pelB Signal Peptide Research Articles

Protein Expression and Purification of Romiplostim and Analysis of Its Secretory Production Using an In Silico Investigated Signal Peptide in E. Coli.

Romiplostim is a thrombopoietin receptor agonist approved for the treatment of immune thrombocytopenia. It is produced by recombinant DNA technology in Escherichia coli. Many researchers have studied the periplasmic or extracellular production of recombinant proteins in E. coli by using signal peptide sequences due to its advantages compared to intracellular production. In this study, the effect of the pelB signal peptide on Romiplostim production was analyzed. The nucleotide sequence of Romiplostim was codon optimized for expression in E. coli BL21. For analysis of the effect of the pelB signal peptide, pET-22b (+) and pET-15b plasmids were used. The probability of signal peptide cleavage and pathway was predicted by using the SignalP 5.0 program, and expression, purification, and biological activity of the recombinant protein were analyzed. In-silico analysis predicted the correct cleavage of the pelB signal peptide. However, the experimental results showed intracellular accumulation of the protein in fusion with this signal peptide without any detectable protein band in periplasmic or extracellular spaces. The in-vivo experiment of purified protein without signal peptide exhibited a significant increment in platelets compared to the control group. Romiplostim was expressed in E. coli with and without signal peptide. The latest one showed suitable in-vivo bioactivity. Despite the results of in-silico prediction, the pelB signal peptide could not transport the protein into the periplasm or extracellular environment in the experimental condition. Trying different signal peptides and more in-silico analysis might be helpful for the efficient secretion of the Romiplostim protein.

Active secretion of a thermostable transglutaminase variant in Escherichia coli

BackgroundStreptomyces mobaraenesis transglutaminase (smTG) is widely used to generate protein crosslinking or attachment of small molecules. However, the low thermostability is a main obstacle for smTG application. In addition, it is still hard to achieve the secretory expression of active smTG in E. coli, which benefits the enzyme evolution. In this study, a combined strategy was conducted to improve the thermostability and secretory expression of active smTG in E. coli.ResultsFirst, the thermostable S. mobaraenesis transglutaminase variant S2P-S23V-Y24N-S199A-K294L (TGm1) was intracellularly expressed in pro-enzyme form in E. coli. Fusing the pro-region of Streptomyces hygroscopicus transglutaminase (proH) and TrxA achieved a 9.78 U/mL of intracellular smTG activity, 1.37-fold higher than the TGm1 fused with its native pro-region. After in vitro activation by dispase, the TGm1 with proH yielded FRAPD-TGm1, exhibiting 0.95 ℃ and 94.25% increases in melting temperature and half-life at 60 ℃ compared to FRAP-TGm1 derived from the expression using its native pro-region, respectively. Second, the TGm1 with proH was co-expressed with transglutaminase activating protease and chaperones (DnaK, DnaJ, and GrpE) in E. coli, achieving 9.51 U/mL of intracellular FRAPD-TGm1 without in vitro activation. Third, the pelB signal peptide was used to mediate the secretory expression of active TGm in E. coli, yielding 0.54 U/mL of the extracellular FRAPD-TGm1. A script was developed to shuffle the codon of pelB and calculate the corresponding mRNA folding energy. A 1.8-fold increase in the extracellular expression of FRAPD-TGm1 was achieved by the Top-9 pelB sequence derived from the coding sequences with the lowest mRNA folding energy. Last, deleting the gene of Braun’s lipoprotein further increased the extracellular yield of FRAPD-TGm1 by 31.2%, reached 1.99 U/mL.ConclusionsThe stabilized FRAPD-smTG here could benefit the enzyme application in food and non-food sectors, while the E. coli system that enables secretory expression of active smTG will facilitate the directed evolution for further improved catalytic properties. The combined strategy (N-terminal modification, co-expression with chaperones, mRNA folding energy optimization of signal peptide, and lipoprotein deletion) may also improve the secretory expression of other functional proteins in E. coli.

Developing of specific monoclonal recombinant antibody fused to alkaline phosphatase (AP) for one-step detection of fig mosaic virus.

