Immobilization of beta-galactosidase for application in organic chemistry using a chelating peptide

The strong interaction of hexa-histidine fusion proteins with metal chelate adsorbents was utilized to immobilize beta-galactosidase with a hexa-histidine peptide at the N-terminus to the Ni(2+)-nitrilotriacetic acid adsorbent. The fusion protein was cloned and expressed in Escherichia coli. The purified soluble fusion protein showed the same specific activity as the purified beta-galactosidase and retained 64 percent of its beta-galactosidase activity when bound to the adsorbent. To demonstrate the potential of the immobilized beta-galactosidase in organic chemistry, allyl-beta-D-galactosidase was synthesized from lactose and allyl alcohol on a gram scale. The same enzyme preparation was reused in three subsequent batches to prepare the model compound with high yield. (c) 1993 John Wiley & Sons, Inc.     

Plasmid retention and gene expression in suspended and biofilm cultures of recombinant Escherichia coli DH5alpha(pMJR1750)

Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5alpha(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5alpha(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h(-1) to 0.35 h(-1) and the beta-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h(-1), about 36% of that without IPTG, and the beta-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction.In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5beta(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The beta-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively. (c) 1993 John Wiley & Sons, Inc.     

A eukaryotic expression vector for the study of nuclear localization signals

We describe a new vector designed to produce β-galactosidase fusion proteins which can be used to assess subcellular localization of target peptide fragments or proteins in eukaryotic cells. The vector was constructed in such a way as to produce the peptide of interest in fusion via a short linker of proline residues to the N terminus of the reporter protein. Efficiency of the transport machinery is optimized using this particular protein fusion construction. This vector has potential uses for readily testing putative nuclear localization sequences and identifying their crucial amino-acid residues.     

Response dynamics of 26-, 34-, 39-, 54-, and 80-kDa proteases in induced cultures of recombinant Escherichia coli

Several researchers have demonstrated that the presence of a heterologous protein in recombinant Escherichia coli elicits a response similar to the heat-shock response, which includes enhanced protease expression. The present work detects, quantifies, and characterizes intracellular protease activity in E. coli that are "shocked" by the induction of a recombinant protein, CAT, which is an endogenous protein in some E. coli strains. A novel, sodium dodecyl sulfate gelatin poly-acrylamide gel electrophoresis (SDS-GPAGE) method is used to detect, quantify, and characterize the presence of these proteases. A hypothesis is proposed which links the amplified protease activity to a temporary depletion of specific amino acid pools, and a stringent-like stress response. (c) 1993 John Wiley & Sons, Inc.     

Foreign gene expression (beta-galactosidase) during the cell cycle phases in recombinant CHO cells

Recombinant mammalian cultures for heterologous gene expression typically involve cells traversing the cell cycle. Studies were conducted to characterize rates of accumulation of intracellular foreign protein in single cells during the cell cycle of Chinese hamster ovary (CHO) cells transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the lacZ gene for bacterial beta-galactosidase (a nonsecreated protein). The lacZ gene was under the control of the constitutive cytomegalovirus promoter. These normally attachment-grown cells were adapted to suspension culture in 10(-7) M methotrexate, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (beta-galactosidase) content of single living cells. Expression of beta-galactosidase as a function of cell cycle phase was evaluated for cells in the exponential growth phase, early plateau phase, and inhibited traverse of the cell cycle during exponential growth. The results showed that the beta-galactosidase production rate is higher in the S phase than that in the G1 or G2/M phases. Also, when cell cycle progression was stopped at the S phase by addition of aphidicolin, beta-galactosidase content in single cells was higher than that in exponential phase or plateau phase cells and increased with increasing culture time. Although the cells did not continue to divide after aphidicolin addition, the production of beta-galactosidase per unit volume of culture was very similar to that in normal exponential growth. (c) 1993 John Wiley & Sons, Inc.     

Preparation of recombinant six-histidine-tagged human LECT2, a chemotactic protein to neutrophils, in Escherichia coli

LECT2 is a chemotactic protein to neutrophils. A recombinant six-histidine-tagged human LECT2, (His)₆-LECT2, was expressed in E. coli using a pET21a(+) vector. The (His)₆-LECT2 was purified from the soluble fraction in E. coli as a single band in sodium dodesyl sulfate/polyacrylamide gel electrophoresis using three steps of column chromatography with Ni²⁺-charged nitrilo-triacetic acid (Ni-NTA) agarose, DEAE-Sepharose, and CM-Sepharose. The purified (His)₆-LECT2 was yielded with 96 μg from the soluble fraction of 1,500 ml culture of E. coli. The circular dichroism spectrum of (His)₆-LECT2 showed the folded structure, which is rich in β-sheet structure and rare in α-helix. This revised version was published online in June 2006 with corrections to the Cover Date.     

