Galactose can be an inducer for production of therapeutic proteins by auto-induction using E. coli BL21 strains

Recently lactose mediated auto-induction in Escherichia coli has gained a lot of interest because higher protein titer could be achieved without the need to monitor growth and add inducer at the proper time. In this study a high level therapeutic protein production by auto-induction was observed in E. coli BL21 using either T7 or tac promoters in the modified Luria Bertani (mLB) medium containing soy peptone instead of tryptone in Luria Bertani (LB) medium. Based on medium analysis and spiking experiments it was found that 0.4 mM galactose from the soy peptone caused the auto-induction. E. coli cultures induced by galactose can saturate at considerably higher density than cultures induced by IPTG. Galactose is not consumed by E. coli BL21. Finally it has been demonstrated that auto-induction can be effectively used in fed-batch fermentation for the industrial production of a therapeutic protein. The principle of galactose mediated auto-induction should be able to apply to high throughput microplates, shake flasks and fed-batch fermentors for clone screening and therapeutic protein expression in E. coli gal(-) strains such as most commonly used BL21.     

Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system

Bacteriophage T7 lysozyme, a natural inhibitor of T7 RNA polymerase, can reduce basal activity from an inducible gene for T7 RNA polymerase and allow relatively toxic genes to be established in the same cell under control of a T7 promoter. Low levels of T7 lysozyme supplied by plasmids pLysS or pLysL, which are compatible with the pET vectors for expressing genes from a T7 promoter, are sufficient to stabilize many target plasmids and yet allow high levels of target protein to be produced upon induction of T7 RNA polymerase. Higher levels of lysozyme supplied by plasmids pLysE or pLysH reduce the fully induced activity of T7 RNA polymerase such that induced cells can continue to grow and produce innocuous target proteins indefinitely. Different configurations of the expression system can maintain several different steady-state levels of target gene expression. The presence of T7 lysozyme has the further advantage of facilitating the lysis of cells in preparing extracts for purification of target gene products.     

Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination

Protocols have been developed and applied for the high-throughput production of [U-15N]- or [U-13C-, U-15N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B12, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of approximately 5, an average wet cell mass yield of approximately 9.5 g L(-1), and an average yield of approximately 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both 15N and 13C at levels greater than 95%. 1H-15N heteronuclear single quantum correlation spectroscopy as well as 13C; 15N-edited spectroscopy showed that the purified [U-15N]- and [U-13C, U-15N]-labeled proteins were suitable for structure analysis.     

Low temperature and glucose enhanced T7 RNA polymerase-based plasmid stability for increasing expression of glucagon-like peptide-2 in Escherichia coli

The high activity of T7 RNA polymerase has made the T7 RNA polymerase-based expression system very powerful for high-level expression of recombinant protein. However, the overactivity of T7 RNA polymerase would also bring about negative effects on plasmid stability and protein production, especially when expressing a toxic protein. If the latter role is dominant, it is necessary to adopt some measures to attenuate the activity or the amount of T7 RNA polymerase in the cells. Apart from the stringent regulation by inserting some genes reducing the amount or the activity of T7 RNA polymerase into plasmids, optimizing the culture conditions would be another way. In this work, we have studied the effects of various culture conditions on the plasmid stability and the target protein yield including selective pressure, culture temperature, toxicity of the target protein and the catabolite repression caused by glucose. The results have indicated that adding antibiotic after induction has little effect in increasing plasmid stability, but inducing expression at low temperature and adding glucose to the medium improved the plasmid stability and the protein yield to a large extent.     

Heterologous protein production in Escherichia coli using the propionate-inducible pPro system by conventional and auto-induction methods

We examined expression of two plant genes encoding coclaurine N-methyltransferase (CMT) and norcoclaurine synthase (NCS) in Escherichia coli from the Salmonella enterica prpBCDE promoter (P_prpB) and compared it to that from the strongest IPTG-inducible promoter, P_T7. In contrast to our previous study showing slightly higher production of green fluorescent protein (GFP) from the pPro system compared to that from the T7 system, production of two plant proteins CMT and NCS from P_prpB was 2- to 4-fold higher than that from P_T7. Unlike P_T7, expression from P_prpB did not reduce cell growth even when highly induced, indicating that this propionate-inducible system is more efficient for overproduction of proteins that result in growth inhibition. In an auto-induction experiment, which does not require monitoring the culture or adding inducer during cell growth, the pPro system exhibited much higher protein production than the T7 system. These results strongly indicate that the pPro system is well-suited for overproduction of recombinant proteins.     

