Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis.

The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-deltaFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-deltaFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-deltaFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14-18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities.

A study of the influence of the hydrophobic core residues of yeast iso-2-cytochrome c on phosphate binding: a probe of the hydrophobic core-surface charge interactions.

To gain insight into the role of hydrophobic core-surface charge interactions in stabilizing cytochrome c, we investigated the influence of hydrophobic core residues on phosphate binding by mutating residues in yeast iso-2-cytochrome c to those corresponding to iso-l-cytochrome c in various combinations. Heat transition of ultraviolet CD was followed as a function of pH in the presence and absence of phosphate. Thermodynamic parameters were deduced. It was found that the I20V/V43A/M98L mutation in the hydrophobic core, whose locations are remote from the putative phosphate sites, modulates phosphate interactions. The modulation is pH dependent. The I20V/ M98L and V43A mutation effects are nonadditive. The results lead to a model analogous to that of Tsao, Evans, and Wennerstrom, where a domain associated with the ordered hydrophobic core is sensitive to the fields generated by the surface charges. Such an explanation would be in accord with the observed difference in thermal stability between iso-2 and horse cytochromes c.

CRTF is a novel transcription factor that regulates multiple stages of Dictyostelium development.

During aggregation, Dictyostelium establish nanomolar oscillation waves of extracellular cAMP, but as development progresses, cells become responsive to higher, non-fluctuating concentrations of cAMP. The regulation of the promoter responsible for expression of cAMP receptor subtype 1, CAR1, during aggregation reflects these signaling variations. Transcription of CAR1 from the early, aggregation promoter is activated by cAMP pulsing, but is repressed by continuous exposure to micromolar concentrations of cAMP. Deletion and mutation analyses of this promoter had defined an element essential for cAMP-regulated expression, and mobility shift assay, DNA crosslinking and DNase I footprinting experiments had identified a nuclear protein (CRTF) with zinc-dependent sequence binding specificity. In our study, CRTF was purified to homogeneity, peptides were sequenced and full-length cDNAs were obtained. The deduced CRTF protein is approximately 100 kDa with a C-terminal, zinc finger-like motif required for DNA binding; CRTF purified from cells, however, represents only a 40 kDa C-terminal fragment that retains DNA-binding activity. As might have been predicted if CRTF were essential for the regulation of CAR1, crtf-null strains fail to develop under standard conditions or to exhibit induced expression of CAR1 or other cAMP-regulated genes. Furthermore, crtf-nulls also fail to sporulate, even under conditions that bypass the dependence on early cAMP signaling pathways. In addition, early developmental events of crtf-null strains could be rescued with exogenous cAMP treatment, constitutive expression of CAR1 or co-development with wild-type cells; however, these treatments were insufficient to promote sporulation. This suggests a cell-autonomous role for CRTF during late development that is separate from its capacity to control CAR1 expression. Finally, ablation of CRTF promotes a precocious induction of certain cAMP-dependent gene expression pathways. We suggest that CRTF may function to help insulate distinct pathways from simultaneous and universal activation by cAMP. CRTF, thus, exhibits multiple complex and independent regulatory functions during Dictyostelium development.

The efficacy of a Salmonella typhi Vi conjugate vaccine in two-to-five-year-old children.

