Recombinant Protein Production by the Baculovirus-Insect Cell System in Basal Media Without Serum Supplementation

The production of β-galactosidase by Sf9 cells infected with recombinant Autographa californica nucleopolyhedrovirus (AcNPV) was investigated in shake-flask culture using two serum-free basal media: Grace's medium and TNM-FH (Grace's medium supplemented with lactalbumin hydrolysate and yeast extract). At the time of infection, cells grown in serum-supplemented TNM-FH were transferred into fresh basal media without adaptation. The absence of serum depressed the β-galactosidase yield considerably in Grace's medium, but to a much lesser extent in TNM-FH, where it reached around 2/3 of the level obtained in TNM-FH supplemented with 10% fetal bovine serum (FBS). While both lactalbumin hydrolysate and yeast extract promoted β-galactosidase production, their removal by medium replacement on post-infection day 1 gave a β-galactosidase yield nearly equal to that obtained in their continuous presence. Supplementation of basal media with phosphatidic acid (PA) from egg yolk lecithin, which has been shown to enhance cell growth and recombinant protein production in serum-free culture of Chinese hamster ovary (CHO) cells, was also effective in increasing β-galactosidase yield. Elevating the multiplicity of infection (MOI) from 2 to 10 plaque-forming units per cell (pfu/cell) also resulted in an increase in product yield. These results provide information important to the development of cost-effective serum-free culture technology for use in large-scale production of recombinant proteins by the baculovirus-insect cell system.     

Cloning and characterization of a β-galactosidase encoding region in Lactobacillus coryniformis CECT 5711

A chromosomal DNA fragment of 7.8 kb from Lactobacillus coryniformis CECT 5711 was cloned in Escherichia coli K-12 and was found to express a functional β-galactosidase. Nucleotide sequence analysis showed that this fragment contained two partially overlapping genes, the lacL (1,881 bp) and the lacM (960 bp), that encode the subunits of a heterodimeric β-galactosidase, with estimated molecular masses of 72,129 and 35,233 Da, respectively. Other three incomplete open reading frames showing homology to another β-galactosidase, an α-galactosidase, and a galactokinase, respectively, were also found. The L. coryniformis β-galactosidase was overproduced in E. coli by using an isopropyl-β-d-thiogalactopyranoside (IPTG) expression system. Two new proteins with an estimated M _r s of approximately 72,000 and 35,000 appeared upon induction with IPTG, and extracts of the recombinant E. coli strain showed β-galactosidase activity.     

Gene delivery into neuronal and glial cells by using a replication-deficient adenovirus vector: prospects for neurological gene therapy

We have used a recombinant adenovirus vector (E1−) expressing β-galactosidase to explore a novel mechanism with which to transfer genes into cells of the central nervous system (CNS). The replication-deficient adenovirus vector expressing β-galactosidase (RAd35) was propagated on a permissive helper cell line (293 cells). High level protein expression from the human cytomegalovirus immediate early promoter (hCMV IE) was obtained in a target cell population of RAd35 infected cultured neuronal and glial cell lines. Light microscopy showed that over 50% of the glial cells studied expressed β-galactosidase. Following retinoic acid treatment, RAd35 infected cell lines ND7/23, NG108 and NTera2, showed β-galactosidase expression in up to 90% of the cells. In addition, these cells showed morphological evidence of differentiation into neurons. This pattern of β-galactosidase expression was also observed in primary rat cerebella granule neuron cultures. In vivo studies were performed in Balb/c mice following direct intracranial injections of RAd35 into the brain. Cell sections showed a localised staining in the brain at the site of injection of the virus. Non-replicating adenovirus vectors are therefore highly efficient systems for delivering a transgene into brain cells. However, their broad cell tropism may limit their applications for genetic disorders in which a specific cell type is to be targeted for gene therapy. To address this problem, we have constructed adenovirus vectors which contain specific neuronal promoters and are currently assessing in vitro expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Maximal Expression of Recombinant cDNAs in COS Cells for Use in Expression Cloning

In the process of establishing an expression cloning system for cell surface receptors we examined parameters which influence the expression of foreign genes in COS cells. The bacterial β-galactosidase gene was chosen as a reporter gene, since it permits the determination of (i) the fraction of cells transfected as well as (ii) the total activity of the synthesized enzyme in parallel experiments. This renders it possible to calculate the enzyme activity per individual cell. In transfected COS cells, the plasmid pXMgal directed a 20- and 10-fold higher β-galactosidase activity than pCH110 and pCDLgal, respectively. DEAE-dextran-mediated DNA uptake and protoplast fusion were found to result in higher expression rates than lipofection and electroporation. A coincubation of the cells with chloroquine during the DEAE-dextran transfection protocol caused, as reported, an increase of β-galactosidase positive cells but considerably reduced the total β-galactosidase activity. However, a 10% DMSO shock at the end of the transfection procedure simultaneously increased the number of transfected cells and the total β-galactosidase activity, thus maintaining the high expression per single cell. Using these optimized conditions, COS-1 cells expressed higher amounts of recombinant protein than COS-7 cells.     