Feline Calicivirus Capsid Protein Expression and Capsid Assembly in Cultured Feline Cells

The capsid protein of feline calicivirus (FCV) was expressed by using plasmids containing cytomegalovirus, simian virus 40, or T7 promoters. The strongest expression was achieved with the T7 promoter and coinfection with vaccinia virus expressing the T7 RNA polymerase (MVA/T7pol). The FCV precursor capsid protein was processed to the mature-size protein, and these proteins were assembled in to virus-like particles.     

Tryptophan promoter derivatives on multicopy plasmids: a comparative analysis of expression potentials in Escherichia coli

A collection of variant plasmids expressing either Escherichia coli galactokinase or human serum albumin under the control of several E. coli trp promoter derivatives were constructed and studied for both efficiency of expression and regulation by tryptophan. Several variables, including the length of the upstream region, tandem duplications of a core promoter, and the insertion of the trp repressor trpR gene onto the expression vector, were studied. It is shown that derivatives containing sequences upstream from the -35 region or multiple copies of the trp promoter produce twofold higher levels of protein than plasmids with a minimal trp promoter truncated at -40. We show that the expression of a heterologous protein such as albumin can be significantly improved (13% vs. 7% of total proteins) if both the upstream trp promoter region, which enhances promoter strength, and an intact trpR gene, are included on the plasmids.     

Broad host range, regulated expression system utilizing bacteriophage T7 RNA polymerase and promoter

An IPTF-regulated broad host range expression system was constructed using compatible broad host range plasmids, the T7 RNA polymerase, and T7 promoter sequences. The system is implemented by the coexistence of two plasmids. The first contains the T7 RNA polymerase gene under the control of lacl or lacl(q) genes and lacUV5 promoter. The second encodes the T7 promoter upstream of a multicloning site. IncP1 or IncP4 T7 promoter plasmids, and IncP1, IncP4 or IncW T7 RNA polymerase plasmids were constructed. The expression from the IncP1 promoter plasmids in the presence of the IncP4 polymerase plasmids was tested by in vivo lacZ fusions and vivo labeling of proteins. In this combination, the use of lac(q) improves the regulation levels in Escherichia coli, whereas, in Pseudomonas phaseolicola, a 28.5-fold regulation was obtained with lacl, Although the level of lacZ expression from the T7 promoter in P. phaseolicola is low compared with E. coli, it is similar to levels obtained with the pm promoter in Pseudomonas putida when the differences in the copy number of the expression vectors are taken into consideration (c) 1993 Wiley & Sons, Inc.     

Reliable scale-up of membrane protein over-expression by bacterial auto-induction: From microwell plates to pilot scale fermentations

The production of well-ordered crystals of membrane proteins for structural investigation by X-ray diffraction typically requires extensive crystallization trials and may involve the screening of multiple detergents, lipids and other additives. Purification of sufficient amounts of protein for such trials is hampered by the fact that even when over-expressed, membrane proteins represent only a small percentage of the total protein content of bacteria. Fermentation-scale cultures of cells are therefore usually required. To maximize the efficiency and reduce the cost of such cultures, in the UK Membrane Protein Structure Initiative we have systematically investigated the use of auto-induction as an alternative to induction of expression with isopropyl-β-D-thiogalactoside. We report here the benefits of first optimizing expression on a multiwell plate scale by systematically varying the concentrations of glucose, glycerol, lactose and succinate present in the auto-induction medium. For subsequent scale-up, comparison of isopropyl-β-D-thiogalactoside induction in shake-flasks with auto-induction in shake-flasks and in 1L fermenters without and with control of pH and aeration revealed that highest yields of target protein were obtained using the latter culture conditions. However, analysis of the time-course of expression highlighted the importance of choosing the correct time for harvest. The high yields of target protein that can be obtained in a single batch by auto-induction, performed on a 30 l scale in a fermenter, obviate batch-to-batch variations that can add an unwanted variable to crystallization screening experiments. The approach described should therefore be of great utility for membrane protein production for structural studies.     

