Recovery of bovine lysozyme from transgenic sugarcane stalks: extraction, membrane filtration, and purification.

Increased industrial use of sugarcane (Saccharum spp. hybrid) for food and bioenergy has led to considerable improvements in its genetic transformation, which allowed the development of not only pest- and herbicide-resistant lines but also lines expressing high-value bioproducts and biopolymers. However, the economic benefits of using inexpensive transgenic plant systems for the production of industrial proteins could be offset by high downstream processing costs. In this work, transgenic sugarcane expressing recombinant bovine lysozyme (BvLz) was used to evaluate the feasibility of extraction and fractionation of recombinant proteins expressed in sugarcane stalks. Three pH levels (4.5, 6.0 and 7.5) and three salt concentrations (0, 50, and 150 mM NaCl) were tested to determine BvLz and total protein extractability. Two extraction conditions were selected to prepare BvLz extracts for further processing by cross-flow filtration, a suitable method for concentration and conditioning of extracts for direct applications or prior to chromatography. Partial removal of native proteins was achieved using a 100 kDa membrane but 20-30 % of the extracted BvLz was lost. Concentration of clarified extracts using a 3 kDa membrane resulted in twofold purification and 65 % recovery of BvLz. Loading of concentrated sugarcane extract on hydrophobic interaction chromatography (HIC) resulted in 50 % BvLz purity and 69 % recovery of BvLz.

Plant molecular farming: systems and products.

Plant molecular farming is a new and promising industry involving plant biotechnology. In this review, we describe several diverse plant systems that have been developed to produce commercially useful proteins for pharmaceutical and industrial uses. The advantages and disadvantages of each system are discussed. The first plant-derived molecular farming products have reached the marketplace and other products are poised to join them during the next few years. We explain the rationale for using plants as biofactories. We also describe the products currently on the market, and those that appear likely to join them in the near future. Lastly, we discuss the issue of public acceptance of molecular farming products.

Improved recovery of active recombinant laccase from maize seed.

Lignolytic enzymes such as laccase have been difficult to over-express in an active form. This paper describes the expression, characterization, and application of a fungal laccase in maize seed. The transgenic seed contains immobilized and extractable laccase. Fifty ppm dry weight of aqueously extractable laccase was obtained, and the remaining solids contained a significant amount of immobilized laccase that was active. Although a portion of the extractable laccase was produced as inactive apoenzyme, laccase activity was recovered by treatment with copper and chloride. In addition to allowing the apoenzyme to regain activity, treatment with copper also provided a partial purification step by precipitating other endogenous corn proteins while leaving >90% of the laccase in solution. The data also demonstrate the application of maize-produced laccase as a polymerization agent. The apparent concentration of laccase in ground, defatted corn germ is approximately 0.20% of dry weight.

Plant-based vaccines: unique advantages.

Numerous studies have shown that viral epitopes and subunits of bacterial toxins can be expressed and correctly processed in transgenic plants. The recombinant proteins induce immune responses and have several benefits over current vaccine technologies, including increased safety, economy, stability, versatility and efficacy. Antigens expressed in corn are particularly advantageous since the seed can be produced in vast quantities and shipped over long distances at ambient temperature, potentially allowing global vaccination. We have expressed the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis virus at high levels in corn, and demonstrate that these antigens delivered in the seed elicit protective immune responses.

Purification and characterization of lymphocyte chymase I, a granzyme implicated in perforin-mediated lysis.

One mechanism of killing by cytotoxic lymphocytes involves the exocytosis of specialized granules. The released granules contain perforin, which assembles into pores in the membranes of cells targeted for death. Serine proteases termed granzymes are present in the cytotoxic granules and include several chymases (with chymotrypsin-like specificity of cleavage). One chymase is selectively reactive with an inhibitor, Biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, that blocks perforin lysis. We report the purification and characterization of this chymase, lymphocyte chymase I, from rat natural killer cell (RNK)-16 granules. Lymphocyte chymase I is 30 kDa with a pH 7.5 to 9 optimum and primary substrate preference for tryptophan, a preference distinct from rat mast cell chymases. This chymase also reacts with other selective serine protease inhibitors that block perforin pore formation. It elutes by Cu2+-immobilized metal affinity chromatography with other granzymes and has the N-terminal protein sequence conserved among granzymes. Chymase I reduces pore formation when preincubated with perforin at 37 degrees C. In contrast, addition of the chymase without preincubation had little effect on lysis. It should be noted that the perforin preparation contained sufficient residual chymase activity to support lysis. Thus, the reduction of lysis may represent an effect of excess prolytic chymase I or a means to limit perforin lysis of bystander cells. In contrast, other chymases and granzyme K were without effect when added to perforin during similar preincubation. Identification of the natural substrate of chymase I will help resolve how it regulates perforin-mediated pore formation.

