Separation and purification of useful proteins using hydrogel ultrafiltration

The hydrogel process is a different form of ultrafiltration and has been used to separate biological molecules. In this study, the gel pore size was predicted by pulse NMR technique and neural network using a data base obtained from gel filtration chromatography and diffusion experiment. Recombinant alkaline phosphatase expressed in insect cells was concentrated 1.5 times by hydrogel ultrafiltration by swelling at 20°C and collapsing at 35°C at 53–65% separation efficiency and 78–83% enzyme recovery. Wild and recombinantAutographa californica nuclear polyhedrosis viruses (AcNPV) were also concentrated 1.4 and 1.6 times of the feed solution at 48.5 and 60.0% separation efficiency, respectively. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of AcNPV and recombinant proteins from insect cells.     

Concentrating Autographa californica nuclear polyhedrosis virus and recombinant alkaline phosphatase from insect cells using a temperature-sensitive hydrogel

Recombinant alkaline phosphatase expressed in insect cells was concentrated by a factor of one and half times at a separation efficiency of 54.2% using hydrogel ultrafiltration. Enzyme concentration was confirmed by SDS-PAGE as well as by spectrophotometric measurement. Wild and recombinant Autographa californica nuclear polyhedrosis viruses (AcNPV) were concentrated 1.4 and 1.6 times of the feed solution at 48.5 and 60.0% separation efficiency, respectively. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of AcNPV and recombinant proteins from insect cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Concentrating rotavirus and recombinant-enhanced green fluorescent protein from E. coli using a pH-sensitive hydrogel

A rotavirus and a recombinant-enhanced green fluorescent protein from E. coli were concentrated 1.7 times and 1.5 times, respectively, by ultrafiltration at 37°C and pH 7 using a pH-sensitive hydrogel, poly(N-isopropylacrylamide-co-N, N-dimethylaminopropyl methacrylamide). Recoveries were 77% and 69%, respectively, and separation efficiencies were 58% and 44%, respectively. The concentration increase of the protein was confirmed by SDS-PAGE.     

Hydrogel ultrafiltration of recombinant alkaline phosphatase from baculovirus-infected Spodoptera frugiperda

Recombinant alkaline phosphatase expressed in insect cells was concentrated 1.5 times by hydrogel ultrafiltration by swelling at 20°C and collapsing at 35°C at 53-65% separation efficiency and 78-83% enzyme recovery. Enzyme entrapment between gel particles and attachment of enzymes to the gel surface was the main reason for the poor separation efficiency and low enzyme recovery. Exposure of the enzyme at the collapse temperature during repeated swelling-collapsing cycles also decreased separation efficiency to 37% due to thermal deactivation of the enzyme.     

Concentrating alkaline serine protease,subtilisin, using a temperature-sensitive hydrogel

Summary The alkaline serine protease, subtilisin, produced by Bacillus licheniformis was concentrated using hydrogel ultrafiltration. Separation efficiency at 15° and 20°C was 84 % but decreased above 25°C.     

Cloning,production and secretion of β-galactosidase inAcetobacter pasteurianus

The gene coding for β-galactosidase fromEscherichia coli was cloned into plasmid pACT71 containing the replicon from plasmid pAC1 fromAcetobacter pasteurianus. E. coli MC4100,E. coli JM105,E. coli LE392.23 andA. pasteurianus 3614 were transformed with the recombinant plasmid pACB815. Cells were cultivated in LB, YPG and M media supplemented with glucose, glycerol, lactose or ethanol and β-galactosidase activity was detected in the cells and in the cultivation medium. The best substrate for production of β-galactosidase was lactose. To release β-galactosidase fromA. pasteurianus cells amino acids were added to the cultivation medium. The highest secretory activity was achieved using 1.5% glycine after 36 h of cultivation in the M medium.     