Present study was performed to develop a fusion recombinant monoclonal antibody for one-step and accurate detection of FMV with a specific single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) named as scFv(FMV-NP)-AP. The gene encoding-specific scFv recombinant antibody binding to nucleocapsid protein of Fig Mosaic Virus (FMV-NP) was fused to upstream of AP gene and integrated in pET26b bacterial expression vector. As vector contain pelB signal peptide, the expressed protein is secreted into periplasmic compartment. Recombinant fusion protein was produced in transformed E. coli following induction by IPTG. Extraction and purification of fusion protein was performed under denatured condition. The results of SDS-PAGE and western blot analysis indicated high integrity and purity with a single band protein with expected size of 72kDa. The total yield of purified scFv(FMV-NP)-AP fusion protein estimated around 0.5-1mg/l cultured medium. Subsequent colorimetric analysis confirmed presence of alkaline phosphatase activity in prepared scFv-AP fusion protein. Specificity of generated recombinant fusion antibody against cognate antigen and the native virus presented in infected plant extracts was assessed by ELISA, western blot and dot blot assays. Results revealed that scFv(FMV-NP)-AP is able to detect the presence of FMV in infected fig plants. The novel approach, implementing specific recombinant fusion antibody developed in this research, leads to one-step detection of FMV in plants by avoiding the use of chemical enzyme-labeled secondary antibodies.

Simultaneous production of 2'-fucosyllactose and difucosyllactose by engineered Escherichia coli with high secretion efficiency.

Difucosyllactose (Di-FL) has strong antimicrobial activity against various pathogens, including group B Streptococcus, identified as the leading cause of neonatal sepsis. In this study, we sought to develop Escherichia coli as a microbial cell factory for efficiently producing Di-FL as well as 2'-fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, by utilizing the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway. The biosynthetic pathway for producing fucosylated oligosaccharides via the salvage pathway requires two enzymes, l-fucokinase/GDP-l-fucose phosphorylase (FKP) from Bacteroides fragilis and α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori. To decrease the intracellular accumulation of 2'-FL while increasing substrate accessibility to FKP and FucT2, we evaluated whether extracellular secretion of FKP and FucT2 would enhance the production of fucosylated oligosaccharides. Among various engineered strains constructed in this study, the ΔLFAR-YA/FF+P-PLA2 strain expressing phospholipase A2 (PLA2 ) from Streptomyces violaceoruber, whose native signal peptide was replaced with the PelB signal peptide (P-PLA2 ), could secrete both FKP and FucT2 into the culture medium. Notably, it was observed that FKP and FucT2 present in the extracellular fraction could catalyze the synthesis of Di-FL from lactose and fucose. As a result, a batch fermentation with the ΔLFAR-YA/FF+P-PLA2 strain resulted in the production of 1.22± 0.01gL-1 Di-FL and 0.47± 0.01gL-1 2'-FL, whereas the control strain could only produce 0.65± 0.01gL-1 2'-FL. This study highlights the benefits of extracellular secretion of enzymes to improve biotransformation efficiency, as the transport of substrates and/or products across the cell membrane is limited.

Functional expression and extracellular secretion of Clostridium thermocellum Cel48S cellulase in Escherichia coli via the signal recognition particle-dependent translocation pathway

As the only glycoside hydrolase family 48 member in Clostridium thermocellum, the exoglucanase Cel48S plays a crucial role in the extremely high activity of the cellulosome against crystalline cellulose. Although the importance of Cel48S in the hydrolysis of crystalline cellulose has been widely accepted, an efficient production system has not yet been established because Cel48S is usually expressed in Escherichia coli within inactive inclusion bodies. For unstable proteins like Cel48S, translocation across the inner membrane can be more advantageous than cytoplasmic production due to the presence of folding modulators in the periplasm and the absence of cytoplasmic proteases. In this study, we evaluated whether the production of Cel48S in the periplasmic space of E. coli could enhance its functional expression. To do so, we attached the PelB signal peptide, which mediates post-translational secretion, to the N-terminal end of Cel48S (P-Cel48S). The PelB signal peptide allowed catalytically active Cel48S to be successfully produced in the culture medium. In addition, we investigated the role of an alternative co-translational pathway on the extracellular production of Cel48S, finding that co-translational secretion yielded a specific activity of recombinant Cel48S of 135.1 ± 10.0 U/mg cell in the culture medium, which was 2.2 times higher than that associated with P-Cel48S expression. Therefore, we believe that our approach has potential applications for the cost-effective conversion of lignocellulosic biomass and the industrial production of other unstable proteins.

