Secretion Vector Research Articles

고초균에서 흰이빨참갯지렁이 페리틴 단백질의 분비 및 사료 효율성

Ferritin is known to regulate iron metabolism and maintain iron in a variety of the eukaryotic organisms. The region encoding the mature ferritin (0.47 kb, H-type) of Periserrula leucophryna was amplified using the designed primers including restriction enzyme site and termination codon and subcloned in frame to the pRBAS secretion vector containing the signal sequence, RBS, and promoter of amylase gene (E. coli-Bacillus shuttle vector), resulting in recombinant pRBAS-PLF vector. Recombinant ferritin (18 kDa) was correctly processed and secreted from Bacillus subtilis LKS strain harboring the pRBAS-PLF vector and quantitatively analyzed by SDS-PAGE and western blot, respectively. Secretion of the ferritin was optimized by culture conditions (host, medium, temperature, nitrogen source) in 3 L batch culture and 5 L jar fermenter. Finally. the ferritin was largely produced using 50 L fermenter as the following conditions; at <TEX>$30^{\circ}C$</TEX>, 150 rpm, 1 vvm in Bacillus subtilis LKS using PY medium. The secreted ferritin was maximally measured (approximately 177.6 ug/ml) when the cell density reached to 14.4 at <TEX>$OD_{600}$</TEX> (20 h incubation). The iron binding activity was confirmed by Perls' staining in 7.5% non-denaturing gel, indicating that the multimeric ferritin (composed of 24 subunits) was formed in the culture broth after secretion. Biologically, the culture broth and powder type containing ferritin were tested for possibility as feed additive in chicken broiler. As a result, the ferritin stimulated the growth of chick broil and improved feed efficiency and production index.

A novel salt-inducible vector for efficient expression and secretion of heterologous proteins in Bacillus subtilis

Bacillus subtilis is commonly used as a host for heterologous protein production via plasmid-based expression system. In order to improve product safety, avoid carbon catabolite repression and lower production cost, a novel salt-inducible vector, pSaltExSePR5, was developed based on a natural plasmid of Lactobacillus plantarum BCC9546. Salt-inducible promoter opuAA and a DNA fragment encoding a signal peptide of subtilisin E (SubE) were sequentially added to the core shuttle vector to facilitate expression and secretion of a target protein in B. subtilis. To evaluate the effectiveness of this system under salt induction, a protease gene from Halobacillus sp. without its native signal sequence was inserted in the pSaltExSePR5 plasmid downstream of SubE signal sequence and transformed into B. subtilis WB800. Protease activities from cell-free supernatants of the recombinant bacteria cultures induced with 0.5–6% NaCl were analyzed. The highest protease activity of 9.1U/ml was obtained after induction with 4% NaCl, while the non-induced culture exhibited activity of 0.128U/ml. The results demonstrated that pSaltExSePR5 provides an alternative vector for efficient and simple production of heterologous proteins in B. subtilis with a safer and more economic inducer.

A food-grade fimbrial adhesin FaeG expression system in Lactococcus lactis and Lactobacillus casei.

Enterotoxigenic Escherichia coli (ETEC) infection is the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factor in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Lactic acid bacteria (LAB) are attractive tools to deliver antigens at the mucosal level. With the safety of genetically modified LAB in mind, a food-grade secretion vector (pALRc or pALRb) was constructed with DNA entirely from LAB, including the replicon, promoter, signal peptide, and selection marker alanine racemase gene (alr). To evaluate the feasibility of the system, the nuclease gene (nuc) from Staphylococcus aureus was used as a reporter to be expressed in both Lactococcus lactis and Lactobacillus casei. Subsequently, the extracellular secretion of the fimbrial adhesin FaeG of ETEC was confirmed by Western blot analysis. These results showed that this food-grade expression system has potential as the delivery vehicle for the safe use of genetically modified LAB for the development of vaccines against ETEC infection.

Heterologous Expression of Mannanase and Developing a New Reporter Gene System in Lactobacillus casei and Escherichia coli.

Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were cloned into the expression vector pELX1. The expression patterns of these reporter genes in Lactobacillus casei were investigated by measuring their enzymatic activities and estimating their recombinant protein yields using western blot analysis. Whereas mannanase activity was positively correlated with the accumulation of ManB during growth, GusA activity was not; western blot analysis indicated that while the amount of GusA protein increased during later growth stages, GusA activity gradually decreased, indicating that the enzyme was inactive during cell growth. A similar trend was observed in E. coli JM109. We chose to use the more stable mannanase gene as the reporter to test secretion expression in L. casei. Two pELX1-based secretion vectors were constructed: one carried the signal peptide of the unknown secretion protein Usp45 from Lactococcus lactis (pELSH), and the other contained the full-length SlpA protein from the S-layer of L. acidophilus (pELWH). The secretion of ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the B. pumilus manB gene is a useful reporter gene in L. casei and E.coli.

Expression of Recombinant pET22b-LysK-Cysteine/Histidine-Dependent Amidohydrolase/Peptidase Bacteriophage Therapeutic Protein in Escherichia coli BL21 (DE3)

ObjectivesBacteriophage-encoded endolysins are a group of enzymes that act by digesting the peptidoglycan of bacterial cell walls. LysK has been reported to lyse live staphylococcal cultures. LysK proteins containing only the cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain has the capability to show lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to clone and express LysK-CHAP domain in Escherichia coli BL21 (DE3) using pET22b as a secretion vector. The pET22b plasmid was used, which encoded a pelB secretion signal under the control of the strong bacteriophage T7 promoter. MethodsThe E. coli cloning strains DH5α and BL21 (DE3) were grown at 37°C with aeration in the Luria-Bertani medium. A plasmid encoding LysK-CHAP in a pET22b backbone was constructed. The pET22b vector containing LysK-CHAP sequences were digested with NcoI and HindIII restriction enzymes. Cloning accuracy was confirmed by electrophoresis. The pET22b-LysK plasmid was used to transform the E. coli strain BL21. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1mM to induce T7 RNA polymerase-based expression. Finally, western blot confirmed the expression of target protein. ResultsIn this study, after double digestion of pEX and pET22b vectors with HindIII and NcoI, LysK gene was cloned into two HindIII and NcoI sites in pET22b vector, and then transformed to E. coli DH5α. Cloning was confirmed with double digestion and analyzed with agarose gel. The recombinant pET22b-LysK plasmid was transformed to E. coli BL21 and the expression was induced by IPTG. The expression was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting method. Observation of a 28.5 kDa band confirmed LysK protein expression. ConclusionIn the present study, LysK-CHAP domain was successfully cloned and expressed at the pET22b vector and E. coli BL21 (DE3).

Subcloning of NGF Gene into pSecTag2/Hygro Secretory Vector and Expression in PC12 Cell Line

Subcloning of NGF Gene into pSecTag2/Hygro Secretory Vector and Expression in PC12 Cell Line

Cloning andexpression of cDNA encoding growth hormone tambaqui (Colossoma macropomum) in the yeast Pichia pastoris

