-SecA Protein-Targeting Pathway in Bacillus subtilis

Bacillus subtilis and its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria, B. subtilis does not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway into B. subtilis by coexpressing SecB from Escherichia coli together with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of the B. subtilis SecA were replaced by the corresponding part of SecA from E. coli. In vitro pulldown experiments showed that, in contrast to B. subtilis SecA, the hybrid SecA protein gained the ability to efficiently bind to E. coli SecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins by B. subtilis was significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.

Amino acid uptake profiling of wild type and recombinant Streptomyces lividans TK24 batch fermentations

Streptomyces lividans is considered an interesting host for the secretory production of heterologous proteins. To obtain a good secretion yield of heterologous proteins, the availability of suitable nitrogen sources in the medium is required. Often, undefined mixtures of amino acids are used to improve protein yields. However, the understanding of amino acid utilization as well as their contribution to the heterologous protein synthesis is poor. In this paper, amino acid utilization by wild type and recombinant S. lividans TK24 growing on a minimal medium supplemented with casamino acids is profiled by intensive analysis of the exometabolome (metabolic footprint) as a function of time. Dynamics of biomass, substrates, by-products and heterologous protein are characterized, analyzed and compared. As an exemplary protein mouse Tumor Necrosis Factor Alpha (mTNF-α) is considered. Results unveil preferential glutamate and aspartate assimilation, together with glucose and ammonium, but the associated high biomass growth rate is unfavorable for protein production. Excretion of organic acids as well as alanine is observed. Pyruvate and alanine overflow point at an imbalance between carbon and nitrogen catabolism and biosynthetic fluxes. Lactate secretion is probably related to clump formation. Heterologous protein production induces a slowdown in growth, denser clump formation and a shift in metabolism, as reflected in the altered substrate requirements and overflow pattern. Besides glutamate and aspartate, most amino acids are catabolized, however, their exact contribution in heterologous protein production could not be seized from macroscopic quantities. The metabolic footprints presented in this paper provide a first insight into the impact and relevance of amino acids on biomass growth and protein production. Type and availability of substrates together with biomass growth rate and morphology affect the protein secretion efficiency and should be optimally controlled, e.g., by appropriate medium formulation and substrate dosing. Overflow metabolism as well as high biomass growth rates must be avoided because they reduce protein yields. Further investigation of the intracellular metabolic fluxes should be conducted to fully unravel and identify ways to relieve the metabolic burden of plasmid maintenance and heterologous protein production and to prevent overflow.

New Approach to Achieve High-Level Secretory Expression of Heterologous Proteins by Using Tat Signal Peptide

The twin-arginine translocation (Tat) pathway is an attractive route for secretory production of heterologous proteins in E. coli. In this study, we investigated the potential use of Tat signal peptide from S. coelicolor to improve secretory expression. The results showed that Tat signal peptide (ssDagA) could effectively secrete active Green fluorescent protein (GFP) to periplasm. When the rare codons of signal sequence were optimized, the expression and secretion yield of GFP improved by about 2-3 folds as detected qualitatively by western blotting and fluorescent analysis. The increase of translation rate could be explained by the unstability of mRNA secondary structure. In summary, our strategy could provide a new approach for high-level secretory expression of heterologous proteins in E. coli.

Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria

In contrast to the general protein secretion (Sec) system, the twin-arginine translocation (Tat) export pathway allows the translocation of proteins across the bacterial plasma membrane in a fully folded conformation. Due to this feature, the Tat pathway provides an attractive alternative to the secretory production of heterologous proteins via the Sec system. In this study, the potential for Tat-dependent heterologous protein secretion was compared in the three Gram-positive bacteria Staphylococcus carnosus, Bacillus subtilis, and Corynebacterium glutamicum using green fluorescent protein (GFP) as a model protein. In all three microorganisms, fusion of a Tat signal peptide to GFP resulted in its Tat-dependent translocation across the corresponding cytoplasmic membranes. However, striking differences with respect to the final localization and folding status of the exported GFP were observed. In S. carnosus, GFP was trapped entirely in the cell wall and not released into the supernatant. In B. subtilis, GFP was secreted into the supernatant, however, in an inactive form. In contrast, C. glutamicum effectively secreted active GFP. Our results clearly demonstrate that a comparative evaluation of different Gram-positive host microorganisms is a crucial step on the way to an efficient Tat-mediated secretory production process for a desired heterologous target protein.

N-Glycosylation of secretion enhancer peptide as influencing factor for the secretion of target proteins from Saccharomyces cerevisiae

hIL-1β-derived polypeptide, when fused to the N-terminal end of target proteins, exerts a potent secretion enhancer function in Saccharomyces cerevisiae. We investigated the effect of N-glycosylation of the secretion enhancer peptide on the secretion of target proteins. The N-terminal 24 amino acids (Ser5-Ala28) of human interleukin 1β (hIL-1β) and interleukin 1 receptor antagonist (IL-1ra) were used as secretion enhancer for synthesizing recombinant human granulocyte-colony stimulating factor (rhG-CSF) from S. cerevisiae. The mutation of potential N-glycosylation site, by substituting Gln for either Asn7 of N-terminal 24 amino acids of hIL-1β (Asn7Gln) or Asn84 of IL-1ra (Asn84Gln), resulted in a dramatic reduction of rhG-CSF secretion efficiency. In contrast, the mutant containing an additional N-glycosylation site on the N-terminal 24 amino acids of hIL-1β (Gln15Asn) secreted twice as much rhG-CSF into culture media as wild type hIL-1β. These results show that N-glycosylation of the secretion enhancer peptide plays an important role in increasing the secretion efficiency of the downstream target proteins. The results also suggest that judicious choice of enhancer peptide and the control of its glycosylation could be of general utility for secretory production of heterologous proteins from S. cerevisiae.