Constitutive Expression System Research Articles

Preparation of high activity yeast whole cell bioctalysts by optimization of intracellular production of recombinant Rhizopus oryzae lipase

Yeast whole cell biocatalysts, which intracellularly overproduced a recombinant lipase with a pro-sequence from Rhizopus oryzae IFO4697 (rProROL) were constructed, and the content of active lipase in Saccharomyces cerevisiae cells was maximized by optimizing the cultivation procedure. rProROL was overproduced intracellularly under the control of the 5′-upstream region of the isocitrate lyase gene of Candida tropicalis ( UPR-ICL) as the inducible system and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter as the constitutive expression system. Enhancement of expression level of ProROL gene at the initial cultivation phase inhibited rProROL accumulation in yeast cells both in GAPDH promoter system by using high glucose concentration at 30 °C and in UPR-ICL system by using non-fermentable carbon sources. The highest intracellular lipase activity of 350.6 IU/l was obtained in the inducible UPR-ICL system with an initial glucose concentration of 0.5% at 30 °C. To prepare the efficient whole cell biocatalyst by intracellular overproduction of lipase, utilization of inducible UPR-ICL and the optimization of cultivation conditions such as temperature, carbon source and its initial concentration are important.

Novel high-level constitutive expression system, pHCE vector, for a convenient and cost-effective soluble production of human tumor necrosis factor-α

A new E. coli expression system, the pHCE-IIB vector, using a constitutively expressing HCE promoter derived upstream from the d-amino acid aminotransferase (d-AAT) gene of Geobacillus toebii, was developed for the high-level expression of foreign proteins without induction. The expression efficiency of recombinant human tumor necrosis factor-α (rhTNFα) using pHCE-IIB was compared with that of the pET14B vector under the control of a T7 promoter induced by IPTG. The total amount of rhTNFα produced by the pHCE system was approximately twice that produced by the pET system under optimal fermentation conditions thereby demonstrating the convenience and economical advantage of the new constitutive pHCE-IIB expression vector over the conventional pET expression vector.

Evaluation of the tetracycline- and ecdysone-inducible systems for expression of neurotransmitter receptors in mammalian cells.

Establishing a stable cell line that expresses a particular protein of interest is often a laborious and time-consuming experience. With constitutive expression systems, a gradual loss of the highly expressing clones over a given time span and/or a severe counter-selection due to toxicity of the expressed protein for the host cell line are major drawbacks. In both cases, inducible expression systems offer a valuable alternative. Over the years, many regulated expression systems have been developed and evaluated. In the present study, we compare the efficiency, the advantages and the drawbacks of a tetracycline- and an ecdysone-inducible system for expression of the reporter protein chloramphenicol acetyltransferase and of different G-protein-coupled serotonin (5-HT) receptors. A high level of expression of different 5-HT receptors was obtained with the tetracycline-inducible system. In the cell line L929, which stably expresses the tetracycline-responsive transactivator, a maximum ligand binding of 20,000 and 9500 fmol/mg protein was measured for the h5-HT(1B) and h5-ht(1F) receptors, respectively. In the HEK293rtTA cell line, levels of 15,700, 3000, and 9100 fmol bound ligand/mg protein were obtained for the h5-HT(1B), h5-ht(1F) and h5-HT(4b) receptors, respectively. These high expression levels remained stable for several months of continuous culture. Although the ecdysone-inducible expression system was useful for tightly regulated expression, the levels were far lower than those obtained with the tetracycline system (e.g. 640 fmol bound ligand/mg protein for the h5-ht(1F) receptor in HEK293EcR).

Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter

High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase ( AOX1) promoter. However, the time taken to reach peak product concentration is usually very long (∼240 h). In this paper, we describe the expression of HBsAg in P. pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase ( GAP) promoter. Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose. Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant. However, this was offset by continuous antigen production by the GAP clone. In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure. The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones. The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg. The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen. These particles were sensitive to thiol reagents. We also explored the possibility of secreting the GAP expressed HBsAg in P. pastoris. In-frame fusion of the Saccharomyces cerevisiae α-factor secretion signal under the constitutive GAP promoter resulted in secretion of ∼20 nm HBsAg particles as evidenced by electron microscopy. However, the levels of secreted HBsAg particles were very low, presumably due to the inherent hydrophobicity of the HBsAg molecule and the consequent propensity for membrane association. Our studies show that secretion is not a good strategy for expression of HBsAg in P. pastoris. The data also suggests that intracellular production of HBsAg under the GAP promoter using multicopy expression cassettes can indeed serve as an effective alternative to the AOX1 promoter. Further, the GAP promoter based system obviates the need to use and extensively monitor methanol during recombinant antigen production. Finally, this constitutive system has the potential for continuous culture wherein several batches of recombinant protein-containing biomass can be harvested from a single initial fermentation.

