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.     

Direct expression in E. coli of a functionally active protein A--snake toxin fusion protein

We constructed a recombinant expression plasmid encoding a protein A--neurotoxin fusion protein. The fused toxin is directly expressed in the periplasmic space of Escherichia coli and can be purified in the milligram range by a single immuno-affinity step. The LD50 values of the fused toxin and native toxin are 130 and 20 nmol/kg mouse respectively. The Kd values characterizing their binding to the nicotinic acetylcholine receptor (AcChoR) are respectively 4.8 +/- 0.8 and 0.07 +/- 0.03 nM. In contrast, the fused and native toxins are equally well recognized by a toxin-specific monoclonal antibody which recognizes the AcChoR binding site. The lower toxicity of the fused toxin might result, therefore, from a steric hindrance, due to the presence of the bulky protein A moiety (mol. wt = 31 kd) rather than to a direct alteration of the 'toxic' site. The fused toxin is more immunogenic than native toxin, since 1 nmol of hybrid toxin and 14 nmol of native toxin give rise to comparable titers of antitoxin antibodies which, furthermore, are equally potent at neutralizing neurotoxicity. The work described in this paper shows that the use of fused toxins may be of paramount importance for future development of serotherapy against envenomation by snake bites.     

Expression screening of fusion partners from an E. coli genome for soluble expression of recombinant proteins in a cell-free protein synthesis system

While access to soluble recombinant proteins is essential for a number of proteome studies, preparation of purified functional proteins is often limited by the protein solubility. In this study, potent solubility-enhancing fusion partners were screened from the repertoire of endogenous E. coli proteins. Based on the presumed correlation between the intracellular abundance and folding efficiency of proteins, PCR-amplified ORFs of a series of highly abundant E. coli proteins were fused with aggregation-prone heterologous proteins and then directly expressed for quantitative estimation of the expression efficiency of soluble translation products. Through two-step screening procedures involving the expression of 552 fusion constructs targeted against a series of cytokine proteins, we were able to discover a number of endogenous E. coli proteins that dramatically enhanced the soluble expression of the target proteins. This strategy of cell-free expression screening can be extended to quantitative, global analysis of genomic resources for various purposes.     

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.     

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.     

Characterization of a functional recombinant rat liver aldehyde dehydrogenase: expression as a non-fusion protein in E. coli

A cDNA encoding a rat liver inducible aldehyde dehydrogenase carried in a pUC8 plasmid is expressed in E. coli as a dimeric enzyme molecule functionally and physically identical to the authentic rat enzyme. The cDNA appears to be transcribed using the lac promoter, but is translated from an initiator codon 174 base pairs from the 5' end of the cDNA. The aldehyde dehydrogenase polypeptide is not produced as a fusion protein. This is the first example of the production by E. coli of a catalytically active, multimeric eukaryotic protein which is not a fusion protein.     

Purification of fusion ferritin from recombinant E. coli using two-step sonications

Fusion ferritin, combined by heavy chain ferritin (21 kDa) and light chain ferritin (19 kDa), was expressed in recombinant E. coli. The fusion ferritin was easily purified by two-step sonications as well as gel filtration chromatography. SDS-gel electrophoresis showed a single band of 38 kDa with heavy and light chains. MALDI-TOF MS gave a molecular weight of fusion ferritin was 38 kDa. The specific activity and yield of purified fusion ferritin are 0.41 Fe³⁺ mg mg^–1 of protein and 66%. Those values are larger than the previous ones of 0.2 Fe³⁺ mg mg^–1 (Kim et al. 2001).     

EGF-SEA融合蛋白在大肠杆菌中的表达和纯化

根据基因库中查到的金黄色葡萄球菌肠毒素A(SEA)基因序列和人体表皮生长因子(EGF)基因序列进行密码子优化,以适于大肠杆菌表达.人工合成SEA基因与EGF基因.将两目的基因克隆至原核表达栽体pFT22b中,经测序验证表明成功构建了重组表达质粒pET22b-EGF-SEA.将构建好的pET22b-EGF-SEA质粒转化大肠杆菌BL21(DE3),经IPTG诱导进行表达;SDS-PAGE分析表明融合基因EGF-SEA在大肠杆菌BL21(DE3)中以包涵体的形式得到了高效表达,产物相对分子质量约为44kDa,与理论值大小一致.包涵体经洗涤,变性、复性后用His Bind Kit进行分离纯化,所得蛋白纯度≥95%.高纯度EGF-SEA融合蛋白的获得为进一步研究其生物学活性及肿瘤治疗奠定了基础.     