Alphaviruses as Tools in Neurobiology and Gene Therapy

Abstract

The broad host range and superior infectivity of alphaviruses have encouraged the development of efficient expression vectors for Semliki Forest virus (SFV) and Sindbis virus (SIN). The generation of high-titer recombinant alphavirus stocks has allowed high-level expression of a multitude of nuclear, cytoplasmic, membrane-associated and secreted proteins in a variety of different cell lines and primary cell cultures. Despite the viral cytopathogenic effects, functional assays on recombinant proteins are possible for a time-period of at least 24 hours post-infection. The high percentage (80–95%) of primary neurons infected with SFV has allowed localization and functional studies of recombinant proteins in these primary cell cultures. Through multiple infection studies the interaction of receptor and G protein subunits has become feasible. Establishment of efficient scale-up procedures has allowed production of large quantities of recombinant protein. Potential gene therapy applications of alphaviruses could be demonstrated by injection of recombinant SIN particles expressing β-galactosidase into mouse brain. Tissue/cell specific infection has been achieved by introduction of an IgG-binding domain of protein A domain into one of the spike proteins of SIN. This enabled efficient targeting of infection to human lymphoblastoid cells.     

Heterologous expression of a gene encoding a thermostable β-galactosidase from <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis>

A genomic DNA library screen yielded the nucleotide sequence of a 12 kb fragment containing a gene (2067 bp) coding a thermostable β-galactosidase from Alicyclobacillus acidocaldarius ATCC 27009. The β-galactosidase gene was expressed in Pichia pastoris, and up to 90 mg recombinant β-galactosidase/l accumulated in shake flask cultures. Using o-nitrophenyl-β-d-galactopyranoside as a substrate, the optimum pH and temperature of the purified recombinant β-galactosidase were 5.8–6.0 and 70°C, respectively. The enzyme retained 90% of its activity when heated at 70°C for 30 min. Approximately 48% of lactose in milk was hydrolyzed following treatment with the recombinant enzyme over 60 min at 65°C.     

Gene transfer in bovine blastocysts using replication-defective retroviral vectors packaged with gibbon ape leukemia virus envelopes

With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat β-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli β-galactosidase marker gene in bovine target cells. By co-culture of bovine blastocysts and virus-producing cells, or by culture of embryos in the medium harvested from virus-producing cells, we transferred the E. coli β-galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV-based replication-defective retroviruses encapsidated with GaLV envelope proteins. The infection was confirmed by the expression of the E. coli β-galactosidase gene under a β-actin internal promoter. In addition, co-culture of ICM cells with virus-producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes. © 1993 Wiley-Liss, Inc.     

The specific inhibition of the expression of the lactose operon by D,L-threo-phenylserine inEscherichia coli

Phenylserine, one of the phenylalanine analogues, is incorporated into proteins ofEscherichia coli and replaces the natural amino acid. The incorporation results in the inhibition of the synthesis of both inducible and constitutive β-galactosidase. The rate of the synthesis of β-galactosidase specific m-RNA is only slightly influenced by phenylserine, the steady-state level being decreased by about 40%. The m-RNA formed in the present of the analogue functions normally and its translation after the removal of the inhibitor results in the formation of normal β-galactosidase. The character of the inhibition of the enzyme synthesis by phenylserine is similar to that caused by chloramphenicol. However, phenylserine specifically inhibits only the synthesis of β-galactosidase, whereas other cell proteins are synthesized. No protein immunologically cross-reacting with the antiserum against normal β-galactosidase is formed by inducible ánd constitutiveEscherichia coli strains. The active transport is completely inhibited as the cells induced in the presence of phenylserine do not accumulate¹⁴C-TMG. It follows from the results that phenylserine inhibits both the formation of TMG-specific permease and the synthesis of the active molecule of β-galactosidase inEscherichia coli.     

