Versatile Escherichia coli Expression Vectors for Production of Truncated Proteins

Several expression vector plasmids containing the tac promoter, the rrnBT₁T₂ terminator, and the pUC ori sequence were constructed. Some of them, the pES series, have a start codon in all three reading frames and multiple cloning sites downstream of the tac promoter and have stop codons also in all three frames and additional stop codons accompanying restriction sites. They are designed for versatile expression of truncated proteins which are produced by deleting portions of the inserted DNA.     

Temperature-regulated expression of the tac/lacI system for overproduction of a fungal xylanase in Escherichia coli

 Temperature-regulated expression of recombinant proteins in the tac promoter (P_tac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the P_tac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42°C were about 4.5 times higher than those of the cells grown at 23°C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl β-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated P_tac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacI ^q, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the λP_L system, the P_tac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best λP_L-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated P_tac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated P_tac system can be used for overproduction of some non-toxic recombinant proteins. Received: 27 June 1995/Received revision: 13 September 1995/Accepted: 30 September 1995     

Development of an<Emphasis Type="Italic">E. coli</Emphasis> expression cassette for the efficient production of a harmful protein

In order to produce a harmful protein more efficiently, this expression cassette, dubbed pCol-MICT, is directed by the colicin promoter, and was constructed by the insertion of a rrnBT₁T₂ fragment of pEXP7, and a MxelnteinCBD fragment of pTXB3, into pSH375. To test whether harmful proteins, including proteolytic enzymes, could be effectively produced by this cassette, the carboxypeptidase (CPase)Taq gene was inserted into the pCol-MICT cassette to yield pCol-CPaseTaq-MICT.E. coli W3110 cells harboring pCol-CPaseTaq-MICT produced a large quantity of this enzyme, as much as 47.2 mg of purified from per liter of culture, when cultured in the presence of mitomycin C (0.4 μg/mL). This indicates that the colicin promoter-controlledE. coli expression cassette was able to produce almost 8 times of protein than the conventionaltac promoter-based system, and that this cassette may be useful in the synthesis of other harmful proteins.     

Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16

Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P _lac , P _tac , or P _BAD derived from Escherichia coli, or promoter regions of phaC1 (P _phaC ) or phaP1 (P _phaP ) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P _lac , P _tac , P _phaC , and P _phaP mediated constitutive gene expression, among which P _tac was the strongest promoter. lacI-P _tac was not thoroughly functional even after addition of isopropyl-β-d-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-P _BAD could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-P _phaP exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-P _phaP for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.     

High level expression of human lipocortin (annexin) 1 in Escherichia coli

Summary Human lipocortin (annexin) 1, a member of the annexin family of phospholipid binding proteins, has previously been expressed in E. coli (Huh et al., 1990). To improve the expression level of lipocortin 1 in E. coli, several expression vectors containing either the P_L or the P_trc promoter were constructed. The highest expression level, up to 20 % of the total E. coli proteins, was obtained with plasmid pHT3. Plasmid pHT3 contains the pUC origin, the lipocortin 1 cDNA under P_trc promoter, and the lacI gene.     

Stable transformation ofArabidopsis with thebar gene using particle bombardment

A plasmid pARK 22 harbouring thebar gene encoding phosphinothricin acetyltransferase (PAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator was constructed and introduced into root sections ofArabidopsis thaliana using the pneumatic particle gun. The root sections that had been bombarded with this plasmid gave four to eight times higher yield of drug-resistant calluses than those sections bombarded with pCaMVNEO or pCH, which respectively contain the neomycin phosphotransferase and hygromycin phosphotransferase genes. Among a number of primary transformant (T₀) plants obtained from independent bialaphos-resistant calluses, three were studied by Southern blot hybridization and PAT enzyme activity analyses, confirming the stable integration of the foreign gene into theArabidopsis genome and its expression in plants. The progeny analysis showed transmission of the foreign gene and its expression in up to the T₂ generation. Some of the T₁ progeny showed morphological abnormalities. Thus, thebar gene can be used effectively to allow selection of transgenicA. thalianna plants.     

pHsh vectors,a novel expression system of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the large-scale production of recombinant enzymes

Gene expression system Hsh is developed to increase enzyme production and to decrease the cost in the induction of gene expression in Escherichia coli. The vectors of Hsh system were constructed by combining a synthesized heat-shock promoter with a synthesized terminator and an origin of replication derived from pUC19 in which the expression of foreign genes was regulated by an alternative sigma factor, σ³² of E. coli. In comparison, the Hsh promoter gave a 2.4-fold higher production for xynIII gene encoding a xylanase than existing heat-shock inducible promoter p _L, 1.2-fold and 3-fold production for xar gene encoding a arabinosidase than trc and T₇ promoter, respectively. The flow-in-heat technique created a rapid rise in temperature for effective induction of gene expression in bioreactor scale.     

