Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Expression and characterization of Pichia etchellsii β-glucosidase in Escherichia coli

The β-glucosidase enzyme is important as the terminal enzyme involved in hydrolysis of cellobiose and short-chain cellodextrins generated during enzymatic cellulose degradation. Under controlled reaction conditions the enzyme also displays cello-oligosaccharide synthesizing ability (based on either the thermodynamic or kinetic approach). We present here the purification of the enzyme β-glucosidase (BGL) of Pichia etchellsii from recombinant pBG55 Escherichia coli clone. The kinetic parameters, substrate specificity and oligosaccharide synthesizing ability of the purified enzyme are also reported. The purified 200-kDa protein (tetramer of 50 kDa) was identified as a broad-substrate-specificity enzyme exhibiting increased temperature and glucose tolerance compared to the native yeast enzyme. Temperature directed substrate specificity for aryl β,1–4 linkage, and β(1–2), β(1–4), β(1–6) and β(2-1) linkages in various natural disaccharides was observed. Glycosylation of the enzyme was found to be unimportant for enzyme activity. With both cellobiose and glucose, oligosaccharide synthesis was detected. The implications of this information with regard to cellulose hydrolysis and oligosaccharide synthesis are discussed.     

Expression of a β-galactosidase gene from Lactobacillus sake in Escherichia coli

Summary A -galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. sake was partially digested with the restriction enzyme Sau3AI, and the 3–6 Kb fragment was ligated to the cloning vector pSP72 digested with BamHI. One E. coli transformant expressing -galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant -galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 50°C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined.     

Expression in Escherichia coli of a cloned β-glucanase gene from Bacillus amyloliquefaciens

Summary The recombinant phage G1 has been identified by screening 700 plaques of a Charon 4A library, containing DNA of Bacillus amyloliquefaciens, for phage clones directing the hydrolysis of lichenan in Escherichia coli, as indicated by haloes surrounding plaques on lichenan agar. The gene coding for an endo--1.3–1.4-glucanase was recloned within a 3.6 kb EcoRI fragment into the EcoRI site of plasmid pBR322, in both orientations.The location and extent of the bgl gene on the 3.6 kb Bacillus DNA insert was estimated by insertion mutagenesis with transposon Tn5 and restriction mapping of Tn5 insertions within or near to the bgl gene.The -glucanase synthesized by E. coli harbouring plasmids pEG1 or pEG2 was shown to accumulate mainly in the periplasmic space but -glucanase activities were also detected extracellulary and in the cytoplasm. The molecular weight of the enzyme synthesized in E. coli harbouring pEG1 was estimated by SDS-polyacrylamide gel electrophoresis to be about 24000. It was shown that the level of bgl gene expression in E. coli varies about 10-fold, depending on the orientation of the 3.6 kb DNA-fragment cloned within the EcoRI site of pBR322. After insertion of HindIII-DNA fragments from phage into the HindIII site of the -glucanase-high-expression plasmid pEG1, we obtained clones also showing an approximately 10-fold reduction in -glucanase activites. It was thus concluded that on plasmid pEG1 the leftward acting Ap^r (PI) promotor of plasmid pBR322 strongly increases the expression in E. coli of the cloned B. amyloliquefaciens bgl gene.Abbreviations Ap ampicillin, Km, kanamycin - kd kilodalton - kb kilobase pairs - moi multiplicity of infection - pfu plaque forming units - SDS sodium dodecylsulphate - Tc tetracycline     

Expression in Escherichia coli of cDNA Encoding Barley β-Amylase and Properties of Recombinant β-Amylase

To express the cloned β-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had β-amylase activity that produced β-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley β-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn’t change throughout the incubation. But Western blot analysis found that one β-amylase having a molecular weight of about 56,000 was synthesized. The recombinant β-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS–PAGE but showed heterogeneity on isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2–5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombiant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27–29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the K_m values for maltohexaose and maltoheptaose. The pI of recombiant barley β-amylase calculated by Genetyx Version 9 based on the presumed amino acid sequence was 5.60, but the real pIs were 5.20–6.13. Therefore, some post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences in the pI and the K_m values for maltohexaose and maltoheptaose between the barley and the recombinant β-amylases.     