HIV—1 P24截短体与gp41截短体的融合蛋白在大肠杆菌中的表达、纯化和鉴定

用基因重组技术将截短的HIV－1 p24基因和gp41基因连接成嵌合基因,插入质粒pGEX-4T3,构建成重组表达质粒pGEX-F。将pGEX-F转化大肠杆菌BL21。经IPTG诱导表达,pGEX-F在大肠杆菌BL21中获得了高效表达。融合蛋白P24－gp41经Glutathione-Sepharose4B亲和层析纯化后,用间接ELISA和免疫印迹检测HIV抗体阳性血清和正常人血清,P24－gp41只与HIV抗体阳性血清反应,证明获得的融合蛋白P24－gp41有很强的抗原特异性和免疫反应性,具有较高的应用价值。     

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。     

High-level expression of recombinant human FK-binding protein from a fusion precursor

The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L inEscherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a 30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed inE. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to [³H]FK-506. The fusion construct, CKS-FKBP, was also found to bind [³H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein.     

Mitomycin C stimulates thermally induced recombinant gene expression in Escherichia coli MC strains

The effects of mitomycin C on C1857-controlled recombinant gene expression have been explored in E. coli cultures when the drug was added simultaneously to the thermal induction. A significantly improved yield of homologous, heterologous and chimeric fusion proteins was observed in E. coli MC1061 and GE864 (a MC4100 derivative) thermoinduced cells. This feature was not detected in other E. coli strains and does not involve a gene dosage mechanism but a strain-dependent stimulation of gene expression unrelated to the RecA protease activity.     

Production of recombinant human glucagon in Escherichia coli by a novel fusion protein approach

Summary A novel approach to the production of a human glucagon in E. coli is described. The 29 amino acids of human glucagon and pentapeptide linker containing enzyme processing site were fused at the amino terminus to a 57 residue N-terminal portion of the human tumor necrosis factor-alpha (hTNF-). The fusion protein was expressed in the E. coli cytoplasm at levels up to 30% of the total cell protein. Precipitation of the fusion protein near its isoelectric point, specific enterokinase cleavage at the linker site and subsequent HPLC purification makes this approach suitable for the production of glucagon as well as other relatively small peptides with therapeutic interests.     

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.     

乳糖诱导木聚糖酶基因在大肠杆菌中的可溶性表达

以构建好的大肠杆菌工程菌BL21(DE3)/xylanase为研究对象,研究了以IPTG和乳糖作为诱导剂时重组蛋白的表达规律。在摇瓶发酵条件下研究了诱导剂浓度、诱导时机、诱导培养时间和诱导培养温度对目标蛋白表达的影响。实验结果表明,乳糖作为诱导剂时,重组菌产酶活力33.9 U/mg略高于IPTG作为诱导剂时重组菌产酶活力28.10 U/mg,这为乳糖作为诱导剂应用于重组大肠杆菌生产木聚糖酶提供了参考依据。     

Production of recombinant porcine tumor necrosis factor alpha in a novel E. coli expression system.

DNA sequences encoding porcine tumor necrosis factor alpha (TNF alpha) were reconstructed from a genomic-derived PCR product for expression in Escherichia coli. A synthetic DNA primer containing most of exon III was fused to exon IV sequences by means of PCR. The fused product was then inserted into the novel FLAG vector by restriction and ligation. This initial recombinant construct was propagated in single-strand form through use of a helper phage and subjected to oligonucleotide-directed mutagenesis for the purpose of introducing additional coding sequences from exons II and III. The final construct encoded a fusion protein consisting of the Omp-A signal peptide, a seven-amino acid FLAG peptide and the soluble form of porcine TNF alpha. Bacteria containing this construct produced a protein which was recognized by anti-FLAG monoclonal antibody in Western blots and which was purified by anti-FLAG immunoaffinity chromatography. The purified material was cleaved with enterokinase to remove the FLAG peptide. Both the enterokinase-cleaved form and the uncleaved form were shown to have TNF activity in a WEHI cell cytotoxicity assay.     

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。