Production of a heterologous recombinant protein using fragments of the glyceraldehyde-3-phosphate dehydrogenase promoter from Penicillium camemberti

The biotechnological applications of cheese-ripening fungi have been limited by a lack of genetics tools, in particular the identification and characterization of suitable promoters for protein expression. In this study, the suitability of the glyceraldehyde-3-phosphate dehydrogenase (gpdP) promoter from Penicillium camemberti to drive the production of a recombinant protein was evaluated. The gpdP gene and its promoter were isolated using PCR and Genome Walker. The promoter of gpdP has two regions with high identity to the regulatory elements gpd-box and ct-box previously described in Aspergillus nidulans. Two fragments of the promoter containing the gpd- and ct-box or the ct-box alone were used to drive the in vivo production of recombinant β-galactosidase using A. nidulans as host. Our results indicate that larger fragment containing gpd-box enhances the production of β-galactosidase activity levels respect to ct-box alone, and that both boxes are necessary to obtain maximal enzymatic activity production. The smaller fragment (187 nt) containing the ct-box alone was able to trigger up to 27% of β-galactosidase activity, and to our knowledge this is the smallest fragment from a gpd gene used to produce a recombinant protein. Differences were not observed when glycerol, galactose or glucose were used as carbon sources, suggesting that the promoter activity is carbohydrate-independent. This is the first report in which a Penicillium gpd promoter is used for recombinant protein production. Our results open the way for the future development of a system for recombinant proteins expression in the biotechnologically important cheese-ripening fungus P. camemberti.     

Cloning and expression of a β-galactosidase gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta(?). Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

Proteolytic degradation of fused protein A-β-galactosidase in Escherichia coli

The stability of the fusion protein staphylococcal protein A-E. coli β-galactosidase (SpA-βgal) produced in E. coli has been studied both in cell disintegrate and in purified preparations. SpA-βgal was degraded by a proteolytic cleavage between the two functional parts of the molecule, resulting in one β-galactosidase tetramer and four protein A molecules. Intermediates were detected, namely β-galactosidase containing three, two and one protein A. The β-galactosidase was stable with respect to enzyme activity and molecular weight, while protein A was further degraded. In cell disintegrate the half-life of SpA-βgal was found to be 6 h at 20°C and 1.5 h at 37°C. The protease responsible for initial proteolytic cleavage of SpA-βgal was shown to be cell debris associated.     

RNAi suppression of β-N-acetylglucosaminidase (BmFDL) for complex-type N-linked glycan synthesis in cultured silkworm cells

Glycoproteins have various biological functions including enzymatic activity, protein stability and others. Due to the presence of paucimannosidic N-linked glycans, recombinant proteins from an insect cell expression system may not be suitable for therapeutic use. Because baculovirus expression systems (BESs) are used to produce recombinant proteins, it is of interest to modify the endogenous N-glycosylation pathway in insects to mimic that of mammals. Using a soaking RNAi sensitive cell line, BmN4-SID1, has enabled us to suppress Bombyx mori FDL (BmFDL), an N-linked glycan-specific β-N-acetylglucosaminidase. Western blotting and MALDI-TOF MS demonstrated that the BmFDL depletion almost completely converted the paucimannosidic structures of the recombinant proteins produced by BES into a complex-type structure. This highly efficient, simple and low-cost method can be used for mass production of secretion proteins with complex-type N-linked glycans.     

Assay and characterization of an osmolarity inducible promoter newly isolated from Bacillus subtilis

An osmolarity-sensitive promoter fragment, P23423, isolated from Bacillus subtilis was characterized. The expression of β-galactosidase (β-Gal) driven by P23423 was regulated by osmolarity both in Escherichia coli and B. subtilis. The classical conserved region of this prokaryotic promoter was found within the sequence of the cloned fragment, and the putative promoter was identified as the control element of RNA not coding for protein (a RNA molecule that is not translated into a protein). The efficiency and benefit of this promoter was further demonstrated via osmolarity-induced expression of three other heterologous proteins in E. coli. Thus, this approach provided a simple and inexpensive inducible promoter element for the expression of cloned genes.     