Stringent regulation and high-level expression of heterologous genes in Escherichia coli using T7 system controllable by the araBAD promoter

The recombinant Eschreichia coli strain BL21 (BAD) was constructed to carry a chromosomal copy of T7 gene 1 fused to the araBAD promoter. To further characterize this expression system, strain BL21 (BAD) was transformed with the plasmid containing the carbamoylase gene from Agrobacterium radiobacter driven by the T7 promoter. Upon induction with L-arabinose, recombinant cells produced 100-fold increase in carbamoylase activity in comparison with uninduced cells on M9 semidefined medium plus glycerol. This protein yield accounts for 30% of total cell protein content. In addition, it was found that after 100 generations the plasmid harboring the carbamoylase gene remained firmly stable in strain BL21 (BAD), but its stability dropped to only 20-30% in strain BL21 (DE3), a commercial strain bearing T7 gene 1 regulated by the lacUV5 promoter in its chromosome. In an attempt to enhance the total protein yield, fed-batch fermentation process was carried out using a two-stage feeding strategy to compartmentalize cell growth and protein synthesis. In the batch fermentation stage, the culture was grown on glucose to reach the stationary growth phase. Subsequently, glycerol was fed to the culture broth and L-arabinose was augmented to induce protein production when cells entered the late log growth phase. As a result, a carbamoylase yield corresponding to 5525 units was obtained, which amounts to a 337-fold increase over that achieved on a shake-flask scale. Taken together, these results illustrate the practical usefulness of T7 system under control of the araBAD promoter for heterologous protein production.     

人胰高血糖素样肽-1突变体融合蛋白在大肠杆菌中的自诱导表达优化

考察了在大肠杆菌中自诱导表达人胰高血糖素样肽-1突变体融合蛋白的可行性,并对自诱导培养条件及培养基成分进行优化,以提高蛋白产量。实验结果表明,最优培养基成分为蛋白胨19.17g/L,酵母膏9.59g/L,Na2HPO45.72g/L,KH2PO45.48g/L,(NH4)2SO42.66g/L,NaCl3.33g/L,甘油2%(V/V),葡萄糖0.68g/L,乳糖6.33g/L,MgSO40.24g/L。在温度33°C、接种量1%、pH7、装瓶量20mL/100mL培养条件下,用该最优培养基自诱导表达人胰高血糖素样肽-1突变体融合蛋白的产量可达348.6mg/L。     

Simultaneous stable expression of neomycin phosphotransferase and green fluorescence protein genes in Trypanosoma cruzi

The ribosomal RNA (rRNA) gene promoter was used to construct plasmid vectors that simultaneously express multiple exogenous genes in Trypanosoma cruzi. Vector pBSPANEO expresses neomycin phosphotransferase, and pPAGFPAN expresses both green fluorescent protein and neomycin phosphotransferase from a single promoter. Both vectors require the presence of the rRNA promoter for stable transfection; epimastigotes transfected with pPAGFPAN strongly fluoresced due to green fluorescent protein expression. Intact plasmids were rescued from the T. cruzi-transfected population after >8 mo of culture, indicating stable replication of these vectors. Vectors were integrated into the rRNA locus by homologous recombination and into other loci, presumably by illegitimate recombination. Parasites bearing tandem concatamers of plasmids were also found among the transfectants. Transfectants expressing green fluorescent protein showed a bright green fluorescence distributed throughout the cell. Fluorescence was also detected in amastigotes after infection of mammalian cells with transfected parasites, indicating that the rRNA promoter can drive efficient expression of these reporter genes in multiple life-cycle stages of the parasite. Expression of the heterologous genes was detected after passage in mice or in the insect vector. These vectors will be useful for the genetic dissection of T. cruzi biology and pathogenesis.     

