The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa.

Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa. Prolonged incubation of ATS with factor Xa leads to the highly specific hydrolysis of the peptide bond between residues Arg34 and Val35, implicating this peptide bond as the putative reactive site. We report here the preparation of pure, cleaved (modified) recombinant ATS (rATS) and utilize this material to provide additional proof that the cleaved peptide bond is in fact the reactive site. Modified rATS retains strong inhibitory potency against factor Xa as evidenced by a dissociation constant of 166.3 +/- 9.6 pM; four-fold greater than that of native inhibitor, 43.4 +/- 1.4 pM. Incubation of pure, modified rATS with catalytic amounts of factor Xa results in resynthesis of the hydrolyzed peptide bond, achieving an equilibrium near unity between native and modified inhibitors. Specific removal of the newly formed carboxy-terminal Arg residue from modified rATS by carboxypeptidase B treatment obviates its conversion to native inhibitor coincident with the complete loss of inhibitory activity. These results establish that rATS inhibits factor Xa according to a standard mechanism of serine protease inhibitors and support the contention that the Arg34-Val35 peptide bond constitutes the reactive site.     

Reconstitution of catalytically competent human zeta-thrombin by combination of zeta-thrombin residues A1-36 and B1-148 and an Escherichia coli expressed polypeptide corresponding to zeta-thrombin residues B149-259.

Human zeta-thrombin, a catalytically competent serine proteinase, arises from a single chymotryptic cleavage at Trp-148 in alpha-thrombin to generate two nonconvalently associated polypeptide segments designated zeta 1-thrombin (the 36-residue A-chain disulfide linked to B-chain residues B1-148) and zeta 2-thrombin (B149-259). We report here the expression of recombinant zeta 2-thrombin in Escherichia coli and the reconstitution of catalytically competent zeta-thrombin by combination of zeta 1-thrombin with recombinant zeta 2-thrombin. A DNA fragment encoding zeta 2-thrombin was cloned into a pATH2 expression vector as a trpE-zeta 2 fusion gene, in which a factor Xa cleavage site was inserted between the trpE and the zeta 2-thrombin gene. High-level expression of this fusion protein was achieved under the control of the E. coli trp promoter. The expressed zeta 2-thrombin was liberated from the fusion protein by factor Xa cleavage, reduced with DTT, and purified to homogeneity by reverse-phase HPLC. Oxidation of the reduced zeta 2-thrombin in the presence of 80 microM CuSO4 and 6 M urea at pH 8.15 yielded material that was indistinguishable on HPLC from zeta 2-thrombin isolated by resolution of human zeta-thrombin. Catalytically active zeta-thrombin was generated by combination of recombinant zeta 2-thrombin with zeta 1-thrombin that was isolated by resolution of human zeta-thrombin. Recombinant zeta-thrombin displayed catalytic activities, toward a small chromogenic substrate and fibrinogen, that were similar to those of alpha-thrombin prepared from human blood plasma and zeta-thrombin obtained by treatment of alpha-thrombin with chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factor Xa subsite mapping by proteome-derived peptide libraries improved using WebPICS, a resource for proteomic identification of cleavage sites

Proteomic identification of protease cleavage site specificity (PICS) is a recent proteomic approach for the easy mapping of protease subsite preferences that determines both the prime- and non-prime side specificity concurrently. Here we greatly facilitate user access by providing an automated and simple web-based data-analysis resource termed WebPics (http://clipserve.clip.ubc.ca/pics/). We demonstrate the utility of WebPics analysis of PICS data by determining the substrate specificity of factor Xa from P6-P6', an important blood coagulation protease that proteolytically generates thrombin from prothrombin. PICS confirms existing data on non-prime site specificity and refines our knowledge of factor Xa prime-site selectivity.     

Characterization of a recombinant granzyme B derivative as a "restriction" protease

Blood coagulation factor Xa (FXa) and Thrombin are well-known serine proteases often used for processing of recombinant fusion proteins, but because they are purified from bovine blood or other animal sources, there is a risk of pathogenic contaminants in the preparation of the proteases. We report here the characterization of a recombinant serine protease produced in Escherichia coli, which can be used as a specific and efficient alternative to FXa and Thrombin as processing protease. This recombinant protease is derived from human granzyme B (GrB). The protease is found to be very stable in general, and it performs very well in the cleavage of several different fusion proteins tested and was even found superior to processing by FXa in two cases.     