BACKGROUND: Typhoid fever is common in developing countries. The licensed typhoid vaccines confer only about 70 percent immunity, do not protect young children, and are not used for routine vaccination. A newly devised conjugate of the capsular polysaccharide of Salmonella typhi, Vi, bound to nontoxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA), has enhanced immunogenicity in adults and in children 5 to 14 years old and has elicited a booster response in children 2 to 4 years old. METHODS: In a double-blind, randomized trial, we evaluated the safety, immunogenicity, and efficacy of the Vi-rEPA vaccine in children two to five years old in 16 communes in Dong Thap Province, Vietnam. Each of the 11,091 children received two injections six weeks apart of either Vi-rEPA or a saline placebo. Cases of typhoid, diagnosed by the isolation of S. typhi from blood cultures after 3 or more days of fever (a temperature of 37.5 degrees C or higher), were identified by active surveillance over a period of 27 months. We estimated efficacy by comparing the attack rate of typhoid in the vaccine group with that in the placebo group. RESULTS: S. typhi was isolated from 4 of the 5525 children who were fully vaccinated with Vi-rEPA and from 47 of the 5566 children who received both injections of placebo (efficacy, 91.5 percent; 95 percent confidence interval, 77.1 to 96.6; P<0.001). Among the 771 children who received only one injection, there was 1 case of typhoid in the vaccine group and 8 cases in the placebo group. Cases were distributed evenly among all age groups and throughout the study period. No serious adverse reactions were observed. In all 36 children studied four weeks after the second injection of the vaccine, levels of serum IgG Vi antibodies had increased by a factor of 10 or more. CONCLUSIONS: The Vi-rEPA conjugate typhoid vaccine is safe and immunogenic and has more than 90 percent efficacy in children two to five years old. The antibody responses and the efficacy suggest that this vaccine should be at least as protective in persons who are more than five years old.

Safety and immunogenicity of improved Shigella O-specific polysaccharide-protein conjugate vaccines in adults in Israel.

Data suggest that the O-specific polysaccharide (O-SP) domain of the lipopolysaccharide (LPS) of Shigella species is both an essential virulence factor and a protective antigen and that a critical level of serum immunoglobulin G (IgG) to this antigen will confer immunity to shigellosis. Because covalent attachment of polysaccharides to proteins increases their immunogenicity, especially in infants and in young children, the O-SP of Shigella species were bound to medically useful proteins, and the safety and immunogenicity of the resultant conjugates were confirmed in adults and 4- to 7-year-old children. Succinylation of the carrier protein improved the immunogenicity of Shigella conjugates in mice and increased their yield. Based on these results, a clinical trial of O-SP conjugates of Shigella sonnei and Shigella flexneri 2a bound to succinylated mutant Pseudomonas aeruginosa exotoxin A (rEPAsucc) or native or succinylated Corynebacterium diphtheriae toxin mutant (CRM9 or CRM9succ) was conducted in healthy adults. The conjugates were safe and immunogenic. S. sonnei-CRM9, S. sonnei-CRM9succ, and S. sonnei-rEPAsucc elicited significant rises of geometric mean (GM) IgG anti-LPS within 1 week of injection (P < 0.001). At 26 weeks, the GM anti-LPS levels elicited by these three conjugates were similar and higher than their prevaccination levels (P < 0.0001). GM IgG anti-LPS levels elicited by S. flexneri 2a-rEPAsucc were significantly higher than those elicited by S. flexneri 2a-rCRM9succ at all intervals after injection. At 26 weeks, the levels of IgG anti-LPS in vaccinees were higher than their prevaccination levels (P < 0.0001). The serum antibody responses were specific, as there was no significant rise of anti-LPS to the heterologous O-SP in any vaccinee. Both conjugates elicited statistically significant rises of serum antibodies to the injected carrier protein. At 6 months, these five Shigella conjugates elicited higher fold rises than similar conjugates (D. N. Taylor et al., Infect. Immun. 61:3678-3687, 1993). Based on these data, we chose S. sonnei-CRM9 and S. flexneri 2a-rEPAsucc for evaluation in children.

Production and purification of human menin from Drosophila melanogaster S2 cells using stirred tank reactor.

A process was developed for producing human menin from transformed Drosophila Schneider 2 cells. Protein expression was achieved after inducing the metallothionein promoter by adding copper sulfate to cells growing in suspension in a stirred-tank reactor. Experiments in shake flasks showed that the production of menin was improved when the induction was conducted late in the exponential phase of cell growth at a concentration of 1-2 x 10(7) cells ml(-1), with a copper concentration of 0.2 mM for no more than 24 h. This observation was confirmed by experiments in bench-scale fermentors. Subsequently, a pilot-scale fermentation yielded 1 mg l(-1) culture of purified menin.