Protocols for production of selenomethionine-labeled proteins in 2-L polyethylene terephthalate bottles using auto-induction medium

Protocols have been developed and applied in the high-throughput production of selenomethionine labeled fusion proteins using the conditional Met auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing 125 mg L(-1) selenomethionine, salts and trace metals, other amino acids including 10 mg L(-1) of methionine, vitamins except vitamin B12, and glucose, glycerol, and alpha-lactose. A schematic for a shaker rack that can hold up to twenty-four 2-L polyethylene terephthalate beverage bottles in a standard laboratory refrigerated floor shaker is provided. The growth cycle from inoculation of the culture bottle through the growth, induction, and expression was timed to take approximately 24 h. Culture growth in the auto-induction medium gave an average final optical density at 600 nm of approximately 6 and an average wet cell mass yield of approximately 14 g from 2 L of culture in greater than 150 expression trials. A simple method for visual scoring of denaturing electrophoresis gels for total protein expression, solubility, and effectiveness of fusion protein proteolysis was developed and applied. For the favorably scored expression trials, the average yield of purified, selenomethionine-labeled target protein obtained after proteolysis of the fusion protein was approximately 30 mg. Analysis by mass spectrometry showed greater than 90% incorporation of selenomethionine over a approximately 8-fold range of selenomethionine concentrations in the growth medium, with higher growth rates observed at the lower selenomethionine concentrations. These protein preparations have been utilized to solve X-ray crystal structures by multiwavelength anomalous diffraction phasing.     

Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration

We report the construction of two novel Escherichia coli strains (DH1lacdapD and DH1lacP2dapD) that facilitate the antibiotic-free selection and stable maintenance of recombinant plasmids in complex media. They contain the essential chromosomal gene, dapD, under the control of the lac operator/promoter. Unless supplemented with IPTG (which induces expression of dapD) or DAP, these cells lyse. However, when the strains are transformed with a multicopy plasmid containing the lac operator, the operator competitively titrates the LacI repressor and allows expression of dapD from the lac promoter. Thus transformants can be isolated and propagated simply by their ability to grow on any medium by repressor titration selection. No antibiotic resistance genes or other protein expressing sequences are required on the plasmid, and antibiotics are not necessary for plasmid selection, making these strains a valuable tool for therapeutic DNA and recombinant protein production. We describe the construction of these strains and demonstrate plasmid selection and maintenance by repressor titration, using the new pORT plasmid vectors designed to facilitate recombinant DNA exploitation.     

A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression

Yeasts are attractive organisms for recombinant protein production. They combine highly developed genetic systems and ease of use with reductions in time and costs. We describe an autoselection system for recombinant protein expression in Saccharomyces cerevisiae which increases yields 5-10-fold compared to conditional selection for expression plasmids. Multicopy expression plasmids encoding essential MOB1 or CDC28 genes are absolutely necessary for the viability of host cells with mob1 or cdc28 deletions in their genomes. Such plasmids are stably maintained, even in rich medium, so optimising biomass production and yields of recombinant protein. Plasmid copy numbers are also increased by limiting selective MOB1 and CDC28 gene expression prior to induction. GST- or 6His-tagged proteins are produced for affinity purification and are expressed from a conditional GAL1-10 promoter to avoid potentially toxic effects of recombinant proteins on growth. Autoselection systems for expressing single or pairs of proteins are described. We demonstrate the versatility of this system by expressing proteins from a number of organisms and include several large and problematic products. The in vitro reconstruction of a step in mitotic regulation shows how this expression system can be successfully applied to the detailed analysis of complex metabolic pathways.     

乳糖作为诱导剂对重组目的蛋白表达的影响

将重组粒细胞-巨噬细胞集落刺激因子/白细胞介素3(GM-CSF/IL-3)融合蛋白表达菌BL21(DE3)(pFu)作为研究对象,对于以乳糖作为诱导剂时重组目的产物的诱导表达规律进行了深入的研究。分析比较了不同培养基中,不同生长阶段进行诱导对于产物表达的影响。对诱导所需的乳糖浓度、诱导持续时间长短等因素亦进行了研究。实验结果表明,在对诱导条件进行优化控制的前提下,利用乳糖作为诱导剂可以达到与IPTG类似的诱导效果。随后的研究中,将乳糖作为诱导剂应用于高密度发酵过程。这些研究结果为乳糖作为诱导剂最终应用于重组基因工程药物的工业化生产提供了有益的参考和借鉴。