Expression-secretion Vector Research Articles

Heterologous expression ofMycobacterium tuberculosis Ag85B inPichia pastoris

The DNA fragment encoding matureMycobacterium tuberculosis major secretory protein Ag85B was inserted into thePichia pastoris secretory expression vector pHBM905A, under the control of theAOX1 promoter. The recombinant plasmid pHBM905A-85B linearized bySal I was introduced intoPichia pastoris strain GS115 by PEG1000 transformation method. After phenotype screening and PCR identification, the resulting GS115-pHBM905A-85B strain was cultivated and induced with methanol. The recombinant Ag85B protein in secreted form was attained with molecular weight of 35×103 approximately detected by SDS-PAGE and Western blot. ELISA experiment proved that the protein had good antigen specificity. Secretory expression of recombinantM. tuberculosis Ag85B inP. pastoris will open a door to mass production of the protein in heterologous host and allow ready evaluation of its immunological function.

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase (AP). The VH-linker-VL, namely scFv gene, was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by Sfi I and Not I, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and Not I restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.

Expression and characterization of Gag protein of HIV-1 CN in Pichia pastoris

To express the core protein of HIV-1 of Chinese prevalent strain (HIV-1 CN) in Pichia pastoris, the full-length gag gene was inserted into the secretory expression vector pHILS1. Linearized recombinant plasmid pHILGAG by SalI was electrotransformed into the yeast strain GS115, and the yeast transformants were identified by PCR. To induce the interest protein to be expressed, the PCR positive transformants were inoculated in the medium of BMGY and BMMY, mRNA of the strain was detected by RT-PCR, and the expressed protein was analyzed by SDS-PAGE, Western blotting and thin layer scanning. mRNA (1.3 kb) was amplified by RT-PCR. SDS-PAGE and Western blotting analysis showed that the molecular mass of the expressed protein was 55 kDa, which was similar to the expected value, and the expressed protein could react with McAb to HIV-1 p24. Thin layer scanning analysis demonstrated that the whole amount of the expressed protein was approximately 13% of the soluble protein in the supernatant. The recombinant yeast had good genetic stability. The optimal expression conditions of the engineering yeast were as follows: BMMY medium, 80–90% of dissolved oxygen, 1% methanol, and 3-day-cultivation course. Gag proteins were expressed under the optimal expression condition and purified via gel filtration chromatography. The purity of the interest protein was up to 85%. After the purified proteins were inoculated into BALB/c mice, the anti-HIV-1 antibodies in the immunized mice could be detected by Western blotting.

30] Lactobacilli as vehicles for targeting antigens to mucosal tissues by surface exposition of foreign antigens

Publisher Summary This chapter discusses lactobacilli as vehicles for targeting antigens to mucosal tissues by surfaces exposition for foreign antigens. Lactobacillus strains have a number of properties that make them attractive candidates as delivery vehicles for the presentation to the mucosa of compounds of pharmaceutical interest, in particular vaccines and immunomodulators. Lactobacilli have been used for centuries in fermentation and preservation of food and feed, and are considered GRAS (generally regarded as safe) organisms. Certain strains of Lactobacillus can colonize the gut, and are believed to show healthpromoting activities for humans and animals. Lactobacillus species have the capacity to evoke a mucosal as well as a systemic immune response against epitopes associated with these organisms after oral or nasal administration. These findings and earlier observations indicating that certain Lactobacillus species show a nonspecific immunoadjuvant effect by triggering of macrophages have prompted research aimed at investigating the potential of these organisms to synthesize foreign antigens and present them to the immune system. This chapter presents the construction and evaluation of a series of expression-secretion vectors with strong constitutive or regulatable promoters and efficient translation initiation regions designed for intra- and extracellular expression of homologous and heterologous proteins.