Expression and purification of coronavirus envelope proteins using a modified β-barrel construct

Coronavirus envelope (E) proteins are short (～100 residues) polypeptides that contain at least one transmembrane (TM) domain and a cluster of 2-3 juxtamembrane cysteines. These proteins are involved in viral morphogenesis and tropism, and their absence leads in some cases to aberrant virions, or to viral attenuation. In common to other viroporins, coronavirus envelope proteins increase membrane permeability to ions. Although an NMR-based model for the TM domain of the E protein in the severe acute respiratory syndrome virus (SARS-CoV E) has been reported, structural data and biophysical studies of full length E proteins are not available because efficient expression and purification methods for these proteins are lacking. Herein we have used a novel fusion protein consisting of a modified β-barrel to purify both wild type and cysteine-less mutants of two representatives of coronavirus E proteins: the shortest (76 residues), from SARS-CoV E, and one of the longest (109 residues), from the infectious bronchitis virus (IBV E). The fusion construct was subsequently cleaved with cyanogen bromide and all polypeptides were obtained with high purity. This is an approach that can be used in other difficult hydrophobic peptides.     

Expression of human CYP27B1 in Escherichia coli and characterization in phospholipid vesicles

CYP27B1 is a mitochondrial cytochrome P450 that catalyses the hydroxylation of 25-hydroxyvitamin D3 at the C1α-position to give the hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3. We successfully expressed human CYP27B1 in Escherichia?coli and partially purified this labile enzyme and carried out a detailed characterization of its kinetic properties in a reconstituted membrane environment. The phospholipid concentration did not affect the enzyme activity in the vesicle-reconstituted system, although it was influenced by the phospholipid composition, with the addition of cardiolipin lowering the K(m) for 25-hydroxyvitamin D3. These data are consistent with the enzyme accessing substrate from the hydrophobic domain of the vesicle membrane. Cardiolipin also caused the appearance of inhibition of activity at high substrate concentrations. This substrate inhibition fitted a model for one catalytic and two inhibitory sites on the enzyme for the binding of substrate. The K(m) for human adrenodoxin was observed to decrease with decreasing substrate concentration, with the catalytic efficiency (k(cat) /K(m) ) being largely independent of adrenodoxin concentration. Human CYP27B1 was also active on 25-hydroxyvitamin D(2) and on intermediates of the CYP24A1-mediated inactivation pathway, 24R,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, with all these substrates showing comparable k(cat) values of 50-71?min(-1) , similar to 25-hydroxyvitamin D3. The latter two substrates gave higher K(m) values than that for 25-hydroxy-vitamin D3. The present study shows that human CYP27B1 can be partially purified in an active form with the enzyme displaying high activity towards a range of substrates in a phospholipid vesicle-reconstituted system that mimics the inner-mitochondrial membrane.     

Coexpression of β-mannanase and xylanase genes in Escherichia coli

[目的]β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域.构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价.[方法]通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl.[结果]菌株诱导21h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍.[结论]SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外.此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好.菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础.     

Expression in E. coli and purification of the fibrillogenic fusion proteins TTR-sfGFP and β2M-sfGFP

The possibility of obtaining recombinant fibrillogenic fusion proteins such as transthyretin (TTR) and β2-microglobulin (β2M) with a superfolder green fluorescent protein (sfGFP) was studied. According to the literature data, sfGFP is resistant to denaturating influences, does not aggregate during renaturation, possesses improved kinetic characteristics of folding, and folds well when fused to different polypeptides. The corresponding DNA constructs for expression in Escherichia coli were created. It could be shown that during expression of these constructs in E. coli, soluble forms of the fusion proteins are synthesized. Efficient isolation of the fusion proteins was performed with the help of nickel-affinity chromatography. For this purpose a polyhistidine sequence (6-His-tag) was incorporated into the C-terminus of the sfGFP. We could show that the purified fusion proteins contained full-size sequences of the most amyloidogenic TTR variant, TTR(L55P) and β2M, and also sfGFP possessing fluorescent properties. In the course of fibrillogenesis both fusion proteins demonstrated their ability to form fibrils that were clearly detectable by atomic force microscopy. Furthermore, with the help of confocal microscopy we were able to reveal structures (exhibiting fluorescence) that are formed during fibrillogenesis. Thus, the use of sfGFP has made it possible to avoid formation of inclusion bodies (IB) during the synthesis of recombinant fusion proteins and to obtain soluble forms of TTR(L55P) and β2M that are suitable for further studies.     