Construction and Characterization of Versatile Cloning Vectors for Efficient Delivery of Native Foreign Proteins to the Periplasm ofEscherichia coli

Induction of the wild type cholera toxin operon (ctxAB) from multicopy clones inEscherichia coliinhibited growth and resulted in low yields of cholera toxin (CT). We found that production of wild type CT or its B subunit (CT-B) as a periplasmic protein was toxic forE. coli,but by replacing the native signal sequences of both CT-A and CT-B with the signal sequence from the B subunit ofE. coliheat-labile enterotoxin LTIIb we succeeded for the first time in producing CT holotoxin in high yield inE. coli.Based on these findings, we designed and constructed versatile cloning vectors that use the LTIIb-B signal sequence to direct recombinant native proteins with high efficiency to the periplasm ofE. coli.We confirmed the usefulness of these vectors by producing two other secreted recombinant proteins. First, usingphoAfromE. coli,we demonstrated that alkaline phosphatase activity was 17-fold greater when the LTIIb-B signal sequence was used than when the native leader for alkaline phosphatase was used. Second, using thepspAgene that encodes pneumococcal surface protein A fromStreptococcus pneumoniae,we produced a 299-residue amino-terminal fragment of PspA inE. coliin large amounts as a soluble periplasmic protein and showed that it was immunoreactive in Western blots with antibodies against native PspA. The vectors described here will be useful for further studies on structure–function relationships and vaccine development with CT and PspA, and they should be valuable as general tools for delivery of other secretion-competent recombinant proteins to the periplasm inE. coli.     

Alcohol production from glucose and xylose usingEscherichia coli containingZymomonas mobilis genes

Summary AnEscherichia coli strain containing a recombinant plasmid encoding the pyruvate decarboxylase and alcohol dehydrogenase genes fromZymomonas mobilis metabolized glucose and xylose to near theoretical yields of ethanol. Enzyme activity measurements indicate high expression levels of both plasmid-encodedZymomonas proteins in the recombinantE. coli. The expression inE. coli is under the control of a promoter in theZymomonas sequence upstream of the pyruvate decarboxylase gene. The maximum ethanol level, using 4% glucose as substrate, was 1.8% (w/v) in anaerobic conditions. In aerobic conditions the natural repression ofE. coli alcohol dehydrogenase results in less ethanol production from clones expressing onlyZymomonas pyruvate decarboxylase.     

Purification of recombinant protein by cold‐coacervation of fusion constructs incorporating resilin‐inspired polypeptides

Polypeptides containing between 4 and 32 repeats of a resilin‐inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence‐related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold‐coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein‐poor phase. We show that purification of recombinant proteins by cold‐coacervation can be performed when engineered as a fusion partner to a resilin‐inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time‐consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold‐coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature. Biotechnol. Bioeng. 2012; 109: 2947–2954. © 2012 Wiley Periodicals, Inc.     

过表达亚硝酸盐还原酶的重组大肠杆菌辅助污水短程硝化

【目的】为了体现并突出亚硝酸盐还原酶在污水脱氮以及短程硝化中的重要性,对过表达亚硝酸盐还原酶的大肠杆菌进行了污水脱氮的研究。【方法】通过转化带有亚硝酸盐还原酶基因的重组质粒,将亚硝酸盐还原酶在大肠杆菌中过表达,通过分析重组大肠杆菌的产物研究了该酶的表达及还原亚硝酸盐的情况,通过将该重组菌与已报道的硝化-反硝化细菌或生活污水进行混合培养,研究重组菌用于辅助氨氮去除的短程硝化能力。【结果】重组大肠杆菌能正确表达亚硝酸盐还原酶,OD600=2.0的菌悬液在2 h内还原约1 mmol/L的亚硝酸盐,并产生几乎等量的一氧化氮;重组大肠杆菌与Acinetobacter sp.YF14菌株等比例混合时,12 h能够提高氨氮脱氮效率约(36.0±7.4)%,且在4 h时,最大亚硝酸盐的积累量减少37%;重组大肠杆菌(OD600=1.0)12 h内能够提高污水厂活性污泥的脱氮效率约(31.0±5.7)%,且未检测到亚硝酸盐和硝酸盐的积累;溶氧水平对于亚硝酸盐还原酶重组菌辅助脱氮具有明显的影响,中等溶氧量[(6.4?0.7)mg/L]时脱氮效果最好。【结论】过表达亚硝酸盐还原酶的大肠杆菌可以提高污水脱氮的短程硝化能力。     

Fusion expression of mutated cecropin CMIV in E. coli

A cDNA coding mutated cecropin CMIV fromBombyx mori was synthesized according to its amino acid sequence usingE. coli biased codons. The gene was cloned into the fusion expression vector pEZZ318 and was expressed inE. coli HB101. The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product. The anti-bacterial activities of recombinant cecropin CMIV were recovered after cleavage by chemical method.     