Isolation of a protease-deficient mutant of Bacillus brevis and efficient secretion of a fungal protein disulfide isomerase by the mutant

The efficient production of a thermostable protein disulfide isomerase (PDI) was successfully achieved using the newly isolated protease-deficient mutant, Bacillus brevis 31-OK. Extracellular protease (exoprotease) activity was about a quarter of that in the parent, and the mutant was deficient in at least one of the major exoproteases. The cDNA encoding the fungal PDI was inserted downstream of the signal peptide-encoding region in an expression-secretion vector for B. brevis. Efficient production of PDI was feasible using B. brevis 31-OK as a host and modified signal sequences composed of three leucine residues inserted in the hydrophobic region of the MWP (middle wall protein) signal sequence. The maximal secretion of PDI into the culture medium was 1.1 g/ l, which is about twice that by the parent strain and fifty times greater than the amount of rat and murine PDIs produced by Escherichia coli. The enzymatic properties such as the specific activity and thermal stability of the recombinant PDI are similar to those of natural PDI derived from Humicola insolens mycelia. B. brevis 31-OK was able to maintain its exoprotease activity at a low level throughout the cultivation and is considered to be useful host for production of a protease-sensitive protein and for increase of protein productivity due to stable accumulation.

Expression and secretion of barley α-amylase and A.niger glucoamylase inSaccharomyces cerevisiae

cDNAs of barley alpha-amylase andA. niger glucoamylase were cloned in oneE. coli-yeast shuttle plasmid resulting in the construction of expression secretion vector pMAG15. pMAG15 was transformed intoS. cerevisiae GRF18 by protoplast transformation. The barley alpha-amylase andA. niger glucoamylase were efficiently expressed under the control of promoter and terminator of yeast PGK gene and their own signal sequence. Over 99% of the enzyme activity expressed was secreted to the medium. The recombinant yeast strain, S.cerevisiae GRF18 (pMAG15), hydrolyzes 99% of the starch in YPS medium containing 15% starch in 47 h. The glucose produced can be used for the production of ethanol.

Secretory production of erythropoietin and the extracellular domain of the erythropoietin receptor by Bacillus brevis: affinity purification and characterization.

Bacillus brevis secretes a large amount of cell wall proteins into the culture medium. For construction of Bacillus brevis expression-secretion vectors of human erythropoietin (EPO) and the extracellular domain of mouse erythropoietin receptor (sEPOR), cDNA for each mature form was inserted into a plasmid containing the promoter region and the signal-peptide encoding region of a cell wall protein. Culture supernatants of transformants were affinity purified using a monoclonal antibody-fixed gel for EPO and and EPO-fixed gel for sEPOR. The affinity purification efficiently removed unwanted proteins, giving samples with sufficiently high purity to analyze amino acid sequences of N-terminal regions and biological activities. Combination of this secretory production and affinity purification may facilitate isolation of a large amount of pure EPO and sEPOR, and is useful for further understanding the molecular mechanism of interaction between EPO and EPOR.

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47

Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47.

Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.

The efficient production of human epidermal growth factor by Bacillus brevis.

A system has been developed for the efficient production of heterologous proteins using Bacillus brevis as a host that secretes large amounts of cell wall protein into the medium. The promoter region and signal peptide-encoding region of the cell wall protein gene were used to construct an expression-secretion vector. Bacterial proteins such as amylases can be produced in large amounts by this system (1 g/l or more), but mammalian proteins such as human alpha-amylase are produced at a low level (one or two orders of magnitude less than for bacterial proteins). The highly efficient secretion of human epidermal growth factor (h-EGF, more than 1 g/l) was obtained with B. brevis HPD31 as the host and plasmid pHY481, derived from B. brevis 481, as the vector. Recombinant hEGF was purified easily from the culture supernatant by two steps. Purified hEGF had the identical NH2-terminal amino acid sequence and COOH-terminal amino acid sequence with those of the authentic hEGF, and it was fully active in biological assays. This recombinant hEGF has been shown to be successful for biological wool harvesting (CSIRO, Australia). These results, in combination with previous results, indicate that foreign proteins of diverse origins can be produced efficiently as functional proteins in B. brevis.