Enhancing extracellular production of recombinant proteins in Escherichia coli by co-expressing with a haloarchaeal protein containing a putative LolA-like domain.

Escherichia coli represents one of the most widely used hosts for recombinant protein production, but its limited capacity for producing extracellular proteins is often cited as a drawback. NJ7G_0991 is an extracellular protein of the haloarchaeon Natrinema sp. J7-2 and comprises a signal peptide, a putative LolA-like domain, and a C-terminal domain of unknown function. Here, we found that the full-length (0991) and the C-terminal domain-deletion variant (0991ΔC) of NJ7G_0991, but not its signal peptide-deletion variant (0991ΔS), were efficiently released into the culture supernatant of E. coli without extensive cell lysis as determined by β-galactosidase activity assay. After lysozyme treatment, E. coli cells producing 0991 or 0991ΔC, but not 0991ΔS, were converted from rod-shaped forms to spheres, suggesting that the secretion of 0991 or 0991ΔC into the periplasm leads to an increase of outer membrane permeability of E. coli. A pelB signal peptide was fused to the N-terminus of the LolA-like domain, and the resulting variant PelB-0991ΔC could be released into the culture supernatant of E. coli more efficiently than 0991ΔC. By using PelB-0991ΔC as a co-expression partner, the extracellular production level of a recombinant thermostable subtilase WF146 could be enhanced by up to 14-fold, and the extracellular concentration of an active site variant of WF146 (WF146-SA) reached up to 129 mg/l. To the best of our knowledge, this is the first report on archaeal protein-based co-expression system for extracellular production of recombinant proteins in E. coli. KEY POINTS: • The haloarchaeal protein NJ7G_0991 can be efficiently released into the culture supernatant of E. coli. • The recombinant NJ7G_0991 increases the outer membrane permeability of E. coli. • The LolA-like domain of NJ7G_0991 can be used as a co-expression partner to improve extracellular production of recombinant proteins in E. coli.

Perturbation of the peptidoglycan network and utilization of the signal recognition particle-dependent pathway enhances the extracellular production of a truncational mutant of CelA in Escherichia coli.

Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production.

Activity and Effectiveness of Recombinant hEGF Excreted by Escherichia coli BL21 on Wound Healing in Induced Diabetic Mice.

ContextHuman epidermal growth factor (hEGF) has biological activities and can be used in medicines and cosmetics. A high level of effectiveness of hEGF can be obtained when three disulfide bonds fold perfectly. Extracellular secretion from E. coli BL21 using the PelB signal peptide is a new way to obtain hEGF with a structure that folds appropriately.ObjectThis study aimed to determine the activity and effectiveness of recombinant hEGF excreted by E. coli BL21 on wound healing in induced diabetic mice.MethodsCell proliferation and migration tests were performed on NIH3T3 cells, followed by wound healing tests in induced diabetic mice, along with histological and endotoxin test at various hEGF concentrations (25, 50, and 75 µg/mL).ResultsBased on the results, hEGF at a level of 50 μg/mL showed optimal proliferation and migration activities. Wound healing in induced diabetic mice showed faster-wound closure within 12 days at hEGF 50 and 75 µg/mL with a percentage wound closure of 95% and 98.5%, respectively, which was significant versus control. In the histology test, the number of fibroblasts showed an increase and was significant at hEGF 75 µg/mL compared to the control group. The single test vial (STV) showed that hEGF solution was free of endotoxin.ConclusionRecombinant hEGF produced by extracellular secretion using E. coli BL21 has optimal diabetic wound healing activity through increased fibroblast proliferation.