Synthesis and kinetic studies of diphenyl 1-(N-peptidylamino)alkanephosphonate esters and their biotinylated derivatives as inhibitors of serine proteases and probes for lymphocyte granzymes.

Diphenyl 1-(N-peptidylamino)alkanephosphonate esters are highly reactive, specific, and aqueously stable irreversible inhibitors which can be used to probe the functions of many serine proteases, including the lymphocyte granzymes. We synthesized 16 peptide phosphonates with Ala, Met, Phe, or Val P1 amino acid residues, including two biotinylated derivatives for future functional and biochemical characterization of granzymes. The reactivity of the inhibitors was characterized with human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), bovine chymotrypsin, and the granzymes of natural killer (NK) cells, which include a number of proteolytic activities (Asp-ase, Met-ase, etc.) that cleave peptide substrates with these residues in the P1 position. The reactivity and specificity of the phosphonates depended on the length and sequence of the peptidyl moiety and on the leaving group. Z-Ala-Ala-AlaP(OPh)2 was a good inhibitor of HLE and PPE (k(obsd)/[I] = 38 and 30 M(-1) s(-1), respectively) and had little reactivity with chymotrypsin. Z-Phe-Pro-Phe-P(OPh)2 was a good inhibitor of chymotrypsin (k(obsd)/[I] = 17,000 M(-1) s(-1)) and had little reactivity with the elastases. The leaving group of Z-MetP(OPh-4-Cl)2 made it a more effective chymotrypsin inhibitor than Z-MetP(OPh)2 (k(obsd)/[I] values of 142 and 30 M(-1) s(-1), respectively). With granzymes, the compounds reacted with a fraction of the Met-ase, chymase, and Ser-ase activities and lacked reactivity with Asp-ase and tryptase. Z-MetP(OPh-4-Cl)2 was an excellent inhibitor of Met-ase 1. Bi-Aca-Aca-Phe-Leu-PheP(OPh)2 appears to react specifically with one chymase while leaving other chymases untouched. Perforin-dependent lysis mediated by cytotoxic lymphocyte granules was inactivated by Z-Ala-Ala-AlaP(OPh)2, Z-MetP(OPh-4-Cl)2, Z-Leu-PheP(OPh)2, and Bi-Aca-Aca-Phe-Leu-PheP(OPh)2. The biochemical properties and biological efficacy of these inhibitors make them suitable for cellular and physiological studies of granzyme function.

P-4 and RNKP-7, new granzyme-like serine proteases expressed in activated rat lymphocytes.

Serine proteases (granzymes) in killer lymphocytes are required for lymphocyte cytotoxic granules to lyse target cells. Herein we report the development of a 3-step PCR cloning technique to amplify novel granzyme genes and two new rat granzymes are described. Degenerate oligonucleotide primers were designed based on sequence motifs selectively expressed in granzymes. These motifs flank "delta" regions that are unique for each granzyme. Total RNA of RNK-16 cells or activated splenocytes was amplified by reverse transcriptase-PCR to obtain cDNA fragments of several new granzymes. Gene-specific primers based on these delta regions were then used for 3'-RACE to obtain clones with the 3' gene ends. Reverse (antisense) delta-based or active site serine primers were used with a granzyme 5'-UTR primer to obtain clones extending to the 5' ends. Using this technique, two new cDNAs, RNKP-4 and RNKP-7, which encode granzymes of 248 and 241 amino acids, respectively, were cloned from activated lymphocytes. RNKP-4 is likely the rat equivalent of mouse granzyme C. RNKP-7 is most closely related to granzymes F and G. Modeling of the predicted proteins suggests large/polar P1 (Gln/Asn) specificity for RNKP-4 and large/hydrophobic P1 (e.g., Phe) specificity for RNKP-7. These specific protease activities were found in cytotoxic RNK-16 lymphocyte granules indicating that the two new genes may be translated and stored as active granzymes.

Characterization, application and potential uses of biotin-tagged inhibitors for lymphocyte serine proteases (granzymes).