重组大肠杆菌的分批补料培养方法

在重组大肠杆菌的培养过程中,存在着菌体的高浓度与外源蛋白的高表达这一矛盾,使得重组菌的比生长速率通常远远低于宿主菌,限制了基因工程菌由实验室规模向工业化规模的转变。要实现重组大肠杆菌的高密度培养,最常用和最有效的方法就是分批补料流加培养。     

Induction studies with Escherichia coli expressing recombinant interleukin-13 using multi-parameter flow cytometry

The expression of interleukin-13 (IL13) following induction with IPTG in Escherichia coli results in metabolic changes as indicated by multi-parameter flow cytometry and traditional methods of fermentation profiling (O₂ uptake rate, CO₂ evolution rate and optical density measurements). Induction early in the rapid growth phase was optimal although this led to lower overall biomass concentrations and lower maximum specific growth rates. In contrast, induction in the mid-rapid growth phase was the most detrimental to cell quality as measured by cytoplamsic membrane depolarisation.     

Enhanced beta-galactosidase production by high cell-density culture of recombinant Bacillus subtilis with glucose concentration control

Cell concentration, recombinant protein (beta-galactosidase) level, and the specific enzyme expression level were increased from 19 to 184 g/L, 18.3 to 129 U/mL, and 3.2 to 5.7 U/mg protein, respectively, in fed-batch culture of recombinant Bacillus subtilis when glucose concentration was controlled at 1 g/L as compared with those of conventional fed-batch culture. Glucose concentration of the culture broth was monitored by an automatic on-line glucose analyzer and controlled with a moving identification combined with optimal control (MICOC) strategy. When glucose concentrations were controlled at 10, 1, and 0.2 g/L, accumulated propionic acid concentrations and specific enzyme activities were 18.5, 4.4, and 0.6 g/L and 2.9, 5.7, and 7.1 U/mg protein, respectively. The addition of various concentrations of sodium propionate to the growth medium in batch cultures resulted in a drastic decrease in the growth rate with respect to propionate concentration. The propionic acid was shown to be responsible for cell growth inhibition and enzyme activity reduction in fed-batch culture. (c) 1992 John Wiley & Sons, Inc.     

Charged fusions for selective recovery of beta-galactosidase from cell extract using hollow fiber ion-exchange membrane adsorption

We explored the use of charged fusions for selective recovery of beta-galactosidase from cell extract using a low-cost, easily scaled, fast, charge-based separation technique-ion exchange on hollow fiber ion-exchange membranes (HFIEMs). The additional charges carried by a series of anionic fusion tails allowed selective binding and release of beta-galactosidase from Escherichia coli cell extract using the HFIEM cartridge. The purification factors increased with fusion length. The beta-galactosidase was recovered in active form. For the longest fusion studied, more than sixfold enrichment in specific activity was attained. The specific activity of the recovered fraction is comparable with that of commercial wild-type beta-galactosidase and affinity-purified fusion protein. (c) 1993 John Wiley & Sons, Inc.     

重组大肠杆菌的高密度发酵和甘油生产条件的初步研究

在摇瓶中进行重组大肠杆菌菌株BL21高密度发酵条件的研究,考察了葡萄糖浓度、盐离子浓度、温度、接种量、发酵时间等对该菌株生产甘油的影响。初步确定底物浓度为2.5%,盐离子浓度0.2%,温度为37℃,接种量为2%,经24h的摇瓶发酵,甘油产量最高达6.8g/L。在30L发酵罐实验中、按初步确定的优化条件发酵26h,甘油产量可达46.67g/L,是LB/葡萄糖培养基中甘油产量的2.06倍。     

Predictive tool for recombinant protein production in Escherichia coli shake-flask cultures using an on-line monitoring system

As Escherichia coli (E. coli) is well defined with respect to its genome and metabolism, it is a favored host organism for recombinant protein production. However, many processes for recombinant protein production run under suboptimal conditions caused by wrong or incomplete information from an improper screening procedure, because appropriate on-line monitoring systems are still lacking. In this study, the oxygen transfer rate (OTR), determined on-line in shake flasks by applying a respiration activity monitoring system (RAMOS) device, was used to characterize the metabolic state of the recombinant organisms. Sixteen clones of E. coli SCS1 with foreign gene sequences, encoding for different target proteins, were cultivated in an autoinduction medium, containing glucose, lactose, and glycerol, to identify relationships between respiration activity and target protein production. All 16 clones showed a remarkably different respiration activity, biomass, and protein formation under induced conditions. However, the clones could be classified into three distinct types, and correlations could be made between OTR patterns and target protein production. For two of the three types, a decrease of the target protein was observed, after the optimal harvest time had passed. The acquired knowledge was used to modify the autoinduction medium to increase the product yield. Additional 1.5 g/L glucose accelerated the production process for one clone, shifting the time point of the maximal product yield from 24 to 17 h. For another clone, lactose addition led to higher volumetric product yields, in fact 25 and 38% more recombinant protein for 2 and 6 g/L additional lactose, respectively.     