Transient metabolic modeling of Escherichia coli MG1655 and MG1655 ΔackA-pta, ΔpoxB Δpppc ppc-p37 for recombinant β-galactosidase production

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins, among other reasons because its genetics are far better characterized than those of any other microorganism. To improve the understanding of recombinant protein synthesis in E. coli, the production of a model recombinant protein, β-galactosidase, was studied in response to the constitutive overexpression of the anaplerotic reaction afforded by PEP carboxylase. To this end, an IPTG wash-in experiment was performed starting from a well-defined steady-state condition for both the wild-type E. coli and a mutant with a defective acetate pathway and a constitutively overexpressed ppc. In order to compare the dynamics of the fluxes over time during the wash-in experiment, a method referred to as transient metabolic flux analysis, which is based on steady-state metabolic flux analysis, was used. This allowed us to track the intracellular changes/fluxes in both strains. It was observed that the flux towards fermentation products was 3.6 times lower in the ppc overexpression mutant compared to the wild-type E. coli. In the former on the other hand, the PPC flux is in general higher. In addition, the flux towards β-galactosidase was higher (12.4 times), resulting in five times more protein activity. These results indicate that by constitutively overexpressing the anaplerotic ppc gene in E. coli, the TCA cycle intermediates are increasingly replenished. The additional supply of these protein precursors has a positive result on recombinant protein production.     

Antitumor Activity of an Alkali Extract of Clostridium saccharoperbutylacetonicum Cells

Bacillus thuringiensis var. israelensis produces 130-kDa proteins which are toxic to mosquito larvae. The ISRH4 gene encoding 1,180 amino acids of the 130-kDa insecticidal protein was fused with lac Z′ on a plasmid, pUC19, and sequentially deleted from the C-terminus to construct a series of deletion mutants. All the deletion mutant genes directed the production of truncated ISRH4 proteins fused with the α-complementing fragment of β-galactosidase in Escherichia coli cells in the presence of isopropyl β-d-thiogalactopyranoside. Analysis of the mosquito larvicidal activity of deletion mutant proteins revealed that the N-terminal 29 amino acids and the C-terminal 485 amino acids could be removed without loss of the activity.     

A 4.3 kb Smad7 promoter is able to specify gene expression during mouse development

Members of transforming growth factor-β (TGF-β) superfamily play important roles in diverse biological functions including early development. These extracellular factors exert their effects by interacting with membrane receptors followed by signal transduction by a group of Smad proteins. Smad7 is an inhibitory Smad protein that specifically antagonizes TGF-β and activin signaling. To characterize the developmental role of Smad7, a transgenic mouse model was generated using a 4.3 kb mouse Smad7 promoter driving β-galactosidase expression. In these mice, the Smad7 promoter defined a restrictive expression pattern of β-galactosidase in a tightly regulated temporal and spatial manner. The β-galactosidase gene was transiently expressed in the cardiovascular structures including heart cushion tissues and the endothelium of major arteries at E11.5 to E12.5. Through E12.5 to E17.5, β-galactosidase expression was prominently detected in the epithelium of developing cochlea and nasolacrimal duct. In addition, it was temporally expressed in trigeminal ganglion, the skeletal muscles surrounding major joints, primordium of the jaws, as well as genital tubercle. These studies indicated that the 4.3 kb Smad7 promoter contains sufficient regulatory elements to define controlled gene expression during mouse development.     

Cooperation between two ClpB isoforms enhances the recovery of the recombinant β-galactosidase from inclusion bodies

Bacterial ClpB is a molecular chaperone that solubilizes and reactivates aggregated proteins in cooperation with the DnaK chaperone system. The mechanism of protein disaggregation mediated by ClpB is linked to translocation of substrates through the central channel within the ring-hexameric structure of ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and the truncated ClpB80 (ClpBΔN), which does not contain the N-terminal domain. The functional specificity of the two ClpB isoforms and the biological role of the N-terminal domain are still not fully understood. Recently, it has been demonstrated that ClpB may achieve its full potential as an aggregate-reactivating chaperone through the functional interaction and synergistic cooperation of its two isoforms. It has been found that the most efficient resolubilization and reactivation of stress-aggregated proteins occurred in the presence of both ClpB95 and ClpB80. In this work, we asked if the two ClpB isoforms functionally cooperate in the solubilization and reactivation of proteins from insoluble inclusion bodies (IBs) in Escherichia coli cells. Using the model β-galactosidase fusion protein (VP1LAC), we found that solubilization and reactivation of enzymes entrapped in IBs occurred more efficiently in the presence of ClpB95 with ClpB80 than with either ClpB95 or ClpB80 alone. The two isoforms of ClpB chaperone acting together enhanced the solubility and enzymatic activity of β-galactosidase sequestered into IBs. Both ClpB isoforms were associated with IBs of β-galactosidase, what demonstrates their affinity to this type of aggregates. These results demonstrate a synergistic cooperation between the two isoforms of ClpB chaperone. In addition, no significant recovery of the β-galactosidase from IBs in ΔclpB mutant cells suggests that ClpB is a key chaperone in IB protein release.