Syntheses of recombinant yellowtail and flounder growth hormones in Escherichia coli.

For syntheses of recombinant yellowtail and flounder growth hormones (r-yGH and r-fGH) in E. coli, expression plasmids were constructed. The expression level of r-yGH and r-fGH in the host cells were very high, reaching 15 and 8% of the total protein, respectively. These product proteins were accumulated in inclusion bodies in the cells. The recombinant hormones were isolated from the pellets ina glutathione reduction/oxidation buffer. The refolded hormones were further purified by DEAE-Toyopearl 650M chromatography to homogeneity. The purified r-yGH and r-fGH were composed of 188 and 174 amino acid residues, respectively, having amino-terminal sequences starting with methionine. The recombinant hormones had potent growth-promoting activities on juvenile rainbow trout Salmo gairdneri in a dose-dependent manner.     

Recombinant flounder growth hormone from Escherichia coli: overexpression, efficient recovery, and growth-promoting effect on juvenile flounder by oral administration

An efficient production method for recombinant flounder growth hormone (r-fGH) from Escherichia coli was developed and the biological activity of purified r-fGH was examined using juvenile flounder. The use of bicistronic construction in the expression plasmid resulted in the production of over 40% of the E. coli cellular protein as r-fGH. The r-fGH was recovered from cell lysates following inclusion body washing, solubilization and refolding in sodium dodecylsulfate (SDS) solution, and removal of contaminated proteins with secondary butanol treatment. The SDS content in purified r-fGH solution was adjusted to appropriate levels by diafiltration. More than 47% of the r-fGH was recovered from the E. coli cell lysates and the purity of recovered r-fGH was 98%. The oral administration of purified r-fGH to juvenile flounder, once a week for 4 weeks at a dosage of 40 μg r-fGH g⁻¹ fish body weight, resulted in significant increases both in weight and length. These results of overexpression, simple purification with high recovery yield and purity, and good growth-promoting activity of the r-fGH suggest that the production scheme described in this study is useful for the potential application of r-fGH in fish farming.     

Expression and Purification of Recombinant lysostaphin in Escherichia coli

Summary A 1.5 kb plasmid-encoded lysostaphin gene fragment of Staphylococcus staphylolyticus was amplified by polymerase chain reaction (PCR) and cloned in Escherichia coli by using plasmid pET29b(+) as an expression vector. By optimizing culture conditions, the activities of lysostaphin were expressed as 66 %, 30 %, and 4 % in extracellular, intracellular, and periplasmic fractions of recombinant E. coli, respectively. The enzyme was purified to homogeneity by using a simple one-step fractionation on bacterial cells of lysostaphin-resistant Staphylococcus aureus mutant. The recombinant enzyme had an Mr of approximate 27 kDa, and its bacteriolytic activity was indistinguishable to the authentic lysostaphin purified from Staphylococcus staphylolyticus.     

Expression of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> endo-β-glucanase II in silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis> L. by using BmNPV/Bac-to-Bac expression system and its bioactivity assay

The silkworm, Bombyx mori, was used to produce recombinant endo-β-glucanase II (rEGII). The EGII gene (egl2) was cloned from the cellulolytic fungus Trichoderma reesei and inserted into B. mori nucleopolyhedrovirus (BmNPV) genome using BmNPV/Bac-to-Bac expression vector. For expression of rEGII, both the BmN cells and B. mori larvae were infected with the recombinant virus. The putative rEGII yield was about 386 μg per larva and the enzyme activity of the purified rEGII was approx 352 U/mg of rEGII. The optimal activity of this purified protein was observed at 55°C and pH 4, respectively.     

重组结核分枝杆菌Hsp16.3的纳米金免疫传感器检测Hsp16.3抗体

克隆和表达结核分枝杆菌热休克蛋白16.3(Hsp16.3),建立纳米金免疫传感器检测结核病患者血清Hsp16.3抗体.PCR扩增hsp16.3基因,构建重组表达质粒pQE30-hsp16.3,表达和纯化Hsp16.3,Western blot分析其反应原性;晶种生长法制备金纳米棒并连接Hsp16.3,建立纳米金免疫传感...     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.     