A fast and simple approach to optimize the unit operation high pressure homogenization - a case study for a soluble therapeutic protein in E. coli

Escherichia coli is one of the most commonly used host organisms for the production of recombinant biopharmaceuticals. E. coli is usually characterized by fast growth on cheap media and high productivity, but one drawback is its intracellular product formation. Product recovery from E. coli bioprocesses requires tedious downstream processing (DSP). A typical E. coli DSP for an intracellular product starts with a cell disruption step to access the product. Different methods exist, but a scalable process is usually achieved by high pressure homogenization (HPH). The protocols for HPH are often applied universally without adapting them to the recombinant product, even though HPH can affect product quantity and quality. Based on our previous study on cell disruption efficiency, we aimed at screening operational conditions to maximize not only product quantity, but also product quality of a soluble therapeutic protein expressed in E. coli. We screened for critical process parameters (CPPs) using a multivariate approach (design of experiments; DoE) during HPH to maximize product titer and achieve sufficient product quality, based on predefined critical quality attributes (CQAs). In this case study, we were able to gain valuable knowledge on the efficiency of HPH on E. coli cell disruption, product release and its impact on CQAs. Our results show that HPH is a key unit operation that has to be optimized for each product.     

Expression of UreB and HspA of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in silkworm pupae and identification of its immunogenicity

For mass production of urease B subunit (UreB) and heat shock protein A subunit (HspA) of Helicobacter pylori with Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) and to determine whether they could be used as an oral vaccine against H. pylori, besides, to determine the time course of expressed recombinant protein and the optimum acquisition time directly through green fluorescence, HspA and enhanced green fluorescence protein (EGFP) genes were cloned into vector pFastBacDual to form donor vector pFastBacDual-(EGFP) (HspA), UreB gene was cloned into vector pFastBacDual to form donor vector pFastBacDual-UreB,then they were transformed into E. coli BmDH10Bac to obtain the recombinant Bacmid-(EGFP) (HspA) and Bacmid-UreB respectively. They were used to transfect BmN cells and generated the recombinant baculovirus BmNPV-(EGFP) (HspA) and BmNPV-UreB. Using these recombinant baculovirus BmNPV-(EGFP) (HspA) and BmNPV-UreB inoculated the silkworm pupae, a recombinant HspA and UreB protein were expressed in silkworm pupae, which were around 13 and 62 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. After oral immunization of mice, serum specific IgG antibodies against HspA and UreB in vaccine group were much higher than that in mock and native silkworm powder control groups. The results indicated that the expressed recombinant HspA and UreB in silkworm pupae would possess good immunogenicity. In addition, when EGFP and HspA proteins were expressed, a direct correlation between the increase in intensity of fluorescence and HspA concentration.     

High cell density continuous culture ofEscherichia coli producing penicillin acylase

Summary We studied high cell density continuous culture (HDCC) of a recombinant (E. coliHB101 (pPAKS2)) and a mutant (E. coli ATCC 11105) strains ofE. coli producing penicillin acylase(PA). Using pure oxygen, high cell density up to 95 g/l was obtained without significant inhibition by a main byproduct, acetic acid. The operation was simple and productivity was several times higher than those of conventional batch and continuous culture. Dissolved oxygen level and CO₂ concentration were important variables, and glucose concentration was naturally regulated in HDCC.     

A hybridization assay specific for ribosomal RNA from Escherichia coli

Summary Ribosomal RNA fromEscherichia coli forms specific hybrids with heterologous DNA fromVibrio metschnicovii. The conditions for such hybridization are described, in which the amount of mRNA fromE. coli binding to theVibrio DNA is about 3% of the amount which hybridizes to the same amount of DNA fromE. coli.     

Secretion of recombinant proteins from E. coli

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.     