Cytotoxic lymphocytes and natural killer cells are able to kill their target cells in minutes. The death of the target cell occurs after the release of cytoplasmic granules from the effector cell. These granules contain the pore-forming protein perforin and serine proteases (granzymes). To date 10 genes encoding lymphocyte granzymes have been discovered; of these only four have been purified and characterized for their substrate specificity. Several are predicted to have a common chymase, like specificity which is found in the granule extracts. Others may need to be enriched as active enzymes before they can be evaluated for substrate hydrolysis. Due to the limitations of detection by substrate hydrolysis, a more sensitive method for the detection of dilute granules was needed. We report the differing reactivities of seven biotin (Bi)-tagged isocoumarin (IC) inhibitors for Asp-ase, chymase, tryptase and Met-ase granzymes. The inhibitors contained different substituents at their no. 3 position: methoxy (OMe), ethoxy (OEt), propoxy (OPr) or 2-phenylethoxy (OEtPh) groups. The OMe group conferred general reactivity, whereas the OEtPh group conferred selective reactivity with chymase granzymes. The inhibitors that contained the longest aminocaproyl (Aca) spacers between the biotin-tag and the isocoumarin ring mediated the most stable granzyme inactivation. These inhibitors were the most effective at blocking lysis of red blood cells by the granule extracts. The inhibitors were used in protein blotting experiments where the biotin was detected with an avidin-enzyme complex. Over 10 granzymes were labelled by the inhibitor Bi-Aca-Aca-IC-OMe. The inhibitors detected granzymes when they were not readily detected by substrate hydrolysis.

Flow cytometric analysis of T-independent antigen binding to dinitrophenyl-specific cells.

Binding of Ag to membrane Ig (mIg) can lead to either activation or desensitization of the B cell. For thymus-independent (TI) Ags the nature and concentration of the Ag determines what type of signal is delivered to the cell. These Ags are capable of directly activating B lymphocytes and are an important model system for the study of mechanisms involved in B cell responses. In this study, we quantified TI Ag binding and B cell receptor involvement as functions of TI Ag structure, concentration, and epitope density. Various epitope densities of two structurally different TI Ags, DNP-polymerized flagellin (pol) and DNP-dextran (dex), were labeled with tetramethylrhodamine isothiocyanate (TRITC) and reacted with DNP-specific murine splenic B lymphocytes and with cells of a cloned DNP-specific cell line. The amount of Ag bound to the cell surface at various doses was measured directly by flow cytometry. For each Ag and dose, FITC-labeled DNP-L-papain was used to quantitate receptor sites not occupied by Ag. Approximately 5% receptor occupancy was observed for immunogenic doses of Ag. Higher Ag concentrations that can induce tolerance caused a substantial increase in the fraction of occupied receptors. This suggests that tolerogenic responses result from an overly restrictive cross-linking of surface receptors. By comparing these data to previously published data on biologic activity of the Ags, we are able to more clearly define those conditions of Ag binding that lead to B cell activation.

Chymase-directed serine protease inhibitor that reacts with a single 30-kDa granzyme and blocks NK-mediated cytotoxicity.

Cytotoxic NK and T lymphocytes kill virally infected cells within minutes without causing damage to themselves or bystander cells. One mechanism of killing involves exocytosis of granules containing serine proteases and perforin. Serine protease inhibitors block killing of target cells mediated by the cytotoxic lymphocytes. There are at least five different serine protease activities in cytolytic granules. Ten different serine protease sequences have been identified with the use of cDNA-specific clones. It is not known whether only one or several of these serine proteases are essential for cytolytic activity. In this study we show that an irreversible serine protease inhibitor, biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, selectively inhibits a chymotrypsin-like (chymase) serine protease activity of rat RNK-16 granule extracts. Under the same conditions, only one 30-kDa (reduced) band was detected on protein blots. Furthermore, only one of three chymase peaks separated by hydrophobic interaction chromatography was inhibited. When this granzyme was inhibited, granule-mediated lysis of erythrocytes was diminished. NK cell killing was completely blocked when biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2 was added to cytotoxicity assays at 200 microM with rat splenocytes as effectors. By confocal fluorescence microscopy, we show that this inhibitor localizes to distinct regions within RNK-16 cells and rat NK cells. Inhibitor treatment of intact cells inactivated the chymase activity and reduced lysis found in their dense organelles. Together these data indicate that biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2 inhibits a granule chymase that is essential to cytolytic activity of NK cells.

