In vivo monitoring of intracellular expression of human interleukin-2 using green fluorescent protein fusion partner in Pichia pastoris

The use of Pichia pastoris for protein production was simplified by creation of fusion proteins containing green fluorescent protein (GFP) and the product of interest. Human interleukin-2 (hIL-2) was used as a model product: GFP enabled clear identification of fusion protein expression and, more importantly, the quantification of hIL-2. Although GFP fusions for bioprocess monitoring have been demonstrated in other hosts, this is its first use in P. pastoris.     

Enhanced activity of Candida rugosa lipase modified by polyethylene glycol derivatives

The derivatives of polyethylene glycol (PEG) were prepared by reacting PEG with propylene oxide to enhance its hydrophobicity and introduce a branched structure. The PEG derivatives were activated with cyanuric chloride and used to modify the lipase fromCandida rugosa. The maximum specific activity of lipase modified with the PEG derivatives was about 2-fold of that modified with PEG for the esterification of oleic acid and lauryl alcohol in hexane.     

Expression of recombinant actin 5C from Drosophila in the methylotrophyc yeast Pichia pastoris

A system of expression for the foreign actin gene in yeast cells Pichia pastoris has been developed. As a target protein, the Drosophila cytoplasmic actin 5C, which has 90% homology to the β-actin of higher eukaryotes, was used. In the present work, in order to develop conditions for biosynthesis of the target protein in yeast cells and a purification procedure for the recombinant protein, a GFP-actin fusion protein containing green fluorescent protein (GFP) as a fusion tag was expressed and purified. The size and survival of P. pastoris cells producing recombinant protein were characterized and shown to depend on the accumulation of recombinant actin. The purified fusion protein was used to obtain a polyclonal antibody necessary for testing for recombinant actin.     

Enhancement of Candida rugosa lipase activity in non-aqueous media by co-lyophilization with amphiphiles from aqueous solution

Modified Candida rugosa lipase was co-lyophilized with two gemini-type amphiphiles, l- and d-2-(3-bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl -propionylamino)-pentanedioic acid didodecyl ester or dodecanoic acid 2-[(3-bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl -propionyl)-(2-dodecanoyloxy-ethyl)-amino]-ethyl ester. Enzymatic activities of the modified lipases in the transesterification between racemic 2,2-dimethyl-1,3-dioxolane-4-methanol and vinyl butyrate in cyclohexane were enhanced as much as by 37-78, 1.5–5- and 41–83-fold of magnitude relative to that of native enzyme, respectively. The lack of significant enhancement of the enzymatic activity, only in the case of the d-isomeric amphiphile-modified lipase, was considered from the topological view of the amphiphile.     

Purification and characterization of two isoforms from Candida rugosa lipase B

Two isoforms of Candida rugosalipase B (LB1 and LB2) were purified by anionic exchange chromatography. The lipases had the same N-terminal sequence, carbohydrate content and pH and thermal stability but different pIs and significant differences in their activities against different p-nitrophenol esters and triacylglycerides.     

Scale-up fermentation of recombinant <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using the GAP promoter

The high-cell-density fermentation of Candida rugosa lipase in the constitutive Pichia pastoris expression system was scaled up from 5 to 800 l in series by optimizing the fermentation conditions at both lab scale and pilot scale. The exponential feeding combined with pH-stat strategy succeeded in small scale studies, while a two-stage fermentation strategy, which shifted at 48 h by fine tuning the culture temperature and pH, was assessed effective in pilot-scale fermentation. The two-stage strategy made an excellent balance between the expression of heterogeneous protein and the growth of host cells, controlling the fermentation at a relatively low cell growth rate for the constitutive yeast expression system to accumulate high-level product. A stable lipase activity of approximately 14,000 IU ml⁻¹ and a cell wet weight of ca. 500 g l⁻¹ at the 800-l scale were obtained. The efficient and convenient techniques suggested in this study might facilitate further scale-up for industrial lipase production.     

High lipase production by Candida rugosa is associated with G1 cells. A flow cytometry study

Growth of C. rugosa on three different culture media was analysed by laser flow cytometry to evaluate physiological growth conditions allowing effective lipase production. The highest productivity was associated with an increased proportion of cells in the G1 phase and was independent of the effect of the medium on lipase formation.     

Expression and characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel production

The Rhizopus oryzae lipase containing prosequence was expressed in Pichia pastoris. Recombinant lipase subunit showed a molecular mass of 32 kDa. The maximum activity of recombinant lipase obtained from Mut(s) recombinant was 90 IU/ml. The enzyme was stable in broad ranges of temperatures and pH, with the optimal temperature at 35 °C and pH 7.0. The crude recombinant R. oryzae lipase can be directly used for the transesterification of plant oils at high-water content of 60-100% (w/w) based on oil weight. The addition of 80% water to the transesterification systems resulted in the yield of methyl ester of 95%, 94% and 92% after 72 h using soybean oil, Jatropha curcas seed raw oil and Pistacia chinensis seed raw oil as raw material, respectively. These results indicate that the recombinant lipase is an effective biocatalyst for enzymatic biodiesel production.     

