重组人载脂蛋白AI米兰突变体在大肠杆菌中的高表达

用RT-PCR法从人肝总RNA库中克隆出人载脂蛋白Al的cDNA序列,再通过重叠PCR将载脂蛋白AI的第179位精氨酸密码子突变成半胱氨酸密码子,即载胎蛋白AI米兰突变体基因。将此目的基因克隆至表达载体pQE30,重组质粒转化JMl09宿主菌,经表达试验筛选出高表达克隆;工程菌经诱导后表达出含6个氨基酸前肽的载脂蛋白AI米兰突变体。表达产物主要以可溶形式存在,但也有部分为包涵体。     

Tunable recombinant protein expression with E. coli in a mixed-feed environment

Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both d-glucose and l-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of l-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.     

Protein expression in E. coli minicells by recombinant plasmids.

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.     

Ultra-high expression of a thermally responsive recombinant fusion protein in E. coli

Elastin-like polypeptides (ELPs) are recombinant peptide-based biopolymers that contain repetitive sequences enriched in glycine, valine, proline, and alanine. Because of the unusually large fraction of these amino acids in ELPs as compared to other cellular proteins, we hypothesized that intracellular pools of these amino acids can be selectively depleted and limit protein yields during expression. In this study, we examined how culture conditions and individual medium components affect protein yields by monitoring cell growth and protein expression kinetics of E. coli expressing an ELP tagged with a green fluorescent protein (GFP). By determining the underlying principles of superior fusion protein yields generated by the hyperexpression protocol, we further improved protein yields through the addition of glycerol and certain amino acids such as proline and alanine and found that amino acid concentrations and the type of basal medium used strongly influenced this beneficial effect. Surprisingly, amino acids other than those that are abundant in ELPs, for example, asparagine, aspartic acid, glutamine, and glutamic acid, also enhanced protein yields even in a nutrient-rich medium. Compared to commonly used Luria-Bertani medium, the protein yield was improved by 36-fold to the remarkable level of 1.6 g/L in shaker flask cultures with a modified medium and optimized culture conditions, which also led to a 8-fold reduction in the cost of the fusion protein. To our knowledge, this is the highest yield of an ELP-fusion protein purified from E. coli cultured in shaker flasks. This study also suggests a useful strategy to improve the yields of other ELP fusion proteins and repetitive polypeptides.     

Optimized conditions for high-level expression and purification of recombinant human interleukin-2 in E. coli

Interleukin-2 (IL-2), a potent cytokine has been used in anti-cancer therapy for over a decade now. IL-2, originally identified as a growth factor for T lymphocytes is a 15 kDa hydrophobic glycoprotein that induces the activation, clonal proliferation and differentiation of T and B-lymphocytes and enhances the cytotoxicity of monocytes and natural killer (NK) cells. Here, we report a simple method for the cloning, high-level expression and purification of IL-2 protein, which can be easily extended to other bioactive therapeutic proteins. The IL-2 gene was amplified from human spleen cDNA and cloned in a prokaryotic (E. coli) expression system. An optimal expression of the IL-2 protein was determined by varying the expression conditions like temperature, inducer concentration and duration of induction. The protein was expressed as inclusion bodies and a panel of reagents including detergents, urea and guanidine hydrochloride were used to solubilize it. After solubilization, the protein was renatured and subjected to a single step gel-filtration chromatography to yield immunobioactive IL-2 protein with > 99% purity.     

High level expression and simple purification of recombinant human granulocyte colony-stimulating factor in E. coli

Summary A human granulocyte colony-stimulating factor (hG-CSF) gene was synthesized and inserted into a trp expression vector for over-expression in E. coli. A strong expression vector was constructed, and a simple purification procedure including in vitro refolding was established. The final productivity of hG-CSF was 500~600mg per l culture, and the purified hG-CSF showed the proliferation of neutrophils in vivo assays.     

Bacterial expression systems for recombinant protein production: E. coli and beyond

Escherichia coli expression system continues to dominate the bacterial expression systems and remain to be the preferred system for laboratory investigations and initial development in commercial activities or as a useful benchmark for comparison among various expression platforms. Some new developments in overcoming its shortcomings are reviewed in this paper, including antibiotics-free selection plasmids, extracellular production, and posttranslational modifications. The ability for E. coli to make mg glycosylated proteins promises even broader applications of the E. coli system in the future. Significant progresses have also been made over the past few years in alternative bacterial expression systems. Notably, the Lactoccocus lactis system has proven to be a viable choice for membrane proteins. Additionally, several Pseudomonas systems were developed and achieved product titers comparable to E. coli systems. Other bacterial systems such as Streptomyces, coryneform bacteria, and halophilic bacteria offer advantages in some niche areas, providing more choices of bacterial expression systems for recalcitrant proteins.     