Recombinant human hemoglobin: Expression and refolding of β-globin fromEscherichia coli

A plasmid analogous to the one described by Nagai and Thogersen (Nature,309, 810–812, 1984) has been constructed for the expression of globins inE. coli. Induction with nalidixic acid produces high yields of a fusion protein, NS1-FX-β-globin, where NS1 represents 81 residues of a flu virus protein and FX represents a blood-clotting Factor Xa recognition sequence, Ile-Glu-Gly-Arg. This fusion protein is readily solubilized in 50 mM NaOH and remains in solution when thepH is adjusted to 8.6. Under these conditions, the fusion protein is hydrolyzed by activated Factor X, giving authentic β-globin which can be folded in the presence of cyanohemin and native α-chains to produce a tetrameric hemoglobin with the functional properties of natural human hemoglobin.     

Activity and stability of Escherichia coli β-galactosidase in cosolvent systems

The kinetic parameters of E.coli -galactosidase were not altered by the addition of 2-propanol or ethyl acetate (1.6% v/v). While ethylene glycol (1.6% v/v) doubled the values of both KM (0.29 mM) and kcat (1393 s–), tetraethyleneglycol-dimethylether (Tetraglyme,1.6% v/v) preserved KM, but decreased kcat. At 50°C all the cosolvents dramatically shortened the enzymatic half life, and so did Tetraglyme and 2-propanol at 28°C. At 28°C, both ethyl acetate and ethylene glycol stabilised the enzyme 9- and 6-fold respectively. This fact, together with the activation effect of ethylene glycol may lead to practical applications. © Rapid Science Ltd. 1998     

Optimization of recombinant β-NGF expression in Escherichia coli using response surface methodology

Human nerve growth factor a member of the neurotrophin family can be used to treat neurodegenerative diseases. As it has disulfide bonds in its structure, periplasmic expression of it using appropriate signal sequence is beneficial. Therefore, in this work β-nerve growth factor (β-NGF) was expressed in Escherichia coli using pET39b expression vector containing DsbA signal sequence. In an initial step, the effect of isopropyl β-D-1-thiogalactopyranoside (IPTG) and lactose concentration as inducer on protein production was investigated using response surface methodology. Then the effect of different postinduction time and temperature on protein production was studied. Our results indicated that the highest β-NGF production was achieved with 1?mM IPTG and low concentrations of lactose (0–2% w/v), low cultivation temperature of 25°C and postinduction time of 2?hr. Also following β-NGF purification, bioassay test using PC12 cell line was done. The biological activity of the purified β-NGF showed a similar cell proliferation activity with the standard recombinant human β-NGF. In conclusion, the results indicated an optimized upstream process to obtain high yields of biologically active β-NGF.     

Production of Bioactive Human β-defensin-3 in Escherichia coli by Soluble Fusion Expression

A codon optimized mature human β-defensin-3 gene (smHBD3) was synthesized and fused with TrxA to construct pET32-smHBD3 vector, which was transformed into E. coli BL21(DE3) and cultured in MBL medium. The volumetric productivity of fusion protein reached 0.99 g fusion protein l⁻¹, i.e. 0.21 g mature HBD3 l⁻¹. Ninety-six percentage of the fusion protein was in a soluble form and constituted about 45% of the total soluble protein. After cell disruption, the soluble fusion protein was separated by affinity chromatography and cleaved by enterokinase, and then the mature HBD3 was purified by cationic ion exchange chromatography. The overall recovery ratio of HBD3 was 43%. The purified mature HBD3 demonstrated antimicrobial activity against E. coli. Revisions requested 13 December 2005; Revisions received 24 January 2006     

Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

Expression in Escherichia coli and characterization of β-xylosidases GH39 and GH-43 from Bacillus halodurans C-125