Large-scale production of biologically active human keratinocyte growth factor-2

A rapid and efficient expression and purification system has been developed for large-scale production of biologically active recombinant human keratinocyte growth factor-2 (rhKGF-2). The gene encoding human KGF-2 was cloned into the expression vector pET3c and transformed into Escherichia coli BL21(DE3)/pLys S. Under optimal conditions in a 30-l fermentor, the average bacterial yield and the average expression level of rhKGF-2 of three batches were up to 732 g and 32%, respectively. The recombinant protein was purified by cation exchange and heparin-affinity chromatography. One hundred and sixty five milligrams of pure rhKGF-2 was achieved per liter culture. A preliminary biochemical characterization of purified rhKGF-2 was performed by Western blotting and mitogenic activity analysis, and the results demonstrated that purified rhKGF-2 could react with anti-human KGF-2 antibody and stimulate the proliferation of HaCat cells. Xiaoping Wu and Haishan Tian contributed equally to this work.     

Secretory expression and enzymatic characterization of recombinant Agarivorans albus β-agarase in Escherichia coli

Agarase catalyzes the hydrolysis of agar, which is primarily used as a medium for microbiology, various food additives, and new biomass materials. In this study, we described the expression of the synthetic gene encoding β-agarase from Agarivorans albus (Aaβ-agarase) in Escherichia coli. The synthetic β-agarase gene was designed based on the biased codons of E. coli to optimize its expression and extracellular secretion in an active, soluble form. The synthesized agarase gene, including its signal sequence, was cloned into the pET-26 expression vector, and the pET-Aaβ-agarase plasmid was introduced into E. coli BL21-Star (DE3) cells. The E. coli transformants were cultured for high-yield secretion of recombinant Aaβ-agarase in Luria-Bertani broth containing 0.6?mM isopropyl β-D-1-thiogalactopyranoside for 9?h at 37°C. The expressed recombinant Aaβ-agarase was purified by ammonium sulfate precipitation and diethylaminoethyl-sepharose column chromatography, yielding ～10?mg/L Aaβ-agarase. The purified recombinant Aaβ-agarase exhibited optimal activity at pH 7 and 40°C, and its activity was strongly inhibited by Cu²⁺, Mn²⁺, Zn²⁺, and Al³⁺ ions. Furthermore, the K_M and k_cat values for purified Aaβ-agarase were ～0.02?mM and ～45/s, respectively. These kinetic values were up to approximately 15–100-fold lower than the K_M values reported for other agarases and approximately 7–30-fold higher than the k_cat/K_M values reported for other agarases, indicating that recombinant Aaβ-agarase exhibited good substrate-binding ability and high catalytic efficiency. These results demonstrated that the E. coli expression system was capable of producing recombinant Aaβ-agarase in an active form, at a high yield, and with attributes useful in the relevant industries.     

High-level secretory expression, purification and characterization of Ailuropoda melanoleuca growth hormone in Pichia pastoris

Growth hormone is one of the most important hormones, which is involved in many reproductive processes of giant panda Ailuropoda melanoleuca. In this study, the mature peptide of A. melanoleuca growth hormone (AmGH) was successfully expressed and secreted in Pichia pastoris under the control of AOX1 promoter. The expression condition for AmGH in P. pastoris, such as the expression time, pH value and methanol concentration in the BMMY were optimized and the AmGH expression level is about 100 mg/L using GS115 recombinant under optimized condition (96 h of 1.5% methanol induction). The secreted nascent AmGH were purified using ammonium sulfate fractionation. The mature AmGH protein exhibited a molecular mass of approximately 22 kDa on SDS–PAGE. This study would provide a new opportunity for large-scale expression and purification of AmGH, which might facilitate studies on the biological activity of AmGH.     

Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria

The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBD_cenA) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.     

Soluble expression of recombinant human CD137 ligand in <Emphasis Type="Italic">Escherichia coli</Emphasis> by co-expression of chaperones

CD137 ligand (CD137L) is a member of the tumor-necrosis factor superfamily that binds CD137 to provide positive co-stimulatory signals for T cells activation. Co-stimulation through CD137/CD137L has become one of the promising approaches for cancer therapy. Previous reports have shown that CD137L expressed in Escherichia coli resulted in inclusion bodies or low yield. In this study, the effects of five different chaperone teams on the soluble expression of recombinant human CD137L protein were explored and analyzed. The poor expression of CD137L in the cytoplasm of E. coli was improved significantly by co-expression of chaperone GroES-GroEL-Tf. After dual induction and affinity chromatography, purified recombinant CD137L was obtained at a yield of 3 mg protein per liter with purity greater than 98% from original undetectable level. Additionally, the purified recombinant CD137L could bind CD137-positive cells in a dose-dependent manner, markedly promote the growth of activated mice T cells, and elevate the release of IL-2. The present work provides an effective system for soluble expression of functional human co-stimulatory molecule CD137L, which will facilitate the clinical developments of recombinant protein drugs.     