N-Terminal Fused Signal Peptide Prompted Extracellular Production of a Bacillus-Derived Alkaline and Thermo Stable Xylanase in E. coli Through Cell Autolysis.

Xylanases are extensively used as industrial enzymes for its ability of hydrolyzing xylan to oligosaccharides. Here, XynHB, a thermo and alkaline stable xylanase derived from Bacillus pumilus HBP8, was extracellularly produced in E. coli cells through N-terminal-fused signal peptides. We found that the matured XynHB itself could be auto-secreted out of E. coli BL21(DE3) cells at a very low level, and two Sec-pathway signal peptides, PelB and OmpA, and one dual Sec-Tat-pathway signal peptide, FhuD, could effectively prompt its extracellular production up to 12-fold. Our results showed that PelB signal peptide led to the highest extracellular production of XynHB for approximately 54.1μg/mL, and FhuD-fused XynHB possessed the highest specific activity of 1746.0U/mg at 70°C. Meanwhile, our studies also indicated that PelB- and FhuD-fused XynHB might disrupt E. coli cells' periplasm during their secretion process, thus causing cell lysis to facilitate their extracellular production. Moreover, further characterization revealed that the extracellular production of XynHB was not affected by the outer membrane permeability of E. coli cells. Our studies provided an advantageous strategy for the extracellular production of xylanase in E. coli, which may also be used for E. coli autolysis in the future.

Real-time monitoring of rhamnose induction effect on the expression of mpt64 gene fused with pelB signal peptide in Escherichia coli BL21 (DE3).

In this research, Escherichia coli BL21 (DE3) harboring an expression vector constructed with a rhamnose-inducible promoter and a pelB signal peptide was used as a host cell to produce MPT64 protein. The objective of this research was to figure out the optimum time of mpt64 gene expression through real-time monitoring of MPT64 protein production and distribution in host compartments. The mpt64 expression was regulated by the rhamnose presence at a concentration of 4 mM. The real-time isolated protein was monitored using polyacrylamide gel electrophoresis in denaturation condition. Based on real-time monitoring, the MPT64 protein (24 kDa) in the cytoplasm was optimum detected at 24 h after induction. For periplasmic fraction, the protein was detected at 4 h after induction but thinning at 15 h after induction. At 16 h after induction, the MPT64 protein band was found in the medium with increasing concentrations until 24 h. Thus, it can be concluded that the mpt64 gene expression was regulated in the presence of rhamnose as an inducer, and the proteins were shown to be translocated throughout the host cell compartment with different levels of protein accumulation at different times, according to the role of pelB as a signal peptide.

Functional Autodisplay of Phenolic Acid Decarboxylase using a GDSL Autotransporter on Escherichia coli for Efficient Catalysis of 4-Hydroxycinnamic Acids to Vinylphenol Derivatives

Bioproduction of vinylphenol derivatives, such as 4-vinylguaiacol (4-VG) and 4-vinylphenol (4-VP), from 4-hydroxycinnamic acids, such as ferulic acid (FA) and p-coumaric acid (pCA), employing whole cells expressing phenolic acid decarboxylases (PAD) as a biocatalyst has attracted much attention in recent years. However, the accumulation of 4-VG or 4-VP in the cell may cause high cytotoxicity to Escherichia coli (E. coli) and consequently cell death during the process. In this study, we firstly report the functional display of a phenolic acid decarboxylase (BLPAD) from Bacillus licheniformis using a GDSL autotransporter from Pseudomonas putida on the cell surface of E. coli. Expression and localization of BLPAD on E. coli were verified by SDS-PAGE and protease accessibility. The PelB signal peptide is more effective in guiding the translocation of BLPAD on the cell surface than the native signal peptide of GDSL, and the cell surface displaying BLPAD activity reached 19.72 U/OD600. The cell surface displaying BLPAD showed good reusability and retained 63% of residual activity after 7 cycles of repeated use. In contrast, the residual activity of the intracellular expressing cells was approximately 11% after 3 cycles of reuse. The molar bioconversion yields of 72.6% and 80.4% were achieved at the concentration of 300 mM of FA and pCA in a biphasic toluene/Na2HPO4–citric acid buffer system, respectively. Its good reusability and efficient catalysis suggested that the cell surface displaying BLPAD can be used as a whole-cell biocatalyst for efficient production of 4-VG and 4-VP.