Genetic studies involving the search, cloning and expression of genes encoding proteins involved in important physiological processes and advantageous features of the economic standpoint have become the biotechnology research increasingly promising. Due to its zootechnical importance, the encoding gene of growth hormone (GH) of various fish species have been isolated, cloned and expressed in heterologous expression systems. The tambaqui (Colossoma macropomum), Amazonian fish species considered promising for fish farming, has been the subject of extensive genetic research with biotechnology focus. This study aimed to express the cDNA encoding GH tambaqui (tGH) in the methylotrophic yeast Pichia pastoris. The nucleotide sequence of cDNA tGH, previously isolated by Sousa (2009) [1], was optimized for expression in P. pastoris and obtained by chemical synthesis. It was then cloned into the cloning vector pUC19 followed by subcloning into expression and secretion vector pPIC9 with the endonucleases Eco RI and NotI. The recombinant plasmid named pPIC-tGH was linearized and inserted into host by electroporation. Recombinant clones were selected in medium auxotrophic for histidine. The expression of recombinant protein was induced by methanol addition during 96 hours occurred in shake flasks. The culture supernatant was analyzed on SDS-PAGE gel and expression of tGH was confirmed by Western blotting. The analysis of the supernatant revealed in both phenotype Mut+ and Muts the presence of a protein band of approximately 23 kDa, which was confirmed to be the tGH recombinant by Western blotting using monoclonal antibody against histidine tail C- terminal. The expression of tGH started already first 24 hours and was sustained throughout the induction period, lasted up to 120 h. These results are similar to of Li et al. [2], who expressed GH carp (Cyprinus carpio) in P. pastoris and observed that from the first 24 hours of induction the recombinant protein was already present in the medium, and time 72 and 96 hours corresponded to the peak of expression. These results demonstrate that GH tambaqui can be successfully expressed in P. pastoris. The tambaqui, for their economic importance, so far was the first Amazonian fish species to have GH expressed in heterologous system, the first step in the production of GHs of other important species for aquaculture regional and national. In the future, tambaqui recombinant GH should be analyzed for their efficiency in accelerating the growth of tambaqui towards the possible use in aquaculture production, which represent progress and innovation in technologies of cultivation of fish Amazonian.

Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus caseiinhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells

BackgroundLactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB–YVAD). YVAD is a tetrapeptide (tyrosine–valine–alanine–aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1β, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB–YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs.ResultsWe constructed the rCTB–YVAD secretion vector pSCTB–YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB–YVAD was generated by transformation with pSCTB–YVAD. Both the culture supernatant of pSCTB–YVAD-transformed L. casei and purified rCTB–YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB–YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB–YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1β secretion in Caco-2 cells, without affecting cell viability.ConclusionsThe rCTB protein fused to a functional peptide secreted by L. casei can bind to GM1 ganglioside, like rCTB, and recombinant YVAD secreted by L. casei may exert anti-inflammatory effects in the intestine. Therefore, rCTB secreted by L. casei has potential utility as a vector for the delivery of YVAD to IECs.

Novel synthetic signal peptides for the periplasmic secretion of green fluorescent protein in Escherichia coli

New secretion vectors containing synthetic signal peptides were constructed to study the periplasmic translocation of green fluorescent protein (GFP) in Escherichia coli. These constructs encode synthetic signal peptides spA and spD fused to the amino terminal end of GFP, and expressed from T7/lac promoter in the BL21DE3 strain by induction with IPTG. The recombinant protein was detected in both the cytoplasmic and periplasmic fractions. Fluorescence analysis revealed that recombinant proteins with signal peptides were not fluorescent, indicating translocation to periplasmic space. In contrast, recombinant proteins without signal peptide were fluorescent. These results indicate that the expressed recombinant proteins were translocated into the periplasm. Therefore, the synthetic signal peptides derived from signal peptides of Bacillus sp. could efficiently secrete the heterologous proteins to the periplasmic space of E. coli.

Expression and secretion of functional recombinant μO-conotoxin MrVIB-His-tag in Escherichia coli

μO-conotoxin MrVIB is a 31-amino acid peptide containing three disulfide bonds isolated from the venom of Conus marmoreus, which is a selective antagonist of voltage-gated sodium channel (VGSC) Nav1.8 and has a long-lasting analgesic activity. Drug development of MrVIB has long been hindered over 15 years by difficult chemical synthesis and oxidative folding. Herein we describe a different approach based on the recombinant expression of gene MrVIB in Escherichia coli. A secretion vector pET22b(+)-MrVIB fused with pelB leader signal peptide and His-tag was constructed, which was transformed into BL21 (DE3) strain of E. coli. The recombinant conotoxin MrVIB-His-tag (rMrVIB-His) was successfully expressed and secreted into the periplasmic space of BL21 (DE3) cells. The pelB leader signal peptide was properly cleaved and three disulfide bonds were also formed properly to yield biological active rMrVIB-His. Folded rMrVIB-His in the periplasmic fraction was isolated with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography (RP-HPLC) and identified by liquid chromatography/mass spectrometry-ion trap-time of flight mass spectrometry (LC/MS-IT-TOF). Biological activity assay of rMrVIB-His showed it had good analgesic effects in three pain models.

Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis

Brazzein is an intensely sweet-tasting protein with high water solubility, heat stability, and taste properties resembling those of carbohydrate sweeteners. In the present study, we describe the expression of the synthetic gene encoding brazzein, a sweet protein in the yeast Kluyveromyces lactis. The synthetic brazzein gene was designed based on the biased codons of the yeast, so as to optimize its expression, as well as on the extracellular secretion for expression in an active, soluble form. The synthesized brazzein gene was cloned into the secretion vector pKLAC2, which contains the yeast prepropeptide signal from the Saccharomycescerevisiae α-mating factor. The constructed plasmid pKLAC2-des-pE1M-brazzein was introduced into the yeast K. lactis GG799. The yeast transformants were cultured for high-yield secretion of the recombinant des-pE1M-brazzein in YPGal medium for 96h at 30°C. The expressed recombinant des-pE1M-brazzein was purified by CM-Sepharose column chromatography and approximately 104mg/L was obtained. The purity and conformational state of the recombinant des-pE1M-brazzein were confirmed using SDS–PAGE, HPLC, and circular dichroism. The identity of the recombinant protein was also confirmed by N-terminal amino acid analysis and taste testing. The purified recombinant des-pE1M-brazzein had an intrinsic sweetness in its minor form, approximately 2130 times sweeter than sucrose on a weight basis. These results demonstrate that the K. lactis expression system is useful for producing the recombinant brazzein in active form at a high yield with attributes useful in the food industry.

Solving the learning parity with noiseʼs open question

The classical cryptographic primitives are constructed on the assumptions that the private key is securely kept and uniformly distributed. Learning parity with noise is a famous problem used to construct several cryptographic primitives. This research studies the open question about the hardness of the learning parity with noise assumption when the secret vector is not uniform and has sufficient min-entropy. The proofs show that the standard learning parity with noise implies that it is secure even if the secret vector is sampled from an arbitrary distribution with sufficient entropy. Furthermore, this paper shows that the symmetric encryption scheme from learning parity with noise is secure even if the secret key has min-entropy at least k.

Expression of recombinant human neutrophil Cathepsin G in Pichia pastoris

Cathespin G (CatG), a serine protease found in the azurophil granules of neutrophils, participates in killing engulfed microorganisms. CatG has dual specificity for chymotrypsin‐like and tryspin‐like substrates. CatG is a poorly understood enzyme and is currently only commercially available as mature enzyme purified from human sources. The yeast Pichia pastoris is being used to express CatG to study its dual specificity and its C‐terminal processing. The full length (C‐terminus present) human CatG amino acid sequence was modified to remove one glycosylation site and eight dibasic sites to avoid potential cleavage by yeast kexin protease. The construct was engineered to have an N‐terminal 6‐His‐cytochrome B5 (CytB5) heme binding fusion domain linked to the modified human CatG by an enterokinase cleavage site for activation. The amino acid sequence was used to generate a codon‐optimized gene that was placed in the pPICzα secretion vector. After transforming Pichia pastoris strain X‐33, 48 Zeocin‐resistant clones were screened for relative levels of CatG activity. Recombinant CatG has been partially purified from fermentation media by nickel affinity chromatography and its activity has been confirmed by assays using synthetic substrates. Supported by a Student Faculty Collaborative Grant from the ETSU Honors College and ETSU Office of Research and Sponsored Programs and by NHLB grant R15HL091770.