Cloning, Overexpression and Purification of Bacillus subtilis Elongation Factor Tu in Escherichia coli

To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His6 tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.

Cloning, overexpression and purification of Bacillus subtilis elongation factor Tu in Escherichia coli.

To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His6 tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.

Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters.

Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) promoter and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in a fermentor. Production of Cel7A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N-glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The k(cat) and K(m) values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site-directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.

Mass production of thermostable D-hydantoinase by batch culture of recombinantEscherichia coli with a constitutive expression system

D-Hydantoinase is an industrial enzyme widely used for the synthesis of optically active D-amino acids. A gene encoding thermostable D-hydantoinase of Bacillus stearothermophilus SD-1 has previously been cloned and constitutively expressed by its native promoter in Escherichia coli XL1-Blue (Lee et al., 1996b). In this work, we attempted mass production of the D-hydantoinase by batch culture of the recombinant E. coli using glycerol as a carbon source. The plasmid content in cells increased in proportion to the culture temperature, which resulted in a two- or three-fold increase of the specific D-hydantoinase activity at 37 degrees C compared with that at 30 degrees C. The plasmid was stably maintained over 80 generations. When glycerol was initially added to a concentration of 100 g/L, the final biomass concentration reached about 50 g-dry cell weight/L in a 50 L-scale fermentation, resulting in the specific enzyme production of 3.8 x 10(4) unit/g-dry cell weight in a soluble form. Glycerol-using batch cultivation of recombinant E. coli was found to be a cost-effective process for the mass production of industrially useful D-hydantoinase. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 449-455, 1997.

Characterization of dominant lethal mutations in the yeast plasma membrane H +-ATPase gene

Site-directed mutants of yeast ATPase were studied after introduction of mutant alleles into a yeast strain where these alleles were constitutively expressed and the expression of the wild-type chromosomal ATPase gene was turned off. One objection to this constitutive expression system was made apparent recently, as dominant lethal mutations are lost by gene conversion with the wild-type allele during the process. Here, the phenotypes of the mutant alleles, which were studied in a constitutive expression system, are re-evaluated under conditions in which these site-directed mutants are conditionally expressed. We show that 12 of 25 site-directed mutations previously described are actually dominant lethal alleles. In addition, we show that dominant mutant proteins interfere with transport of wild-type ATPase to the plasma membrane.

Colicins B and Ia as novel counterselective agents in interspecies conjugal DNA transfers from colicin-sensitive Escherichia coli donors to other Gram-negative recipient species

Colicins are powerful counterselective agents in conjugal DNA transfers using colicin-sensitive Escherichia coli donor strains and colicin-resistant recipient species. We have constructed a high-level constitutive expression system for the bacteriocin and immunity genes of channel-forming colicins B and Ia to optimize production of these colicins as molecular biology reagents.

High level expression inEscherichia coliof a fungal gene under the control of strong promoters

A recent report (Patino et al., (1989) FEMS Microbiol. Lett. 58, 139–144) described the low level expression, in Escherichia coli, of the Isopenicillin N Synthase (IPNS) gene the Cephalosporiumacremonium under the control of strong promoters. We report here our work on the expression of the IPNS gene. Plasmids containing the IPNS gene under the control of the trp or trc promoters directed synthesis of high levels of active IPNS in E. coli. Constitutive and inductive high level IPNS expression systems have been developed. Importantly, the expression vectors do not encode β-lactamase so IPNS activity can be determined directly by biological assays. Analysis by nmr verified that the IPNS produced from these expression systems catalysed the conversion or δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (LLD-ACV) to isopenicillin N in high yield.

High level expression in Escherichia coli of a fungal gene under the control of strong promoters

A recent report (Patino et al., (1989) FEMS Microbiol. Lett. 58, 139–144) described the low level expression, in Escherichia coli, of the Isopenicillin N Synthase (IPNS) gene the Cephalosporium acremonium under the control of strong promoters. We report here our work on the expression of the IPNS gene. Plasmids containing the IPNS gene under the control of the trp or trc promoters directed synthesis of high levels of active IPNS in E. coli. Constitutive and inductive high level IPNS expression systems have been developed. Importantly, the expression vectors do not encode β-lactamase so IPNS activity can be determined directly by biological assays. Analysis by nmr verified that the IPNS produced from these expression systems catalysed the conversion or δ-( l-α-aminoadipoyl)- l-cysteinyl- d-valine (LLD-ACV) to isopenicillin N in high yield.