Use of a design of experiments approach to optimise production of a recombinant antibody fragment in the periplasm of Escherichia coli: selection of signal peptide and optimal growth conditions

Production of recombinant proteins such as antibody fragments in the periplasm of the bacterium Escherichia coli has a number of advantages, including the ability to form disulphide bonds, aiding correct folding, and the relative ease of release and subsequent capture and purification. In this study, we employed two N-terminal signal peptides, PelB and DsbA, to direct a recombinant scFv antibody (single-chain variable fragment), 13R4, to the periplasm via the Sec and SRP pathways respectively. A design of experiments (DoE) approach was used to optimise process conditions (temperature, inducer concentration and induction point) influencing bacterial physiology and the productivity, solubility and location of scFv. The DoE study indicated that titre and subcellular location of the scFv depend on the temperature and inducer concentration employed, and also revealed the superiority of the PelB signal peptide over the DsbA signal peptide in terms of scFv solubility and cell physiology. Baffled shake flasks were subsequently used to optimise scFv production at higher biomass concentrations. Conditions that minimised stress (low temperature) were shown to be beneficial to production of periplasmic scFv. This study highlights the importance of signal peptide selection and process optimisation for the production of scFv antibodies, and demonstrates the utility of DoE for selection of optimal process parameters.

Enhanced Solubility of Anti-HER2 scFv Using Bacterial Pelb Leader Sequence

Single chain Fragment variable (scFv) is an antibody fragment consisting variable regions of heavy and light chains. scFvs enhance their penetrability into tissues while maintaining specific affinity and having low immunogenicity. Insoluble inclusion bodies are formed when scFvs are expressed in reducing bacterial cytoplasm. One strategy for obtaining functionally active scFv is to translocate the scFv into the oxidized environment of the periplasm where the possibility for disulfide bond formation is increased. This can be achieved by cloning the gene in a vector containing N-terminal pelB leader peptide that export foreign proteins to the periplasmic space. The aim of this study is to evaluate the influence of periplasmic localization using pelB leader peptide on the solubility of anti HER2-scFv. Herein, anti HER2-scFv gene was cloned between NcoI and XhoI sites of pET22-b (+) containing pelB leader peptide and in same sites of pET28-b (+) (without pelB). The expression in BL21 (DE3) was induced using IPTG and was analyzed using SDS-PAGE and Western blot experiment. Then, the solubility of anti HER2-scFvin BL21 (DE3) containing both pET22- and pET28-(anti HER2-scFv) was determined. The results of the present study demonstrated that anti HER2-scFv was expressed by both pET22-b (+) and pET28-b (+) vectors in BL21 (DE3). The proper expression of anti-HER2 scFv was confirmed by appearance of a  28 kDa band in Western blot analysis. The most anti HER2-scFv expression from BL21 containing pET28-(anti HER2-scFv) was achieved when it was induced by 0.25 mM IPTG at 37 C, 24 h post-induction. The ratio of soluble/insoluble anti HER2-scFv was significantly higher in BL21 containing pET22-(anti HER2-scFv) than in that containing pET28-(anti HER2-scFv). Totally, fusion of pelB signal sequence to anti HER2-scFv resulted in solubility enhancement. Therefore, production of functional anti HER2-scFv with proper disulfide bond can be achieved by directing the recombinant protein to periplasmic space using pelB signal peptide in pET22 (+) vector.

Optimization of Secreted Recombinant Human Epidermal Growth Factor Production Using Pectate Lyase B from Escherichia Coli BL21(DE3) by Central Composite Design and Its Production in High Cell Density Culture.