Development of dual-inducible duet-expression vectors for tunable gene expression control and CRISPR interference-based gene repression in Pseudomonas putida KT2440

The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual-inducible duet-expression vectors for P. putida KT2440. A number of inducible promoters (P_lac, P_tac, P_tetR/tetA and P_bad) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.     

Tissue-specific and ABA-regulated Maize GIN gene expression in transgenic tobacco

Summary To study the regulatory functions of the ON promoter region, a ppG1b1GUS construct, consisting of 1402 bp 5 flanking sequence ofGlbl, 1919 by GUS coding sequence, and 283 by 3 NOS terminator, was cloned into a binary vector and introduced into tobacco plants byAgrobacterium-mediated transformation. Histochemical GUS assays of T_o tobacco mature seeds indicate that theGlbl promoter drives GUS expression in ABA treated seeds. Further GUS assays of the T, seeds at different developmental stages revealed that without ABA treatment, theGibl promoter drives GUS expression in immature seeds. The results from both T_o and T₁ tobacco plants indicated thatGlbl-driven GUS expression in tobacco is embryo specific.     

Construction of two Gateway vectors for gene expression in fungi

We report the construction of two Gateway fungal expression vectors pCBGW and pGWBF. The pCBGW was generated by introducing an expression cassette, which consists of a Gateway recombinant cassette (attR1-Cmr-ccdB-attR2) under the control of fungal promoter PgpdA and a terminator TtrpC, into the multiple cloning site of fungal vector pCB1004. The pGWBF is a binary vector, which was generated from the plant expression vector pGWB2 by replacing the CaMV35S promoter with PgpdA. The pGWBF can be transformed into fungi efficiently with Agrobacterium-mediated transformation. The applicability of two newly constructed vectors was tested by generating the destination vectors pGWBF-GFP and pCBGW-GFP and examining the expression of GFP gene in Trichoderma viride and Gibberella fujikuroi, respectively. Combining with the advantage of Gateway cloning technology, pCBGW and pGWBF will be useful in fungi for large-scale investigation of gene functions by constructing the interested gene destination/expression vectors in a high-throughput way.     

Promoter engineering to optimize recombinant periplasmic Fab′ fragment production in Escherichia coli

Fab’ fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab’ fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab’ fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the P_tac promoter, present in the plasmid, pTTOD‐A33 Fab’, to a P_tic promoter which has been shown by others to direct expression at a 35% reduced rate compared to P_tac. We characterized the resultant production trains in which either P_tic or P_tac promoters direct Fab’ fragment expression. The P_tic promoter strain showed a 25?30% reduction in Fab’ expression relative to the original P_tac strain. Reduced Fab’ leakage and increased viability over the course of a fed‐batch fermentation were also observed for the P_tic promoter strain. We conclude that cell design steps such as the P_tac to P_tic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab’ fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840–847, 2016     

Cloning and expression of the α–L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae

 First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1 _P ) and terminator (PGK1 _T ) sequences on a multicopy episomal plasmid. The resulting construct PGK1 _P -abf B-PGK1 _T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively. Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996     

Incident radiation and the allocation of nitrogen within Arctic plant canopies: implications for predicting gross primary productivity