Fluorescent derivatives of diphenyl [1-(N-peptidylamino)alkyl]phosphonate esters: synthesis and use in the inhibition and cellular localization of serine proteases.

Three fluorescein- and one Texas Red-labeled derivatives of [1-(N-dipeptidylamino)alkyl]phosphonate diphenyl esters were synthesized and evaluated as inhibitors of serine proteases. The two fluorophores, FITC and TXR, were attached to the peptide phosphonates via an epsilon-aminocaproyl unit that acts as a spacer group and facilitates the binding of the phosphonate inhibitor to the targeted enzymes. These derivatives are potent and specific inhibitors of chymotrypsin, porcine pancreatic elastase (PPE), and human leukocyte elastase (HLE). FTC-Aca-Phe-Leu-PheP(OPh)2 (3) inhibited chymotrypsin very potently (k(obsd)/[I] = 9500 M-1 s-1) and 600-fold better than it did PPE (k(obsd)/[I] = 16 M-1 s-1). FTC-Aca-Ala-Ala-MetP(OPh)2 (1) was a more effective inhibitor of chymotrypsin (k(obsd)/[I] = 190 M-1 s-1) than PPE and HLE (k(obsd)/[I] = 13 and 22 M-1 s-1, respectively). Only HLE and PPE were inhibited by FTC-Aca-Ala-Ala-AlaP(OPh)2 (2) (k(obsd)/[I] = 41 and 22 M-1 s-1, respectively). The specificity of these inhibitors toward the targeted serine proteases depends on the sequence of the tripeptide portion and was not affected by the presence of the fluorescent label. Trypsin, for instance, was not inhibited by any of these compounds. In some cases, the inhibitory potency was increased by the fluorescent label. For example, chymotrypsin was inhibited by the fluorescent compounds, FTC-Aca-Ala-Ala-MetP(OPh)2 (1) and FTC-Aca-Phe-Leu-PheP(OPh)2 (3), more potently than by the nonfluorescent compounds, Boc-Ala-Ala-MetP(OPh)2 (5) and Z-Phe-Leu-PheP(OPh)2 (7).(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane changes in lipopolysaccharide-stimulated murine B lymphocytes associated with cell activation.

The lateral diffusion of the fluorescent lipid analog 3,3'-dioctadecylindocarbocyanine iodide (DiI) was measured in the membranes of murine B lymphocytes treated with the B cell mitogen lipopolysaccharide (LPS). The mobility of DiI, as measured by fluorescence photobleaching recovery (FPR) techniques, was temperature-dependent with a value of 6.1.10(-9) cm2 s-1 at 37 degrees C. Untreated cells exhibited this diffusion coefficient over 72 h in culture. In contrast, DiI mobility decreased to 2.0.10(-9) cm2 s-1 at 37 degrees C in membranes of LPS-stimulated lymphocytes 24 h following LPS exposure. Interestingly, this decreased lipid lateral diffusion was not accompanied by any change in surface immunoglobulin lateral diffusion which remained essentially unchanged at 3.6-4.3.10(-11) cm2 s-1 over 72 h. To determine whether LPS effects on lipid lateral diffusion were due to insertion of LPS into the cell plasma membrane, we examined TRITC-LPS diffusion in B lymphocytes from LPS-responsive Balb/c and C3Heb/FeJ mice and from hypo-responsive C3H/HeJ mice. DiI and TRITC-LPS mobility decreased more than 50% in LPS-stimulated Balb/c and C3Heb/FeJ cells by 72 h. On C3H/HeJ lymphocytes, there was no change in DiI or TRITC-LPS lateral diffusion throughout the incubation period. These data indicate that B lymphocyte membrane composition is altered in LPS-activated lymphoblasts and that the decreased lateral diffusion of lipid probes does not result from membrane perturbation by LPS insertion into the lipid bilayer. Further, similarities between TRITC-LPS and DiI lateral diffusion suggest that most LPS molecules interact non-specifically with B cell membranes, presumably by acyl chain insertion of the lipid A moiety.

Proteases and lymphocyte cytotoxic killing mechanisms.

A subfamily of serine proteases is uniquely expressed by cytotoxic natural killer lymphocytes and T cells. Protease cleavage of different natural substrates is now implicated in the cytotoxic mechanisms of target cell membrane pore formation, DNA fragmentation and cytostasis.