Plasmodium falciparum Carbonic Anhydrase is a Possible Target for Malaria Chemotherapy

Plasmodium falciparum is responsible for the majority of life-threatening cases of human malaria. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Carbonic anhydrase (CA) is present at high levels in human red cells and in P. falciparum. Existence of at least three isozymes of the α class was demonstrated in P. falciparum and a rodent malarial parasite Plasmodium berghei. The major isozyme CA1 was purified and partially characterized from P. falciparum (PfCA1). A search of the malarial genome database yielded an open reading frame similar to the α-CAs from various organisms, including human. The primary amino acid sequence of the PfCA1 has 60% identity with a rodent parasite Plasmodium yoelii enzyme (PyCA). The single open reading frames encoded 235 and 252 amino acid proteins for PfCA1 and PyCA, respectively. The highly conserved active site residues were also found among organisms having α-CAs. The PfCA1 gene was cloned, sequenced and expressed in Escherichia coli. The purified recombinant PfCA1 enzyme was catalytically active. It was sensitive to acetazolamide and sulfanilamide inhibition. Kinetic properties of the recombinant PfCA1 revealed the authenticity to the wild type enzyme purified from P. falciparum in vitro culture. Furthermore, the PfCA1 inhibitors acetazolamide and sulfanilamide showed good antimalarial effect on the in vitro growth of P. falciparum. Our molecular tools developed for the recombinant enzyme expression will be useful for developing potential antimalarials directed at P. falciparum carbonic anhydrase.     

Cloning of exoinulinase gene from Penicillium janthinellum strain B01 and its high-level expression in Pichia pastoris

Aims: The aim of this study is to improve exoinulinase production by expression of a cloned exoinulinase gene inuA1 (GenBank accession no. JF961344 ) from Penicillium janthinellum strain B01 in Pichia pastoris. Methods and Results: A full‐length cDNA of exoinulinase gene (inuA1) was cloned from P. janthinellum strain B01 using RACE PCR. An open reading frame (ORF) of 2115 bp is interrupted by a single intron of 67 bp. The fragment encodes a signal peptide with 20 amino acids and a mature protein with 684 amino acids. The inuA1 was subcloned to the pPICZαC expression vector and succesfully over‐expressed in Pichia pastoris X‐33. The highest activity of exoinlinase reached 272·8 U ml^?1 in the fermentation liquid. It was c. 11‐fold of that produced by wild‐strain B01. A large amount of fructose was identified after the hydrolysis of inulin with the crude recombinant exoinulinase. The recombinant exoinulinase was purified and characterized. The molecular weight of the purified recombinant exoinulianse was 100 kDa. The mass spectrometry result indicated that the purified protein was indeed recombinant exoinulinase. The optimal pH and temperature of the purified recombinant exoinulianse were 4·5 and 50°C, respectively. Conclusions: An exoinulinase gene of P. janthinellum strain B01 was cloned, sequenced and over‐expressed successfully in P. pastoris. Significance and Impact of the Study: Only a few genes have been cloned from P. janthinellum because its molecular biology is poorly understood. In this study, we cloned and over‐expressed inuA1 gene of P. janthinellum in P. pastoris. This recombinant exoinulinase can be used to hydrolyse inulin to produce fructose and facilitate the biofuel production from inulin resources.     

Zebrafish eggs used as bioreactors for the production of bioactive tilapia insulin-like growth factors

Multiple advantages-including the short generation time, large numbers of fertilized eggs, low cost of cultivation and easy maintenance favor the use of fish as bioreactors for the production of pharmaceutical proteins. In the present study, zebrafish eggs were used as bioreactors to produce mature tilapia insulin-like growth factors (IGFs) proteins using the oocyte-specific zona pellucida (zp3) promoter. The chimeric expression plasmids, pT2-ZP-tIGFs-IRES-hrGFP, in which hrGFP was used as reporter of tilapia IGFs expression, were designed to established Tg (ZP:tIGFs:hrGFP) transgenic lines for the expression of tilapia IGF-1 and IGF-2. Recombinant tilapia IGF-1 and IGF-2 were expressed as soluble forms in cytoplasm of fertilized eggs. The content level of tilapia IGF-1 and IGF-2 were 6.5 and 5.0% of the soluble protein, respectively. Using a simple Ni–NTA affinity chromatography purification process, 0.58 and 0.49 mg of purified tilapia IGF-1 and IGF-2 were obtained, respectively, from 650 fertilized eggs. The biological activity of the purified tilapia IGF-1 and IGF-2 was confirmed via a colorimetric bioassay to monitor the growth stimulation of zebrafish embryonic cells (ZF4), tilapia ovary cells (TO-2) and human osteosarcoma epithelial cells (U2OS). These results demonstrate that the use of zebrafish eggs as bioreactors is a promising approach for the production of biological recombinant proteins.     