Expression and characterization of recombinant human cytochrome c in E. coli

Cytochrome c is a heme protein involved in electron transfer, cell apoptosis, and diseases associated with oxidative stress. Here we expressed human cytochrome c in E. coli and purified it to homogeneity with a yield of 10–15 mg/L. The redox potential of recombinant human cytochrome c was 0.246 V which was measured by cyclic voltammetry. This is similar to that of horse cytochrome c with a value of 0.249 V. The sequential assignment and structural analysis of recombinant human ferrocytochrome c were obtained using multidimensional NMR spectroscopy. On the basis of our NMR studies, the recombinant human cytochrome c produced in E. coli exhibits the same tertiary fold as horse cytochrome c. These results provide evidence that human cytochrome c expressed in E. coli possesses a similar function and structure to that of the horse protein. It is known that cytochrome c plays a role in many human diseases. This study serves as the basis for gaining insight into human diseases by exploring structure and function relationships of cytochrome c to its interacting proteins.     

Transport regulation of recombinant gene expression in E. coli and B. subtilis

Expression kinetics of the lactose (lac) operon in Escherichia coli are reviewed for both wild-type and recombinant cell cultures under chemostatic conditions. A unified model which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression is summarized and supporting data is shown to verify its accuracy. The synthesis of alpha-amylase with a recombinant form of Bacillus subtilis is also reviewed to point out generic features in transport regulation, the lac operon model providing a point of departure. While there are many similarities in the influence of transport on both regulating models, there are also important differences. In a chemostat system, the synthesis of alpha-amylase is nongrowth associated, while beta-galactosidase is a growth-associated enzyme. Nevertheless, transport regulation is an important feature in both instances.     

Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization

Interleukin 15 (IL-15) has shown remarkable biological properties of promoting NK- and T-cell activation and proliferation, as well as enhancing antitumor immunity of CD8(+) T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin-15 (rhIL-15) for clinical manufacturing. Human IL-15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21-AI, E. coli BL21-AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20-L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL-15. Stop codons of the expression construct were further investigated after 15-20% of the purified rhIL-15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21-AI cells containing the rhIL-15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing.     

乳糖诱导耐热木聚糖酶基因在大肠杆菌中表达

考察乳糖代替IPTG诱导耐热木聚糖酶基因在重组大肠杆菌BL21中表达的可行性。分别以IPTG和乳糖作为诱导剂,对诱导时机、诱导剂浓度、诱导持续时间等主要因素进行了分析比较。结果表明,当菌体生长至发酵液吸光值OD600达1．3时加入乳糖其终浓度达29．2 mmol／L,37℃,持续诱导9．5h,木聚糖酶活力达到最高,为3587.5U,是IPTG优化条件下持续诱导7h达到的最高酶活的2．07倍,而成本只有IPTG的1／204。     

Effects of plasmid amplification and recombinant gene expression on the growth kinetics of recombinant E. coli

An experimental study was undertaken to identify and quantitate the effects of plasmid amplification and recombinant gene expression on Escherichia coli growth kinetics. Identification of these effects was possible because recombinant gene expression and plasmid copy number were controlled by different mechanisms on plasmid pVH106/172. Recombinant gene expression of the lactose operon structural genes was under the control of the lac promoter and was activated by the addition of the chemicals, IPTG and cyclic AMP, to the fermentation medium. Plasmid content was amplified in a separate fermentation by increasing culture temperature since the plasmid replicon was temperature-sensitive. A final fermentation was performed in which both plasmid content and recombinant gene expression were induced simultaneously by adding chemicals and raising the culture temperature. Recombinant growth rates were found to be reduced by the expression of high levels of recombinant lac proteins in the chemical induction experiments and by the amplification of plasmid levels in the temperature induction experiment. High expression of recombinant lac proteins following chemical induction was accompanied by a loss in recombinant cell viability. In the plasmid amplification experiment, the recombinant cells did not lose viability but the recombinant product yields were much lower than those achieved in the chemical induction experiments. Combining temperature and chemical induction increased the recombinant product yield by a factor of 4400 but also lowered cellular growth rates by 70%.     

The proteolytic activity of the recombinant cryptic human fibronectin type IV collagenase from E. coli expression

Human plasma fibronectin (pFN) contains a cryptic metalloprotease present in the collagen-binding domain. The enzyme could be generated and activated in the presence of Ca²⁺ from the purified 70-kDa pFN fragment produced by cathepsin D digestion. In this work we cloned and expressed the metalloprotease, designated FN type IV collagenase (FnColA), and a truncated variant (FnColB) in E. coli. The recombinant pFN protein fragment was isolated from inclusion bodies, and subjected to folding and autocatalytic degradation in the presence of Ca²⁺, and yielded an active enzyme capable of digesting gelatin, helical type II and type IV collagen, - and -casein, insulin b-chain, and a synthetic Mca-peptide. In contrast, isolated plasma fibronectin, type I collagen, and the DNP-peptide were no substrates. Both catalytically active recombinant pFN fragments were efficiently inhibited by EDTA, and batimastat, and, in contrast to the glycosylated enzyme isolated from plasma fibronectin, were also inhibited by TIMP-2.     