Context:Human Epidermal Growth Factor (hEGF) is a potential therapeutic protein that has been widely used as a healing agent for various chronic wounds. It induces the proliferation and metabolism of epithelial cells, regenerates skin cells, and validates skin elasticity. In the previous study, recombinant hEGF (rhEGF) had been successfully expressed extracellularly in Escherichia coli (E. coli) BL21 (DE3) using pectate lyase B (PelB) signal peptide. The previous study has shown that the medium concentration and the induction time influenced the production of rhEGF.Aims:Therefore, this study was conducted to optimize the induction time and medium concentration for rhEGF extracellular secretion then followed by scale-up production.Settings and Design:This experiment was carried out using E. coli BL21 (DE3) which contains pD881 plasmid that carries hEGF and PelB gene. Optimization design of induction time and medium concentration were obtained using Central Composite Design (CCD).Methods and Material:The method of study started by the rejuvenation of E. coli culture, extracellular secretion, and optimization in the flask scale then followed by scaled-up production with high-cell density culture in the fermenter.Statistical analysis used:The optimization was carried out using Response Surface Methodology (RSM) and multi regression analysis.Results:This work showed that the multiplication of 1.5-fold medium concentration with induction time 3h after the culture started gave the best result among another condition in this study. Additionally, the rhEGF production in the fermenter scale was identified by SDS-PAGE Tricine and quantified by ELISA, which showed 122.40 μg of the rhEGF per milliliter medium.Conclusions:In respect of the result, we conclude that the optimized condition of extracellular secretion was successfully obtained, and gives higher result before the previous study.

A two-stage glycine supplementation strategy enhances the extracellular expression of sortase A in Escherichia coli

Abstract Sortase A (SrtA) is a transpeptidase widely used in protein engineering. In this study, the enhancement of the extracellular expression of SrtA in Escherichia coli was investigated using a combined strategy based on the PelB signal peptide and chemical additives. First, glycine was identified to be the best additive for promoting the release of SrtA from the periplasm to the medium. Then, the effect of glycine concentration on cell growth and SrtA production was investigated, and a two-stage supplementation strategy was developed in order to control the impairment of cell growth and to achieve the maximum production of secretory SrtA. The results showed that when 0.5% glycine was added to the medium at the beginning of cell growth and 1% glycine was added at the end of the exponential phase, the extracellular yield of SrtA was 228.0 mg/L and the enzyme activity was 100.4 U/mL at the end of fermentation; these values were 5.3- and 8.6-fold higher, respectively, than those attained in the control culture without any additives. This result represents the highest yield of extracellular SrtA ever reported and demonstrates a promising process for the production of SrtA for large-scale industrial application.

Fitting replacement of signal peptide for highly efficient expression of three penicillin G acylases in E. coli.

High level expression of penicillin G acylase (PGA) in Escherichia coli is generally constricted by a complex maturation process and multiple limiting steps. In this study, three PGAs isolated from Providencia rettgeri (PrPGA), Alcaligenes faecalis (AfPGA), and Achromobacter xylosoxidans (AxPGA) were efficiently expressed in E. coli by replacing with applicable signal peptide. Different bottlenecks of the expression process were analyzed for PrPGA, AfPGA, and AxPGA. Subsequently, five efficient signal peptides, including OmpA, pelB, Lpp, PhoA, and MalE, were used to replace the original signal peptides of the PGAs. With respect to AfPGA and AxPGA, translocation was the primary limitation, and the use of pelB signal peptide effectively overcame this barrier. For PrPGA, which was almost not expressed in wild type, the translation initiation efficiency was optimized by replacing with MalE signal peptide. In addition, low temperature (20°C) slowed down the transcription and translation, thereby facilitating the posttranslational process and preventing the formation of inclusion bodies. Furthermore, combined induction with IPTG and arabinose not only enhanced the cell density but also remarkably improved the expression of PGAs. Final specific activities of the three PGAs reached 2100 (PrPGA), 9200 (AfPGA), and 1400 (AxPGA)U/L/OD600, respectively. This simple and robust strategy by fitting replacement of signal peptide might dramatically improve the expression of PGAs from various bacteria, which was significant in the production of many valuable β-lactam antibiotics.