Biological nitrogen and organic matter removal from tannery wastewater in pilot plant operations in Ethiopia

A whole-cell biotransformation system for the conversion of d-fructose to d-mannitol was developed in Escherichia coli by constructing a recombinant oxidation/reduction cycle. First, the mdh gene, encoding mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH), was expressed, effecting strong catalytic activity of an NADH-dependent reduction of d-fructose to d-mannitol in cell extracts of the recombinant E. coli strain. By contrast whole cells of the strain were unable to produce d-mannitol from d-fructose. To provide a source of reduction equivalents needed for d-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH), encoding formate dehydrogenase, was functionally co-expressed. FDH generates the NADH used for d-fructose reduction by dehydrogenation of formate to carbon dioxide. These recombinant E. coli cells were able to form d-mannitol from d-fructose in a low but significant quantity (15 mM). The introduction of a further gene, encoding the glucose facilitator protein of Zymomonas mobilis (GLF), allowed the cells to efficiently take up d-fructose, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM d-mannitol in 17 h. Due to equimolar formation of sodium hydroxide during NAD⁺-dependent oxidation of sodium formate to carbon dioxide, the pH value of the buffered biotransformation system increased by one pH unit within 2 h. Biotransformations conducted under pH control by formic-acid addition yielded d-mannitol at a concentration of 362 mM within 8 h. The yield Y _{D-mannitol/D-fructose}was 84 mol%. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for d-mannitol formation.     

Heat-Stable Enterotoxin Produced by Yersinia enterocolitica Isolated from Patients

Twenty-three strains of Yersinia enterocolitica were isolated from children with gastrointestinal illness and examined for the production of enterotoxins by using both suckling mouse and Chinese hamster ovary (CHO) cell assay systems. Six strains were found to be enterotoxigenic in the suckling mouse assay, but all strains were negative in the CHO cell assay. Enterotoxin was detected in the culture supernatant when organisms were grown at 25 C but not at 37 C. Enterotoxin in a 15-fold concentrated culture supernatant was precipitated by adding absolute ethanol to a concentration of 90%. However, after being dialyzed against distilled water in Spectra/por 6 membrane tubing, it was soluble in 80% acetone. One unit dose of partially purified enterotoxin was 5.0 μg of protein/mouse in the suckling mouse assay. The molecular weight of enterotoxin was between 10,000 and 50,000 daltons as determined by ultrafiltration. It was stable to heat (121 C × 20 min or 100 C × 60 min). These observations indicate that Y. enterocolitica isolated in Japan also produce an enterotoxin similar to the heat-stable enterotoxin of Escherichia coli. However its physicochemical properties seem to be different from those of E. coli.     

A comparative study of glucose conversion to ethanol by Zymomonas mobilis and a recombinant Escherichia coli

Summary Zymomonas mobilis and recombinant Escherichia coli B (pLOI297) were compared in side-by-side batch fermentations using a synthetic cellulose hydrolysate (glucose/salts) medium with pH control at 6.0 and an inoculation cell density of 35–50 mg dry wt. cells/L. At a nominal glucose concentration of 6%, both cultures achieved near maximal theoretical ethanol yields; however, the Z. mobilis fermentation was complete at 13h compared to 33h for the E.coli fermentation. With approx.12% glucose, the Z. mobilis fermentation was complete in 20h with a process yield of 0.49 g ethanol/g added glucose compared to the E. coli fermentation which remained 20% incomplete after 6 days resulting in a process yield of only 0.32 g/g. Nutrient supplementation (10g tryptone/L) resulted in complete fermentation of 12% glucose (pH 6.3) by the recombinant E. coli in 4 days, with a yield of 0.48 g/g.     

Cloning of the gene coding for aromatic amino acid aminotransferase from anE. Coli B strain

Summary AnE. coli B strain showing high activity in the transamination of phenylpyruvate to phenylalanine was used as the DNA source for the construction of a cosmid library inE. coli DG30, a strain which is known to be defective in all three major transaminase genes (aspC, ilvE andtyrB). By complementation analysis, cosmid clones could be identified with inserts carrying atyrB gene. The DNA inserts were further subcloned into pAT153 and thetyrB gene fromE. coli B was found to be similar to the gene reported forE. coli K12. Plasmids containing theE. coli BtyrB gene were transformed into the originalE. coli B strain and the recombinant strains assayed for transaminase activity and plasmid stability.Dedicated to Prof. Dr. Heinz Harnisch on the occasion of his 60th birthday.