Capsaicin hydrolysis by Candida antarctica lipase

Capsaicin was hydrolysed by lipase B from Candida antarctica into vanillylamine and 8-methyl-6-trans-nonenoic acid. Conversions of 70% were obtained after 72 h at 70 °C in water but decreased to only 15% when capsaicin was solubilized in 15% (v/v) ethanol/water after 72 h at 45 °C. No activity occurred in chloroform/water mixtures. According to our knowledge, this is the first report concerning amide hydrolysis by a lipase.     

Secretory expression and characterization of a highly Ca-activated thermostable L2 lipase

Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 °C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca²⁺. L2 lipase showed a preference for medium to long chain triacylglycerols (C₁₀–C₁₆), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.     

Optimization of process variables in castor oil hydrolysis by Candida rugosa lipase with buffer as dispersion medium

In this study, castor oil is hydrolyzed in presence of Candida rugosa lipase, while in the buffer (aqueous) phase as a dispersion medium. The following conditions were used to optimize the process: speed of agitation, initial pH of buffer phase, temperature, and ratio of buffer phase volume to oil weight. The optimal conditions are 1,100 rpm, pH 6.5, temperature 35°C, and 3:1 buffer phase volume to oil weight ratio. Under these described conditions, the reusability of lipase was tested and it was found that nearly 80% of original hydrolysis percentage was achieved after the first recycle.     

High-yield purification and characterization of recombinant human leukotactin-1 in<Emphasis Type="Italic">Pichia pastoris</Emphasis>

The human chemokine, the short version of leukotactin-1 (shLkn-1; molecular weight =7.2 kD and 66 amino acids), was expressed and secreted into a culture medium using the methylotrophic yeast,Pichia pastoris. The recombinant shLkn-1 was purified from the culture supernatant using a simple two-step procedure consisting of cation exchange and reverse phase chromatography (RPC), in which shLkn-1 was highly purified (99.5%) with a high recovery yield of 82.7%. The C-terminal truncated derivative of shLkn-1 was found in the supernatant and was separated by RPC. The physicochemical properties of the purified shLkn-1 were verified to be the same as expected. The biological activity of the purified recombinant shLkn-1 was also quantified using a chemotaxis assay. It was observed that the recombinant shLkn-1 had the maximum migration activity at a concentration of 10 nM, as potent as MIP-1α.     

Isolation of peroxisome-defective CHO mutant cells using green fluorescent protein

The authors constructed a recombinant green fluorescent protein (GFP) (PTS-GFP), which carries peroxisome targeting signal (PTS1 or PTS2) as an additional sequence, by polymerase chain reaction. The gene encoding for the recombinant GFP was constructed into an eukaryotic expression vector, and stable transformant of CHO cell expressing PTS-GFP was isolated, following the transfection of the plasmid encoding for the GFP. Each expressed PTS-GFP appeared to be localized in peroxisomes, because the GFP was observed in cellular structures, as was catalase. The observation proposed a visual screening procedure for isolating peroxisome-defective mutant. Following an enrichment of mutant cells by use of 9-(1′-pyrene)nonanol/ultraviolet irradiation (P9OH/UV) method, five peroxisome-defective mutants were isolated by pursuing the fluorescent signals from GFP. Two mutants (SK24 and SK32) were isolated from CHO cells expressing PTS1-GFP, and three mutants (PT13, PT32, and PT54) were isolated from cells expressing PTS2-GFP. Four mutants, except for PT13, showed cytosolic distributions of both PTS-GFP and catalase. On the other hand, mutant PT13 showed a cytosolic distribution on PTS2-GFP, but a peroxisomal distribution on catalase. Cell fusion analysis between SK24 mutant and other mutants indicated that PT54 mutant is in the same complementation group (CG) as SK24, but that SK32, PT13, and PT32 mutants are in different complementation group(s) from SK24.     

Monitoring a recombinant Pichia pastoris fed batch process using Fourier transform mid-infrared spectroscopy (FT-MIRS)

The use of Fourier transform mid-infrared spectroscopy (FT-MIRS) to predict the concentrations of key analytes in fed-batch cultivations of an industrial strain of Pichia pastoris in a chemical complex medium was investigated. Models for glycerol, methanol (substrates), and product (an heterologous protein) were built, and evaluated. The use of a multi-bounce attenuated total reflectance (HATR) accessory aided spectral acquisition in optically dense samples. Generally, all models were robust and performed well on external validation, using data from processes not present in the original modelling exercise. Substrate models lacked the complexity of some previous IR models, and the models performed adequately even at low analyte concentration (<1 g l^–1). Thus, simultaneous, rapid monitoring of low concentrations of multiple analytes in a complex bioprocess matrix with little or no sample pre-treatment is achievable using ATR FT-MIRS.     