Insulin-like Growth Factor (IGF) Signaling Requires αvβ3-IGF1-IGF Type 1 Receptor (IGF1R) Ternary Complex Formation in Anchorage Independence, and the Complex Formation Does Not Require IGF1R and Src Activation

Integrin αvβ3 plays a role in insulin-like growth factor 1 (IGF1) signaling (integrin-IGF1 receptor (IGF1R) cross-talk) in non-transformed cells in anchorage-dependent conditions. We reported previously that IGF1 directly binds to αvβ3 and induces αvβ3-IGF1-IGF1R ternary complex formation in these conditions. The integrin-binding defective IGF1 mutant (R36E/R37E) is defective in inducing ternary complex formation and IGF signaling, whereas it still binds to IGF1R. We studied if IGF1 can induce signaling in anchorage-independent conditions in transformed Chinese hamster ovary cells that express αvβ3 (β3-CHO) cells. Here we describe that IGF1 signals were more clearly detectable in anchorage-independent conditions (polyHEMA-coated plates) than in anchorage-dependent conditions. This suggests that IGF signaling is masked by signals from cell-matrix interaction in anchorage-dependent conditions. IGF signaling required αvβ3 expression, and R36E/R37E was defective in inducing signals in polyHEMA-coated plates. These results suggest that αvβ3-IGF1 interaction, not αvβ3-extracellular matrix interaction, is essential for IGF signaling. Inhibitors of IGF1R, Src, AKT, and ERK1/2 did not suppress αvβ3-IGF-IGF1R ternary complex formation, suggesting that activation of these kinases are not required for ternary complex formation. Also, mutations of the β3 cytoplasmic tail (Y747F and Y759F) that block β3 tyrosine phosphorylation did not affect IGF1R phosphorylation or AKT activation. We propose a model in which IGF1 binding to IGF1R induces recruitment of integrin αvβ3 to the IGF-IGF1R complex and then β3 and IGF1R are phosphorylated. It is likely that αvβ3 should be together with the IGF1-IGF1R complex for triggering IGF signaling.

Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli.

High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low-speed centrifugation. Following pre-extaction (or washing), protein is extracted from washed pellets using guanidine⋅HCl. The solubilized and unfolded protein is either directly folded or further purified by gel filtration in the presence of guanidine⋅HCl as described in this unit. A support protocol describes the removal of guanidine⋅HCl from column fractions so they can be monitored by SDS-PAGE.

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010

Leuconostoc carnosum 4010 is a protective culture for meat products. It kills the foodborne pathogen Listeria monocytogenes by producing two class IIa (pediocin-like) bacteriocins, leucocin A and leucocin C. The genes for leucocin A production have previously been characterised from Leuconostoc gelidum UAL 187, whereas no genetic studies about leucocin C has been published. Here, we characterised the genes for the production of leucocins A and C in L. carnosum 4010. In this strain, leucocin A and leucocin C operons were localised in different plasmids. Unlike in L. gelidum, leucocin A operon in L. carnosum 4010 only contained the structural and the immunity genes lcaAB without transporter genes lcaECD. On the contrary, leucocin C cluster included two intact operons. Novel genes lecCI encode the leucocin C precursor and the 97-aa immunity protein LecI, respectively. LecI shares 48 % homology with the immunity proteins of sakacin P and listeriocin. Another leucocin C operon lecXTS, encoding an ABC transporter and an accessory protein, was 97 % identical with the leucocin A transporter operon lcaECD of L. gelidum. For heterologous expression of leucocin C in Lactococcus lactis, the mature part of the lecC gene was fused with the signal sequence of usp45 in the secretion vector pLEB690. L. lactis secreted leucocin C efficiently, as shown by large halos on lawns of L. monocytogenes and Leuconostoc mesenteroides indicators. The function of LecI was then demonstrated by expressing the gene lecI in L. monocytogenes. LecI-producing Listeria was less sensitive to leucocin C than the vector strain, thus corroborating the immunity function of LecI.