The potential of Lactobacillus as a carrier for oral immunization: Development and preliminary characterization of vector systems for targeted delivery of antigens

Oral administration of lactobacilli evokes mucosal and systemic immune responses against epitopes associated with these organisms ( Gerritse et al., 1990, 1991). The adjuvant function of different Lactobacillus species was investigated under the conditions of intraperitoneal (i.p.) injection or oral administration. After i.p. injection of trinitrophenylated chicken γ-globulin, high DTK responses were observed with Lactobacillus casei and Lactobacillus plantarum, but low responses with Lactobacillus fermentum and Lactobacillus delbrueckii subsp. bulgaricus. In different experimental model systems L. casei and L. plantarum consistently showed significant adjuvanticity. A series of expression and expression-secretion vectors containing the strong constitutive promoter of the L. casei L-ldh gene or the regulatable promoter of the Lactobacillus amylovorus amy gene ( Pouwels and Leer, 1995) was used for the intracellular, extracellular and surface-bound expression of an influenza virus antigenic determinant fused to Escherichia coli β -glucuronidase. Intracellular expression of the fusion protein amounted to 1–2% of total soluble protein. Lactobacilli synthesizing the fusion protein intracellularly evoked an oral immune response after subcutaneous priming.

Analysis of the Mycobacterium tuberculosis 85A antigen promoter region

A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences. In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis. Insertion and deletion mutations within the promoter region indicated that, unlike most E. coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST.

Efficient production of casoxin D, a bradykinin agonist peptide derived from human casein, by Bacillus brevis.

We efficiently produced a small peptide by the host-vector system using Bacillus brevis as a host. DNA encoding the physiologically functional casoxin D, composed of seven amino acids, was ligated in tandem. An expression-secretion vector containing DNA, which codes for a fusion protein of epidermal growth factor-casoxin D pentamer, was constructed. B. brevis transformed with this plasmid produced about 0.5 g/liter of the fusion protein in the culture supernatant. The fusion protein was purified with ammonium sulfate fractionation from the supernatant and digested with two kinds of proteinases. A peptide well separated by high pressure liquid chromatography was identified as biologically active casoxin D.

Expression of the pheromone 3-encoding gene of Euplotes octocarinatus using a novel bacterial secretion vector.

The pheromone 3-encoding gene (phr3) of Euplotes octocarinatus was expressed in Escherichia coli using a novel expression-secretion vector. The vector, pExSec1, contains a strong and tightly regulated T7 promoter, the corresponding Shine-Dalgarno sequence and the T7 terminator region. Translation starts at the protein A leader sequence followed by the synthetic ZZ sequence of protein A. The expression-secretion modules are embedded in the high-copy-number plasmid vector, pUK21, which carries a kanamycin-resistance marker (KmR). The produced ZZ-pheromone 3 (Phr3) fusion protein was secreted into the culture medium of the host cells. It was isolated by affinity chromatography and was further purified by gel filtration. After refolding with protein disulfide isomerase (PDI), the fusion protein exhibited the same high activity as the native pheromone.

A model system for the investigation of heterologous protein secretion pathways in Lactococcus lactis.

The capacity of recombinant strains of Lactococcus lactis to secrete a heterologous protein was investigated by constructing two expression-secretion vectors (pLET2 and pLET3) for use with a lactococcal gene expression system driven by the highly active T7 RNA polymerase. The vectors incorporated different lactococcal secretion leaders and translation initiation sequences. When tetanus toxin fragment C (TTFC) was used as a test protein, the quantities of TTFC produced by the pLET2-TTFC strain exceeded the rate of secretion of TTFC into the growth medium. However, nearly all of the soluble TTFC associated with the cell (3.4%) was translocated through the cell membrane. The pLET3-TTFC strain did not accumulate TTFC intracellularly and exhibited growth characteristics and viability identical to the growth characteristics and viability of the control strain. This strain secreted approximately 2.9 mg of TTFC per liter into the growth medium after 6 h of growth under test tube conditions. Our results indicate that L. lactis is capable of secreting substantial amounts of heterologous protein and also confirm the findings of other workers that the cell wall may serve as a functional barrier to the diffusion of some secreted proteins into the growth medium.