Arctic vegetation is characterized by high spatial variability in plant functional type (PFT) composition and gross primary productivity (P). Despite this variability, the two main drivers of P in sub‐Arctic tundra are leaf area index (L_T) and total foliar nitrogen (N_T). L_T and N_T have been shown to be tightly coupled across PFTs in sub‐Arctic tundra vegetation, which simplifies up‐scaling by allowing quantification of the main drivers of P from remotely sensed L_T. Our objective was to test the L_T–N_T relationship across multiple Arctic latitudes and to assess L_T as a predictor of P for the pan‐Arctic. Including PFT‐specific parameters in models of L_T–N_T coupling provided only incremental improvements in model fit, but significant improvements were gained from including site‐specific parameters. The degree of curvature in the L_T–N_T relationship, controlled by a fitted canopy nitrogen extinction co‐efficient, was negatively related to average levels of diffuse radiation at a site. This is consistent with theoretical predictions of more uniform vertical canopy N distributions under diffuse light conditions. Higher latitude sites had higher average leaf N content by mass (N_M), and we show for the first time that L_T–N_T coupling is achieved across latitudes via canopy‐scale trade‐offs between N_M and leaf mass per unit leaf area (L_M). Site‐specific parameters provided small but significant improvements in models of P based on L_T and moss cover. Our results suggest that differences in L_T–N_T coupling between sites could be used to improve pan‐Arctic models of P and we provide unique evidence that prevailing radiation conditions can significantly affect N allocation over regional scales.     

High expression vectors for the production of recombinant single-chain urinary plasminogen activator from Escherichia coli

Summary An expression cassette containing a synonymous gene for human single-chain urokinase-type plasminogen activator (Rscu-PA) 5'-flanked by a trp promoter and the Shine-Dalgarno sequence of the xyl A operon of Bacillus subtilis and terminated by the terminators trp A and Tn10 was constructed and inserted into a pBR322 derivative to yield pBF160. When compared to pUK54 trp 207-1 containing the natural scu-PA gene without the Shine-Dalgarno sequence and terminator, the expression efficiency of pBF160 in Escherichia coli strains was improved by one order of magnitude. Replacement of the trp by the tac promoter (pBF171) did not affect expression. Inserting the Shine-Dalgarno sequence and Tn10 terminator into pUK54 trp 207-1 (pWH1320) slightly increased the expression level, whereas elimination of the Shine-Dalgarno sequence and the terminators from pBF160 with almost complete conservation of the synonymous structural gene (pBF191) significantly reduced the expression. Variation of the distance between the Shine-Dalgarno sequence and the start codon between 8 and 10 bp (pBF163) proved irrelevant. In conclusion, poor expression of mammalian genes in E. coli may result from both improperly designed regulatory elements and structural features of the coding region and therefore de-novo synthesis of the gene may be required to obtain satisfactory expression.     

Construction of recombinant K88 DNA with Ptac promoter

The gene encoding K88ab was localized on the 11.6 kbHindIII-HindIII fragment of 74 kb plasmid DNA ofE. coli 7301. The smallest recombinant DNA producing the K88ab antigen was obtained by excision of the 5.15 kbEcoRI-EcoRI fragment from recombinant DNA composed of the 11.6 kb K88ab fragment in the pBR322 vectro. The size of the smallest fragment was 6.5 kb. Expression of the K88ab antigen was controlled by the P1 promoter of the pBR322 vector. Substitution of promoter P_tac for promoter P1 made it possible to achieve expression of the K88ab antigen byE. coli MT. Substitution of promoter P_L for promoter P1 failed to achieve expression of the K88 ab antigen in the recipient strains used.     

Irradiance influences tea leaf (<Emphasis Type="Italic">Camellia sinensis</Emphasis> L.) photosynthesis and transpiration

Rates of net photosynthesis (P _N) and transpiration (E), and leaf temperature (T_L) of maintenance leaves of tea under plucking were affected by photosynthetic photon flux densities (PPFD) of 200–2 200 μmol m⁻² s⁻¹. P _N gradually increased with the increase of PPFD from 200 to 1 200 μmol m⁻² s⁻¹ and thereafter sharply declined. Maximum P _N was 13.95 μmol m⁻² s⁻¹ at 1 200 μmol m⁻² s⁻¹ PPFD. There was no significant variation of P _N among PPFD at 1 400–1 800 μmol m⁻² s⁻¹. Significant drop of P _N occurred at 2 000 μmol m⁻² s⁻¹. PPFD at 2 200 μmol m⁻² s⁻¹ reduced photosynthesis to 6.92 μmol m⁻² s⁻¹. PPFD had a strong correlation with T_L and E. Both T_L and E linearly increased from 200 to 2 200 μmol m⁻² s⁻¹ PPFD. T_L and E were highly correlated. The optimum T_L for maximum P _N was 26.0 °C after which P _N declined significantly. E had a positive correlation with P _N.