重组大肠杆菌合成左旋多巴条件的优化

通过对合成体系各组分对产物形成的影响的研究。分别求得了底物邻苯二酚,丙酮酸及乙酸氨的表观米氏常数,表观底物抑制常数和最适底物浓度,并得出合成左旋多巴的最适反应体系为：1%丙酮酸,1.2%邻苯二酚,2%乙酸铵,0.1?TA,0.2%亚硫酸钠,用氨水调pH至8.0。加入从与合成体系等体积培养液中收获的重组大肠杆菌细胞,于15℃反应16h,L-DOPA的产量为15.36g/L。     

Sensitive model with which to detect athermal effects of non-ionizing electromagnetic radiation

To clarify the potential of non-ionizing electromagnetic radiation to cause biological effects by athermal mechanisms, and to initiate elucidation of those mechanisms, a model system amenable to scrutiny at the molecular level has been designed and characterized. Assessment of beta-galactosidase activity in E. coli JM101 containing the plasmid pUC8 provides a sensitive assay with many important advantages. The ability to examine at the molecular level each of the processes involved in producing beta-galactosidase should permit elucidation of the molecular mechanism(s) that give rises to an observed effect.     

Ion exchange immobilization of changed beta-galactosidase fusions for lactose hydrolysis

The use of charged peptides fused to enzymes for immobilization onto ion-exchange membranes was explored for the enzyme x-galactosidase. The additional charged peptides, containing 1, 5, 11, and 16 aspartates, fused to x-galactosidase, for the most part did not interfere with the kinetic behavior for lactose hydrolysis. There was a 2-fold decline in V(m) for the 16-aspartate fusion, but the others were quite similar to the wild type enzyme (BGWT). BGWT and the fusions all retained approximately 50% of their activities when adsorbed onto ion-exchange membranes. In contrast to BGWT, the enhanced binding strength of the 11 aspartate fusion provided the ability to hydrolyze whey permeate at 0.3 M ionic strength without enzyme leakage, and to immobilize the enzyme directly from diluted cell extract with 83% purity. (c) 1994 John Wiley & Sons, Inc.     

The use of flow cytometry to detect nucleic acids attached to the surface of Escherichia coli in high cell density fed-batch processes

E. coli was grown in an aerobic fed-batch process for the production of a recombinant protein (rhGH). The cells were examined by flow cytometry and PI (propidium iodide) staining. The fluorescence of the PI-stained cells increased with increasing concentrations of DNA in the medium. Furthermore, DNA and RNA attached to the cell could partly be degraded with DNase/RNase and the fluorescence decreased. Formate excretion during the aerobic processes may be due to DNA and possibly also RNA attached to the cell surface, so creating diffusion resistance.     

响应面分析法优化重组大肠杆菌生物合成谷胱甘肽的条件

通过响应面分析法和典型性分析得出重组大肠杆菌酶法合成谷胱甘肽的最优条件:菌体量249 mg/mL,磷酸钾缓冲液145 mmol/L,MgCl243 mmol/L和ATP 34 mmol/L,预测谷胱甘肽最大量为16.50 mmol/L。验证性实验证明在优化条件下,重组大肠杆菌酶法合成谷胱甘肽达16.42 mmol/L。响应面分析还表明,在重组大肠杆菌酶法合成谷胱甘肽各因素中,MgCl2和ATP,以及菌体量与磷酸钾缓冲液之间的交互作用较显著。     

The expression of an R3 lipopolysaccharide-core by pathotypes of Escherichia coli

AIMS: To investigate the incidence of an R3 lipopolysaccharide (LPS)-core amplicon in a range of pathotypes of Escherichia coli, including Verocytotoxin-producing E. coli (VTEC), enteroaggregative E. coli (EAggEC) and enteropathogenic E. coli (EPEC). METHODS AND RESULTS: A total of 100 strains of E. coli belonging to a range of pathotypes, including 41 strains of VTEC, were screened for the genes encoding the R3 LPS-core using PCR. Fifty-four per cent produced an amplicon with the R3 primer set. Of the 41 VTEC, 66% had an R3 LPS-core with a PCR product being observed with all strains belonging to serotypes O26:H11, O111ac:H- and O145:H25. However, 46% of enteroaggregative E. coli and 50% of enteropathogenic E. coli were also shown to have an R3 LPS-core structure. CONCLUSIONS: Strains with an R3 LPS-core are widely distributed within the species E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of E. coli with an R3 LPS-core structure appear not to be associated with a specific pathotype.