Secretion of firefly luciferase in E.coli

A DNA segment carrying the full-length, intronless firefly luciferase gene was inserted into the high expression secretion vector, pIN-III -ompA. Upon induction of gene expression, luciferase activity was detected in extracts prepared from periplasmic fractions. The results indicated that the OmpA signal peptide was able to direct secretion of firefly luciferase across the cytoplasmic membrane. This has important implications for using this luciferase as a reporter in studying protein export and targeting.

Very efficient extracellular production of cholera toxin B subunit using Bacillus brevis.

We have constructed a very efficient synthesis and secretion system for cholera toxin B subunit (CTB) of Vibrio cholerae 569B using Bacillus-brevis. The constructed expression-secretion vector has the multiple promoters and the signal peptide coding region of the mwp gene, a structural gene for one of the major cell wall proteins of B. brevis strain 47, directly followed by the gene encoding the mature CTB. A large amount of mature CTB (1.4 g per liter of culture) was secreted into the medium. It had the same amino terminal amino acid sequence as that of authentic CTB and was fully active in GM1 ganglioside binding assay.

Production of a fungal protein, Taka-amylase A, by protein-producing Bacillus brevis HPD31.

An expression-secretion vector, pMK300, was constructed to express the Aspergillus oryzae Taka-amylase A (Taa) cDNA. The promoter and signal peptide regions of the HWP (a major cell wall protein of Bacillus brevis HPD31) gene on pMK300 were efficiently utilized in B. brevis HPD31 and a large amount of Taa (22 mg/l) was secreted into the medium. The HWP signal peptide utilized for secretion of Taa was correctly processed during the protein transport across the membrane. The enzymatic properties of Taa produced by B. brevis HPD31 were the same as those of the Aspergillus oryzae Taa in several respects; specific activity, thermal and pH stabilities, and temperature and pH optima. These results, in combination with previous results, indicate that B. brevis HPD31 could be used to produce extracellularly foreign proteins of diverse origins as functional proteins.

Tissue-specific expression in the salivary glands of transgenic mice.

Using a DNA construct, named Lama, derived from the murine parotid secretory protein (PSP) gene, we have obtained salivary gland specific gene expression in transgenic mice. Lama is a PSP minigene and allows analysis of the PSP gene 5' regulatory region by transgenesis. We show here that the regulatory region included in Lama with 4.6 kb of 5' flanking sequence is sufficient to direct expression specifically to the salivary glands. The expression level in the parotid gland is only about one percent of the PSP mRNA level, while that of the sublingual gland is near the PSP mRNA level. This suggests significant differences in the PSP gene regulation in the two glands. In addition, Lama is a secretory expression vector in which cDNAs or genomic fragments can be inserted. We demonstrate that the Lama construct can direct the expression of a heterologous cDNA encoding the C-terminal peptide of human factor VIII to salivary glands and that the corresponding peptide is secreted into saliva.

The tetracycline inducible expression of α-amylase in Bacillus subtilis

Abstract In order to construct a tetracycline (Tc) inducible expression-secretion vector system, the α-amylase structural gene of Bacillus subtilis strain 1A412 was isolated, and inserted between the promoter-operator and the terminator region of a Tc-resistant (Tc r ) gene. When the α-amylase gene was expressed in B. subtilis on a plasmid, the expression of the α-amylase gene could be modulated by the Tc concentration. α-Amylase activity was increased approximately 15-fold by adding 500 ng/ml Tc.