Functional correlation in amino acid residue mutations of yeast iso-2-cytochrome c that is consistent with the prediction of the concomitantly variable codon theory in cytochrome c evolution.

Fitch and Markowitz' theory of concomitantly variable codons (covarions) in evolution predicted the existence of functional correlation in amino acid residue mutations among present-day cytochromes c. Mutational analysis was carried out on yeast iso-2-cytochrome c, where hydrophobic core residues I20, M64, L85, and M98 and surface residue L9 were mutated, in selected combinations, to those found in mammalian and bird cytochromes c. The functionality assay is based upon the ability of yeast cells to grow in YPGE medium. Furthermore, experiments on the single M64L and M98L mutations as well as the double M64L/M98L mutation using NMR showed that the effects of these mutations are to perturb the structural integrity of the protein. We identified functional correlation in two cases of a pair of residue mutations, the I20-->V and M98-->L pair and the L9-->I and L85-->I pair. In both cases, only one of the two alternative, putative evolutionary pathways leads to a functional protein and the corresponding pairs of residue mutations are among those found in present-day cytochromes c. Since valine is predicted to be at position 20 in the ancestral form of cytochrome c, the present data provide an explanation for the ancient requirement of leucine rather than methionine in position 98. The present data provide further evidence for the role of those specific atom-atom interactions in directing a pathway in the evolutionary changes of the amino acid sequence that have taken place in cytochrome c, in accordance with Fitch and Markowitz.

Production of HIV-1 gp120 in packed-bed bioreactor using the vaccinia virus/T7 expression system.

The HeLa cell-vaccinia virus system is an attractive method for producing recombinant mammalian proteins with proper post-translation modifications. This approach is especially important for the production of HIV-1 envelope glycoprotein, gp120, since more than half of its total mass is due to carbohydrates. A recombinant vaccinia virus/T7 RNA polymerase expression system was developed to express and produce large amounts of gp120 tagged with six histidine residues. In this system, the expressed T7 RNA polymerase from one virus drives the transcription of the gp120 encoded in the second virus. During the process development phase, the following parameters were studied: infection time, infection duration, multiplicity of infection, ratio of the two viruses, medium composition, and medium replacement strategy during the infection phase. The chosen production method was based on using the packed-bed bioreactor. The HeLa cells were immobilized on fibrous disks (Fibra-Cel) packed in an internal basket positioned in a vertically mixed bioreactor (Celligen Plus), and 25 g of carriers were packed in a 1.6-L (working volume) reactor. The process included a growth stage followed by a production stage. In the growth stage, the bed was perfused with a serum-containing medium, allowing the cells to grow to saturation, and in the production stage, done using serum-free medium, the cells were infected with the two recombinant viruses. The expressed protein was secreted, collected from the culture fluid, and purified. The specific production was found to be between 2 and 3 microg of protein/10(6) cells, and the volumetric production was around 10 mg/50 g carriers.

Vibrio cholerae O139 conjugate vaccines: synthesis and immunogenicity of V. cholerae O139 capsular polysaccharide conjugates with recombinant diphtheria toxin mutant in mice.

Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulated V. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to < or =50. Treatment with 2-ME reduced the titers of 17 of 20 patients to < or =50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned.

Investigation of the TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using (13)C-NMR/MS.

Acetate accumulation is a common problem observed in aerobic high cell density Escherichia coli cultures. A previous report has hypothesized that the glyoxylate shunt is active in a low acetate producer, E. coli BL21, and inactive in a high acetate producer, JM109. To further investigate this hypothesis, we now develop a model for the incorporation of (13)C from uniformly labeled glucose into key TCA cycle intermediates. The (13)C isotopomer distributions of oxaloacetate and acetyl-CoA are first determined using NMR and MS techniques. These distributions are next validated by predicting the NMR spectrum of glutamate. Under steady state isotopic conditions, and with knowledge of the full isotopomer distributions of oxaloacetate and acetyl-CoA, the flux ratios through the TCA cycle and the glycoxylate shunt are obtained with respect to the flux through the PPC anaplerotic shunt. We conclude that in BL21, the glyoxylate shunt is active at 22% of the flux through the TCA cycle, and is inactive in JM109. Further, in BL21, the flux through the TCA cycle equals the flux through the PPC shunt, while in JM109 the TCA cycle flux is only third of the flux through the PPC shunt.