Secretory expression and characterization of a novel peroxiredoxin for zearalenone detoxification in Saccharomyces cerevisiae

Zearalenone (ZEN) is a Fusarium mycotoxin, which is considered to be an oestrogenic endocrine disruptor found to cause severe morphological and functional disorders of reproductive organs in livestock. Increasing attention has been paid to the development of an effective strategy for ZEN decontamination. ZEN is oxidized into smaller estrogenic metabolites by a novel peroxiredoxin (Prx) isolated from Acinetobacter sp. SM04. The Prx coding gene was cloned in a secretory vector pYES2-alpha (pYα) with an alpha (α) signal peptide gene inserted into the multiple cloning site of pYES2. The recombinant Prx was secreted from Saccharomyces cerevisiae INVSc1 after inducing with 2% (w/v) galactose for 72h, and was found to be nearly 20kDa through 12% SDS-PAGE. The expressed amount of recombinant Prx was 0.24mg/mL in the extracellular supernatant. Recombinant Prx showed a gradient increase at the beginning of ZEN degradation. The final ZEN degradation amount was 0.43μg by one unit recombinant Prx after 12h. Furthermore, the temperature, H2O2 concentration, and pH for highest peroxidase activity of recombinant Prx were 80°C, 20mM and 9.0, respectively. When compared with other peroxidases, the thermal stability and alkali resistance of recombinant Prx were much better. The results suggest that recombinant Prx is successfully expressed in S. cerevisiae.

Optimized production of scygonadin in Pichia pastoris and analysis of its antimicrobial and antiviral activities

The crab antimicrobial peptide scygonadin is confirmed to have antimicrobial activity against bacteria and it is probably associated with the reproductive immunity in Scylla paramamosain. To obtain large quantity of scygonadin for further biological assays, a 306bp cDNA sequence encoding the mature peptide of scygonadin was cloned into a secretion vector of pPIC9K, and a high-level of the recombinant scygonadin was achieved in Pichia pastoris. The optimal expression condition was determined as incubation with 0.5% methanol for 48h at 28°C under pH 6.0, and a total of 70mg scygonadin was expressed in 1L culture medium. The recombinant product was purified and 97% pure scygonadin was obtained using immobilized metal affinity chromatography with a yield of 46mg/L. The recombinant scygonadin was confirmed using SDS–PAGE analysis and MS-fingerprinting. P. pastoris-derived scygonadin exhibited relatively higher antimicrobial activities against bacteria than Escherichia coli-derived scygonadin. The antimicrobial activity of the recombinant scygonadin against pathogenic Aeromonas hydrophila showed salt resistant and the killing kinetics of A. hydrophila was time dependent. Besides, the antiviral assay demonstrated that scygonadin could interfere with white spot syndrome virus (WSSV) replication in vitro-cultured crayfish haematopoietic (Hpt) cells. Taken together, this is the first report on the heterologous expression of scygonadin in P. pastoris, and P. pastoris is an effective expression system for producing large quantities of biological active scygonadin for both research and agricultural application.

Bone marrow stromal cells transfected with ciliary neurotrophic factor gene by secretory vector

Bone marrow stromal cells transfected with ciliary neurotrophic factor gene by secretory vector

Molecular Characterization of N-Terminal Pro-sequence of Keratinase Ker P from Pseudomonas aeruginosa: Identification of Region with Chaperone Activity

In silico analysis of keratinase Ker P from Pseudomonas aeruginosa revealed that its full gene of 1,497 bp constituted of a 72-bp signal sequence along with a long 520 bp pro-sequence and 905 bp core region. Position specific multiple sequence alignment of Ker P protein with other distant proteases revealed high variability within their N-terminal regions while the core protein was considerably conserved. Ker P (F1) and its four N-terminal truncations (F2-F5) lacking 72, 177, 405, 507 bp, respectively, were cloned and constitutively expressed as extracellular protein in pEZZ-18 secretory vector with Escherichia coli HB101 as the expression host. Ker P F1, Ker P F2, Ker P F3 and Ker P F4 products were active whereas no keratinolytic activity was obtained in Ker P F5. Further analysis revealed that only 187 bp pro-sequence region is required for correct folding of the protein into its active conformation and, thus, has chaperone-like activity. Further, comparative biochemical characterization revealed that the full-length keratinase Ker P F1 was catalytically more efficient than the truncated forms. Among the truncated enzymes, keratinase Ker P F4 exhibited better thermostability than Ker P F2 with a t(1/2) of >1 h at 60 °C. It also had a higher V (max) and K (m) on casein as compared with Ker P F2. However, no significant variation was observed with respect to kinetics on synthetic substrates.