Development of a generic \u03b2-lactamase screening system for improved signal peptides for periplasmic targeting of recombinant proteins in Escherichia coli

Targeting of recombinant proteins to the Escherichia coli periplasm is a desirable industrial processing tool to allow formation of disulphide bonds, aid folding and simplify recovery. Proteins are targeted across the inner membrane to the periplasm by an N-terminal signal peptide. The sequence of the signal peptide determines its functionality, but there is no method to predict signal peptide function for specific recombinant proteins, so multiple signal peptides must be screened for their ability to translocate each recombinant protein, limiting throughput. We present a screening system for optimising signal peptides for translocation of a single chain variable (scFv) antibody fragment employing TEM1 β-lactamase (Bla) as a C-terminal reporter of periplasmic localisation. The Pectobacterium carotovorum PelB signal peptide was selected as the starting point for a mutagenic screen. β-lactamase was fused to the C-terminal of scFv and β-lactamase activity was correlated against scFv translocation. Signal peptide libraries were generated and screened for β-lactamase activity, which correlated well to scFv::Bla production, although only some high activity clones had improved periplasmic translocation of scFv::Bla. Selected signal peptides were investigated in fed-batch fermentations for production and translocation of scFv::Bla and scFv without the Bla fusion. Improved signal peptides increased periplasmic scFv activity by ~40%.

Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide.

Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.

Extracellular expression of alkali tolerant xylanase from Bacillus subtilis Lucky9 in E. coli and application for xylooligosaccharides production from agro-industrial waste

An alkali tolerant xylanase gene from Bacillus subtilis Lucky9 was cloned and extracellular expressed in E. coli BL21. Xylanase amino acid sequence showed 99% identity with xylanase sequence from Bacillus subtilis 168, and was belonged to glycoside hydrolase family 11. The recombinant E. coli (pET-pelB-xynLC9) containing pelB signal peptide produced extracellular xylanase of 436.5U/mL for 8h, which was used arabinose as extra carbon source and inducer for enhancing extracellular production. The extracellular xylanase was determined by SDS–PAGE with a relative molecular mass of 21kDa. The recombinant xylanase was optimally activity at pH 6.5 and 60°C. The xylanase exhibited 80% residual activity over a broad pH range of 6.0–9.0 for 24h. Thermostability studies showed that xylanase retained 60% residual activity after 2h at 60°C. The main end-products of hydrolysis of beech-wood xylan and corncob by the extracellular xylanase were xylobiose and xylotriose. This extracellular xylanase without purification is a suitable candidate for application in the industrial production of xylooligosaccharides from agro-industrial waste for use as prebiotics.

Expression and production optimization of sucrose isomerase from Pantoea dispersa in Escherichia coli

To improve the yield of sucrose isomerase from Pantoea dispersa UQ68J, we studied the effect of different signal peptides and fermentation conditions on sucrose isomerase expression in Escherichia coli. The gene of sucrose isomerase was optimized and expressed in E. coli BL21 (DE3) with native signal peptide which was named as ORI strain. The total and extracellular enzyme activity was 85 and 65 U/mL in the flask, respectively. The mature protein, which started from the 22th amino acid, was connected with the PelB and OmpA signal peptide to construct P22 and O22 strain, respectively. The total activity of P22 reached 138 U/mL, which was 1.6 times of ORI strain. The total activity of O22 strain was similar to that of ORI strain. Induced by 3.0 g/L lactose, the total activity of P22 strain increased to 168 U/mL. In 3 L fermentor, the effects of glycine concentration and induction time were studied. Induction when the DCW reached 18 g/L (OD₆₀₀=30), with 0.5% glycine, the extracellular enzyme activity reached 1 981 U/mL, and the total enzyme activity reached 2 640 U/mL, which is the highest activity of sucrose isomerase that was expressed in recombinant E. coli.