Recovery of mouse endostatin produced by Pichia pastoris using expanded bed adsorption.

Endostatin, a 20 KDa fragment of collagen XVIII, was shown to have an inhibitory effect on angiogenesis and can potentially be used as a tumor growth suppressor. To obtain the amount needed for testing, the protein was successfully cloned and expressed in Pichia pastoris. At the end of the fermentation process, the concentration of the endostatin in the culture was 50 mg per liter, accompanied by 400 gr per liter (wet weight) of biomass. Before the protein can be captured and purified on a packed bed of heparin-Sepharose, the biomass must be removed. Because of the high biomass concentration, conventional biomass removal techniques like centrifugation or filtration are inefficient and cumbersome. Therefore, the expanded-bed adsorption technique was chosen as an alternative approach. An efficient procedure for the initial recovery and purification of the endostatin was developed. The process utilized a cation- exchanger resin instead of a heparin-based affinity resin, because its dynamic capacity was higher, even though it was affected by the high linear flow on the expanded bed. After adjusting the conductivity, pH and biomass concentration, the complete broth was pumped directly on the expanded-bed matrix (Streamline SP XL). Though the yields of protein are similar, the expanded-bed approach is superior to the packed-bed method for several reasons. The expanded-bed process was shorter (only 8 hours compared to 16 hours for the packed bed), it is cheaper, and the product has higher specific activity (29% compared with 18%). Endostatin produced by the expanded-bed adsorption method showed the expected bioactivity and is currently being tested for its potential as a tumor suppressor.

Continuous production and recovery of recombinant Ca2+ binding receptor from HEK 293 cells using perfusion through a packed bed bioreactor.

The extracellular domain of human parathyroid Ca(2+) receptor was needed in order to study itsstructure and clinical application. The Ca(2+)receptor is a unique member of the G protein-coupledreceptor super-family, expressed in parathyroid andkidney cells where it has been shown to play acritical role in extracellular calcium homeostasis.The desired protein was produced by immobilizing thetransformed HEK 293 cells in a packed-bedconfiguration using a 1.6 l (working volume)bioreactor equipped with a vertical mixing impellerassembly and an internal basket. The process includeda propagation phase followed by a production phase. Inthe propagation phase, lasting approximately 160 h, the bed was perfused with a serum-containingmedium, allowing the cells to grow at a constantgrowth rate to approximately 3 x 10(10). At this point the production phase was begun, replacing themedium with serum-free medium and continuing theperfusion process for additional 350 h. Duringthis phase, the medium was pumped through the packedbed at a rate of 4-6 l per day, keeping theresidual glucose concentration around 1 g l(-1) andcollecting and processing approximately 80 l ofspent medium. This continuous perfusion method of thepacked-bed bioreactor was compared to a repeated batchmethod in which existing medium was replenished whenthe glucose concentration was down to 1 g l(-1). Using this method, serum-free medium was replaced withserum containing medium a few times when a decline inthe glucose consumption was observed. Though mediumconsumption and protein yield are similar in bothmethods (roughly 10 mg l(-1)), there aredifferences related to the ease of operation andprocessing of the produced protein. The continuousperfusion operation was found to be preferable and waschosen as the production strategy.

Syntheses and immunologic properties of Escherichia coli O157 O-specific polysaccharide and Shiga toxin 1 B subunit conjugates in mice.