Fusion with maltose-binding protein (MBP) affects neither RNA N-glycosidase activity nor immunogenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica

A genomic clone encoding mature karasurin-A (KRNA), a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica, was efficiently expressed in E. coli using an expression cassette vector pMAL-c2. The resultant recombinant KRNA fused with maltose-binding protein (MBP) was recovered from the soluble fraction of the bacterial cells and purified to near homogeneity after one round of the affinity chromatography. Neither the karasurin precursor retaining both N- and C-terminal peptides, nor the protein with the N-terminal peptide was successufully produced even as a MBP-fusion. The protein with its C-terminal peptide was over-produced but was recovered in an insoluble fraction. Both the recombinant MBP-KRNA fusion protein and recombinant KRNA with MBP removed were as active as the native KRNA from root tubers. The immunogenicity of the recombinant KRNA was also unaffected by fusion with MBP.     

Characterization of two novel lipase genes isolated directly from environmental sample

Two novel lipase genes (lipJ02, lipJ03) were isolated directly from environmental DNA via genome-walking method. Lipase gene lipJ02 contained an open reading frame (ORF) of 1,425 bp and encoded a 474-amino acids lipase protein, while lipase gene lipJ03 contained an ORF of 1,413 bp and encoded a 470-amino acids lipase protein. The lipase genes were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant P. pastoris were screened via a high-throughput method. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized. The optimum temperature for the purified lipase LipJ02 and LipJ03 was 30 and 35°C, respectively, at pH 8.0. They exhibited similar thermostability, but LipJ02 exhibited better pH stability than LipJ03.     

Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator

The development of specific catalytic inhibitors for the serine protease urokinase-type plasminogen activator (uPA) has been hindered due to difficulties in producing sufficient amounts of active recombinant uPA that is catalytically equivalent to native uPA. The purpose of this study was to develop an efficient system for the expression of recombinant human uPA that exhibits comparable proteolytic activity to that of the native protein. Since post-translational modifications (e.g. glycosylations) of uPA are necessary for efficient proteolytic activity, we have used a mammalian cell line [Chinese hamster ovary (CHO)-S] to express recombinant human uPA. CHO-S cells were selected to stably express full-length recombinant human uPA containing a hexahistidine tag at its C-terminus to permit purification by nickel-based affinity chromatography. Secretion of recombinant uPA into the culture media was confirmed by immunoblotting and the presence of an N-linked glycosylation was confirmed by PNGase sensitivity. Enzymatic activity of purified recombinant uPA was demonstrated using zymography and quantitatively compared to native uPA by kinetic analysis using an uPA-specific substrate. Native uPA and the recombinant uPA demonstrated comparable K_m values (55.7 and 39 μM, respectively). Furthermore, inhibition studies using benzamidine resulted in a K_i of 195 μM for native uPA, while recombinant uPA had a K_i of 112 μM. These data indicate that recombinant human uPA expressed by CHO-S cells is functionally comparable to native uPA.     

Obtaining and refolding a recombinant mannan-binding lectin from the holothurian <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

Our previous studies have shown that the holothurian Apostichopus japonicus produces a mannan-binding lectin (MBL-AJ) that possesses a unique specificity for carbohydrates, which allows its use for cancer antigen detection. In the present work, we report on the isolation of the gene encoding MBL-AJ and its heterologous expression in Escherichia coli cells. Expression of MBL-AJ was carried out under the control of an inducible promoter in the E. coli Top10/pQE-80L expression system. The recombinant MBL-AJ was purified by flow-through column metal-affinity chromatography. Optimal conditions for the refolding of recombinant MBL-AJ were selected. The “sandwich” ELISA method with an antibody against native MBL-AJ was used to determine the values of immunochemical cross reactivity of the native MBL-AJ and its recombinant forms that were obtained in different ways. The extent of immunochemical homology between native and recombinant MBL-AJ obtained under optimal conditions was 69%.     

A digestive beta-glucosidase from the silkworm, Bombyx mori: cDNA cloning, expression and enzymatic characterization

A digestive β-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori β-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori β-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori β-glucosidase to be a single gene. Northern blot analysis of B. mori β-glucosidase gene confirmed larval midgut-specific expression. The B. mori β-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori β-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant β-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori β-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant β-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per μg of recombinant B. mori β-glucosidase. The purified recombinant B. mori β-glucosidase showed the highest activity at 35 °C and pH 6.0, and were stable at 50 °C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori β-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.