To develop xylosidases as tools for the hydrolysis of wheat bran arabinoxylans, two β-xylosidases from Bacillus halodurans C-125 have been cloned and expressed in Escherichia coli. The recombinant (His)₆-tagged enzymes, designated as XylBH39 and XylBH43, were efficiently purified using Ni²⁺-affinity chromatography. Determination of native molecular masses indicated that XylBH43 is dimeric in solution, whereas a similar analysis of XylBH39 did not allow differentiation between the dimeric and trimeric states. Both enzymes had similar pH and temperature optima (pH 7.5 and 55 °C for XylBH39 and pH 8 and 60 °C for XylBH43) and were relatively stable over the pH range of 3.5–8.5. In contrast, XylBH39 was more thermostable. At 60 °C, XylBH39 and XylBH43 displayed approximate half-life values of 2.40 and 0.05 h, respectively. The comparison of the ratio k _cat/K _M revealed that XylBH43 hydrolyzed p-nitrophenyl-β-d-xyloside more efficiently (4.6-fold) than XylBH39. Similarly, while XylBH43 was 18-fold less active on p-nitrophenyl-α-l-arabinofuranoside, XylBH39 was essentially inactive on this substrate. Using either p-nitrophenyl-β-d-xyloside or xylotriose, XylBH39 performed transglycosylation, while xylobiose proved to be a poor substrate for both hydrolysis and transglycosylation. The use of XylBH39 and XylBH43 for the posttreatment of endoxylanase-generated wheat bran hydrolysates revealed that XylBH43 efficiently produced xylose monomers (385 μg/ml after 330 min incubation). Its activity was improved by the simultaneous deployment of an α-l-arabinofuranosidase. Together, these enzymes were able to release 521 μg/ml of xylose after 330 min. This constitutes an approximate yield improvement of 35%.     

Optimization of soluble human interferon-γ production in Escherichia coli using SUMO fusion partner

Interferon-γ (IFN-γ) is a broad-spectrum antiviral glycoprotein that produced by lymphatic T cells and natural killer cells those who had stimulated by antigen. Human IFN-γ (hIFN-γ) often used in clinical research and practice because of its bioactivity, for example, antivirus, antitumor, controlling cell apoptosis, and the strict selectivity. However, due to the difficulties of Escherichia coli expression system meet in protein folding, the hIFN-γ often existed as inclusion body. The production of soluble hIFN-γ can be developed to shorten the production cycle and decrease the cost. In this study, small ubiquitin-related modifier fusion technology was used to express and purify recombinant hIFN-γ. Expression induced by adding 50 mM arginine and 1 % (w/v) glycerol into the culture at 24 °C existed as a soluble form of 70 % in total protein. Finally, about 62 mg recombinant hIFN-γ was obtained from 1 L fermentation culture with no less than 96 % purity. Determined by cytopathic effect inhibition assay, the specific activity of the recombinant hIFN-γ achieved at 7.78 × 10⁵ IU/mL.     

An improved lentiviral system for efficient expression and purification of β-defensins in mammalian cells

β-Defensins are a family of conserved small cationic antimicrobial peptides with different significant biological functions. The majority of mammalian β-defensins are expressed in epididymis, and many of them are predicted to have post-translational modifications. However, only a few of its members have been well studied due to the limitations of expressing and purifying bioactive proteins with correct post-translational modifications efficiently. Here we developed a novel Fc tagged lentiviral system and Fc tagged prokaryotic expression systems provided new options for β-defensins expression and purification. The novel lentiviral system contains a secretive signal peptide, an N-terminal IgG Fc tag, a green fluorescent protein (GFP), and a puromycin selection marker to facilitate efficient expression and fast purification of β-defensins by protein A magnetic or agarose beads. It also enables stable and large-scale expression of β-defensins with regular biological activities and post-translational modification. Purified β-defensins such as Bin1b and a novel human β-defensin hBD129 showed antimicrobial activity, immuno-regulatory activity, and expected post-translational phosphorylation, which were not found in Escherichia coli (E. coli) in expressed form. Furthermore, we successfully applied the novel system to identify mBin1b interacting proteins, explaining Bin1b in a better way. These results suggest that the novel lentiviral system is a powerful approach to produce correct post-translational processed β-defensins with bioactivities and is useful to identify their interacting proteins. This study has laid the foundation for future studies to characterize function and mechanism of novel β-defensins.     

Catabolite repression of β-galactosidase synthesis in Escherichia coli

1. Repression by glucose of β-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i⁻) and permease-less (y⁻) cells as well as in the corresponding i⁺ and y⁺ strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-β-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of β-galactosidase synthesis (e.g. isopropyl thio-β-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-β-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.