Escherichia coli O157 is the major cause of diarrhea-associated hemolytic uremic syndrome (HUS). Strains causing HUS contain either Shiga toxin 1 (Stx1) or Stx2, or both. In adult volunteers, conjugate vaccines of detoxified lipopolysaccharide (LPS) elicited bactericidal antibodies to E. coli O157. Here, the detoxified LPS was conjugated with improved schemes to the nontoxic B subunit of Stx1. Mice injected with these bivalent conjugates elicited both bactericidal antibodies to E. coli O157 and neutralization antibodies to Stx1.

Treatment with succinic anhydride improves the immunogenicity of Shigella flexneri type 2a O-specific polysaccharide-protein conjugates in mice.

Seroepidemiological data and a clinical trial with a Shigella sonnei O-specific polysaccharide (O-SP)-Pseudomonas aeruginosa recombinant exoprotein A (rEPA) conjugate provide evidence that a critical level of immunoglobulin G (IgG) lipopolysaccharide (LPS) antibodies in serum confers protection against shigellosis. We evaluated the immunogenicity of conjugates whose carrier proteins and O-SPs were treated with succinic anhydride (SA), which reacts with amino groups at neutral pH to form amide-linked carboxyls (succinylation). Conjugates were synthesized with either of two genetically inactivated medically useful toxins, the diphtheria protein CRM9 or rEPA, bound to the O-SP of Shigella flexneri type 2a. Conjugates composed of the succinylated protein, succinylated O-SP, or both succinylated components were administered to mice by a clinically relevant scheme, and their levels of serum IgG anti-LPS and anti-proteins were assayed 7 days after the second and third injections. CRM9 served as a more immunogenic carrier than rEPA. Conjugates composed of succinylated components were more immunogenic than the conjugates composed of the native components. SA treatment of both the carrier protein and the O-SP did not confer an advantage over the succinylated protein alone. Conjugates prepared with native proteins, in general, elicited slightly higher levels of IgG protein antibodies than conjugates composed of the SA-treated proteins.

Disruption of the KEX1 gene in Pichia pastoris allows expression of full-length murine and human endostatin.

Endostatin is a potent angiogenesis inhibitor. In order to isolate sufficient quantities of soluble protein for in vivo studies in mice, we expressed murine endostatin in Pichia pastoris. Analysis of the expressed protein by mass spectrometry indicated that the protein was truncated. N-terminal sequence analysis determined that the N-terminus was intact, suggesting that the C-terminal lysine was missing. In Saccharomyces cerevisiae, Kex1p can cleave lysine and arginine residues from the C-terminus of peptides and proteins. We hypothesized that the KEX1 homologue in P. pastoris is responsible for the loss of the C-terminal lysine of endostatin. To test this hypothesis, we cloned and disrupted the P. pastoris KEX1 gene. Although the overall amino acid identity between the P. pastoris and the S. cerevisae Kex1p is only 36%, the amino acid residues involved in the catalytic activity or close to the active residues are highly conserved. Disruption of the KEX1 reading frame allowed expression of murine and human endostatin with the C-terminal lysine. The KEX1 disruption strain may be a useful tool for the expression of other proteins with a C-terminal basic amino acid. Addition of a lysine to the C-terminus of recombinant proteins may protect the C-terminus from degradation by other carboxypeptidases.

Expression, purification, and biochemical characterization of the amino-terminal extracellular domain of the human calcium receptor.

We purified the extracellular domain (ECD) of the human calcium receptor (hCaR) from the medium of HEK-293 cells stably transfected with a hCaR cDNA containing an isoleucine 599 nonsense mutation. A combination of lectin, anion exchange, and gel permeation chromatography yielded milligram quantities of >95% pure protein from 15 liters of starting culture medium. The purified ECD ran as an approximately 78-kDa protein on SDS-polyacrylamide gel electrophoresis and was found to be a disulfide-linked dimer. Its NH2-terminal sequence, carbohydrate content, and CD spectrum were defined. Tryptic proteolysis studies showed two major sites accessible to cleavage. These studies provide new insights into the structure of the hCaR ECD. Availability of purified ECD protein should permit further structural studies to help define the mechanism of Ca2+ activation of this G protein-coupled receptor.