Structure and dynamics of green fluorescent protein

Many marine organisms are luminescent. The proteins that produce the light include a primary light producer (aequorin or luciferase) and often a secondary photoprotein that red shifts the light for better penetration in the ocean. Green fluorescent protein is one such secondary protein. It is remarkable in that it autocatalyzes the formation of its own fluorophore and thus can be expressed in variety of organisms in its fluorescent form. The recent determination of its 3D structure and other physical characterizations are revealing its molecular mechanism of action     

Visualizing the infection process of Xanthomonas campestris in cabbage using green fluorescent protein

The plant pathogen, Xanthomonas campestris NRRL B-1459 was chromosomally tagged with gfp, and the transformant, which was subjected to Southern hybridization showed the presence of gfp in the chromosome. The virulence-related gene of the transformant was not affected by the insertion of gfp. After inoculation into cabbage plants, the infection process was visually studied in planta. Using a fluorescence microscope, the migration and distribution of gfp-labelled bacteria was visualized in real time. As the gfp-labelled cells were easily visualized from the beginning of infection, we observed a time delay of 2 days between distribution of the Xanthomonas cells in cabbage plant and the appearance of visible necrosis.     

Enhanced activity and enantioselectivity of Candida rugosa lipase immobilized on macroporous adsorptive resins for ibuprofen resolution

The lipase from Candida rugosa was immobilized on three commercially available macroporous adsorptive resins for kinetic resolution of ibuprofen. One resin, CRB02, increased the enzyme activity by 50% to 0.027 g g(-1) min(-1). The deactivation constant (0.19 h(-1)) of the immobilized enzyme was half of that of the native enzyme and the enantioselectivity (E = 29.2) of the immobilized lipase was 2.2 times as much as that of the native lipase for the kinetic resolution of ibuprofen with 1-propanol in isooctane at 30 degrees C.     

Agrobacterium tumefaciens-mediated transformation of the legume Astragalus sinicus using kanamycin resistance selection and green fluorescent protein expression

To develop an efficient protocol for the transformation of the legume Astragalus sinicus (Chinese milk vetch), cotyledon segments were infected with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBINm-gfp5-ER which carries the gfp5 gene encoding green fluorescent protein and the kanamycin (Km) resistance gene nptII. The infected explants were cultured on shoot regeneration (SR) medium containing 1.0 mg l^–1 -naphthaleneacetic acid (NAA) and 1.0 mg l^–1 thidiazuron (TDZ). Putative transformed shoots were selected on SR medium containing 75 g ml^–1 Km, 200 g ml^–1 Timentin, and transformation was monitored by observation of GFP expression under a dissecting fluorescence microscope with appropriate filters. The identification of GFP-expressing shoots or callus in combination with Km selection allowed the visual selection of growing transgenic cells and shoots with no escapes. Plants were regenerated from seven independent transgenic events and five plants have set seed. GFP expression segregated in the T₁ seedlings of the two lines tested in a 3 – 1 ratio. In addition to the GFP expression of the transgenic plants, the transgenic nature of individual plants was confirmed by Southern and Western blot analyses.     

Integrated bioprocessing in Saccharomyces cerevisiae using green fluorescent protein as a fusion partner

In this study, we examine the use of green fluorescent protein (GFP) for monitoring a hexokinase (HXK)-GFP fusion protein in Saccharomyces cerevisiae for various events including expression, degradation, purification, and localization. The fusion, HXK-EK-GFP-6 x His, was constructed where the histidine tag (6 x His) would allow for convenient affinity purification, and the enterokinase (EK) cleavage site would be used for separation of HXK from GFP after affinity purification. Our results showed that both HXK and GFP remained active in the fusion and, more importantly, that there was a linear correlation between HXK activity and GFP fluorescence. Enterokinase cleavage studies revealed that both GFP fluorescence intensity and HXK activity remained unchanged after separation of the fusion proteins, which indicated that fusion of GFP did not cause structural alteration of HXK and thus did not affect the enzymatic activity of HXK. We also found that degradation of the fusion protein occurred, and that degradation was limited to HXK with GFP remaining intact in the fusion. Confocal microscopy studies showed that while GFP was distributed evenly in the yeast cytosol, HXK-GFP fusion followed the correct localization of HXK, which resulted in a di-localization of both cytosol and the nucleus. GFP proved to be a useful fusion partner that may lead to the possibility of integrating the bioprocesses by quantitatively following the entire process visually.