人乳头瘤病毒16型早期基因E6、E7的克隆及表达

高危人乳头瘤病毒16型的感染与宫颈癌的发病密切相关。HPV16E6和E7蛋白在大多数HPV16相关宫颈癌及其癌前病变中持续表达,因此E6和E7蛋白可作为制备HPV16相关肿瘤及其癌前病变治疗性疫苗的靶抗原〔2〕。采用套式PCR方法,从宫颈癌患者组织中扩增出E6、E7基因并成功构建了包含E6、E7的表达质粒PET-E6、PET-E7。SDS聚丙烯酰胺凝胶电泳和Western-blotting分析结果表明,外源基因E6,E7在T7启动子控制下可获得稳定表达。     

Escherichia coli from animal reservoirs as a potential source of human extraintestinal pathogenic E. coli

Extraintestinal pathogenic Escherichia coli (ExPEC) are an important cause of urinary tract infections, neonatal meningitis and septicaemia in humans. Animals are recognized as a reservoir for human intestinal pathogenic E. coli, but whether animals are a source for human ExPEC is still a matter of debate. Pathologies caused by ExPEC are reported for many farm animals, especially for poultry, in which colibacillosis is responsible for huge losses within broiler chickens. Cases are also reported for companion animals. Commensal E. coli strains potentially carrying virulence factors involved in the development of human pathologies also colonize the intestinal tract of animals. This review focuses on the recent evidence of the zoonotic potential of ExPEC from animal origin and their potential direct or indirect transmission from animals to humans. As antimicrobials are commonly used for livestock production, infections due to antimicrobial-resistant ExPEC transferred from animals to humans could be even more difficult to treat. These findings, combined with the economic impact of ExPEC in the animal production industry, demonstrate the need for adapted measures to limit the prevalence of ExPEC in animal reservoirs while reducing the use of antimicrobials as much as possible.     

Nitrite inhibition of Vitreoscilla hemoglobin (VHb) in recombinant E. coli: direct evidence that VHb enhances recombinant protein production

Bacteria engineered with the gene (vgb) encoding Vitreoscilla hemoglobin (VHb) typically produce more protein than unengineered cells, and it has generally been assumed that VHb is responsible for this effect. Here, using matched strains of E. coli that bear a recombinant alpha-amylase gene (MK57) or the alpha-amylase gene and vgb (MK79), we provide evidence supporting this assumption. Sodium nitrite (which is known to inhibit heme proteins) was tested over a range of concentrations regarding effects on growth, alpha-amylase production, respiration, and VHb function in MK57 and MK79. Nitrite concentrations were identified at which respiration of cell membranes was inhibited only slightly and to approximately equal degrees in both strains, while whole cell respiration was inhibited to a greater extent and about twice as much in MK79 as MK57. This suggests that these concentrations inhibit VHb while having a much smaller effect on cytochrome oxidase. Direct measurements of VHb showed, in fact, that the same nitrite concentrations greatly decreased the levels of active (ferrous) and, to a somewhat lesser extent, total (ferrous plus ferric) VHb in MK79. Finally, these same nitrite concentrations reversed the advantage regarding alpha-amylase production of MK79 over MK57 seen at 0 mM nitrite, linking the presence of active VHb with the increase in alpha-amylase production.     

High yield expression of recombinant pro-resilin: lactose-induced fermentation in E. coli and facile purification

Resilin is an elastic protein with outstanding material properties: high resilience and a very high fatigue lifetime. We are interested in the production of resilin-like proteins which can be photo-chemically cross-linked to form rubbery biomaterials to be used in a variety of industrial and medicinal applications. A method has been developed for producing soluble recombinant proteins in small scale fermentation equipment using glycerol batch for initial growth and primary induction by IPTG at carbon source depletion, followed by new growth in lactose-induced culture. Recombinant rec1-resilin has been over-expressed in the host strain Escherichia coli BL21(DE3)pLysS at a level of up to 300 mg/l, a greater than 20-fold increase in volumetric productivity, relative to that obtained from conventional IPTG induction in LB medium. The primary induction step before lactose induction in fresh medium resulted in a 2.5- to 3-fold increase of both volumetric productivity and cell specific yield compared to that without primary induction under the same conditions. This method is amenable and suitable for large scale production of soluble resilin-like proteins at a low operating cost. In addition, a simple 'salt precipitation and heat purification' method allowed rapid and efficient downstream processing of a large quantity of soluble recombinant resilin-like proteins. These methods will enable investigation of the structural and functional properties of resilin-like proteins, and the development of highly resilient biomaterials.