Use of an ethanol sensor for feedback control of growth and expression of TBV25H in Saccharomyces cerevisiae.

A process for production of a malaria transmission blocking vaccine candidate under the control of the ADH2 promoter in Saccharomyces cerevisiae was developed. Monitoring and controlling the ethanol concentration during the process is essential for successful expression of the recombinant protein. A simple sensor accomplishing this task has been developed, the principle of its operation is the following: air-flow through silicone tubing submerged in the media picks up ethanol, which is detected by an alcohol sensor that relays a signal to a controller regulating the amount of ethanol added to the culture. The sensor was used successfully in high cell density cultures of various scales.

Characterization of the imitation switch subfamily of ATP-dependent chromatin-remodeling factors in Saccharomyces cerevisiae.

We have identified and characterized two Imitation Switch genes in Saccharomyces cerevisiae, ISW1 and ISW2, which are highly related to Drosophila ISWI, encoding the putative ATPase subunit of three ATP-dependent chromatin remodeling factors. Purification of ISW1p reveals a four-subunit complex with nucleosome-stimulated ATPase activity, as well as ATP-dependent nucleosome disruption and spacing activities. Purification of ISW2p reveals a two-subunit complex also with nucleosome-stimulated ATPase and ATP-dependent nucleosome spacing activities but no detectable nucleosome disruption activity. Null mutations of ISW1, ISW2, and CHD1 genes cause synthetic lethality in various stress conditions in yeast cells, revealing the first in vivo functions of the ISWI subfamily of chromatin-remodeling complexes and demonstrating their genetic interactions. A single point mutation within the ATPase domain of both ISW1p and ISW2p inactivated all ATP-dependent biochemical activities of the complexes, as well as the ability of the genes to rescue the mutant phenotypes. This demonstrates that the ATP-dependent chromatin-remodeling activities are essential for the in vivo functions of both ISW1 and ISW2 complexes.

Use of streamline chelating for capture and purification of poly-His-tagged recombinant proteins.

Expression of recombinant proteins with poly-histidine tags enables their convenient capture and purification using immobilized metal affinity chromatography (IMAC). The 6 x His-tagged protein binds to a chelating resin charged with metal ions such as Ni2+, Cu2+ or Zn2+, and can therefore be separated from proteins which have lower, or no, affinity for the resin. Two recombinant proteins, a malaria transmission-blocking vaccine candidate secreted extracellularly by S. cerevisiae and a modified diphtheria toxin produced intracellularly by E. coli, were expressed with 6 x His tags and could therefore be purified using IMAC. In an effort to further simplify the initial capture of these proteins, an expanded bed adsorption technique using a chelating resin (Streamline Chelating) was introduced. It was possible to capture the intracellular diphtheria protein from E. coli directly after cell lysis, without prior centrifugation or filtration. The extracellular malaria vaccine candidate was also directly captured from a high cell density yeast culture. Detailed information on the experimental work performed, and the capture processes developed, is provided.

Zinc-binding of endostatin is essential for its antiangiogenic activity.

Endostatin is a potent angiogenesis inhibitor in vitro and in vivo. We used the yeast Pichia pastoris to express and purify soluble endostatin. It was discovered that metal chelating agents can induce N-terminal degradation of endostatin. We theorized that a metal was removed from endostatin which changed the conformation and allowed a contaminating protease to degrade the N-terminus. Atomic absorption and amino acid analysis of endostatin purified from Pichia pastoris and mammalian cells showed a 1:1 molar ratio of Zn2+ to protein. Ding et al. have shown that histidines 1, 3, 11, and aspartic acid 76 coordinate the Zn2+ atom (1). An H1/3A double, an H11A, and a D76A single mutant of endostatin were not able to regress Lewis lung carcinoma. We conclude that the ability of endostatin to bind Zn2+ is essential for its antiangiogenic activity.