Structure based design of 4-(3-aminomethylphenyl)piperidinyl-1-amides: novel, potent, selective, and orally bioavailable inhibitors of betaII tryptase

Tryptase is a serine protease found almost exclusively in mast cells. It has trypsin-like specificity, favoring cleavage of substrates with an arginine (or lysine) at the P1 position, and has optimal catalytic activity at neutral pH. Current evidence suggests tryptase beta is the most important form released during mast cell activation in allergic diseases. It is shown to have numerous pro-inflammatory cellular activities in vitro, and in animal models tryptase provokes broncho-constriction and induces a cellular inflammatory infiltrate characteristic of human asthma. Screening of in-house inhibitors of factor Xa (a closely related serine protease) identified beta-amidoester benzamidines as potent inhibitors of recombinant human betaII tryptase. X-ray structure driven template modification and exchange of the benzamidine to optimize potency and pharmacokinetic properties gave selective, potent and orally bioavailable 4-(3-aminomethyl phenyl)piperidinyl-1-amides.     

Large-scale purification and characterization of recombinant tick anticoagulant peptide

Recombinant tick anticoagulant peptide (r-TAP), a potent and specific inhibitor of blood coagulation factor Xa, was purified to > 99% homogeneity at the multi-gram scale. Genetically engineered yeast secreted 200–250 mg/l of the heterologous protein into the medium. Cells were separated from broth by diafiltration and purification was done by two chromatographic steps, both conducive to operation on a large scale. Analysis of the purified protein by several methods indicated that it was > 99% homogeneous and no incompletely processed or truncated proteins were detected. Physico-chemical characterization data of r-TAP show that it exists as a monomer in solution and no evidence of post-translational modification was observed. The purified protein was fully active in inhibiting human coagulation factor Xa.     

Synthesis and expression in Escherichia coli of a gene for kappa-bungarotoxin

A gene which codes for the 66-residue polypeptide of kappa-bungarotoxin has been chemically synthesized by linking together 3 synthetic double-stranded oligonucleotides in a bacterial plasmid. The synthesis incorporated six unique silent restriction sites spaced throughout the gene for use in cassette mutagenesis. Direct expression of the kappa-bungarotoxin polypeptide by itself in Escherichia coli failed to result in a stable product. The toxin polypeptide was stabilized and expressed in E. coli as part of a fusion protein with rat intestinal fatty acid binding protein under control of the nalidixic acid inducible recA promoter. Two fusion protein constructs were prepared that differed only in the cleavage site between the fatty acid binding protein and the toxin polypeptide. One contained a factor Xa cleavage site, and the other, since the toxin itself is devoid of methionine, contained a methionyl residue that served as a cyanogen bromide cleavage site. The fusion proteins were isolated by ion-exchange chromatography and reverse-phase HPLC. The construct containing the factor Xa cleavage site could not be cleaved under nondenaturing conditions. On the other hand, kappa-bungarotoxin was efficiently cleaved from the methionyl fusion protein with CNBr. The toxin polypeptide was isolated by reverse-phase HPLC and ion-exchange chromatography and produced a complete and specific blockade of neuronal nicotinic acetylcholine receptors in chick ciliary ganglia which was indistinguishable from that produced by a comparable amount of venom-purified kappa-bungarotoxin.     

Initiation of the extrinsic pathway of blood coagulation: evidence for the tissue factor dependent autoactivation of human coagulation factor VII

Previous studies demonstrated proteolytic activation of human blood coagulation factor VII by an unidentified protease following complex formation with tissue factor expressed on the surface of a human bladder carcinoma cell line (J82). In the present study, an active-site mutant human factor VII cDNA (Ser344----Ala) has been constructed, subcloned, and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity in a single step from serum-free culture supernatants by immunoaffinity column chromatography. Mutant factor VII was fully carboxylated, possessed no apparent clotting activity, and was indistinguishable from plasma factor VII by SDS-PAGE. Cell binding studies indicated that mutant factor VII bound to J82 tissue factor with essentially the same affinity as plasma factor VII and was cleaved by factor Xa at the same rate as plasma factor VII. In contrast to radiolabeled single-chain plasma factor VII that was progressively converted to two-chain factor VIIa on J82 monolayers, mutant factor VII was not cleaved following complex formation with J82 tissue factor. Incubation of radiolabeled mutant factor VII with J82 cells in the presence of recombinant factor VIIa resulted in the time-dependent and tissue factor dependent conversion of single-chain mutant factor VII to two-chain mutant factor VIIa. Plasma levels of antithrombin III had no discernible effect on the factor VIIa catalyzed activation of factor VII on J82 cell-surface tissue factor but completely blocked this reaction catalyzed by factor Xa. These results are consistent with an autocatalytic mechanism of factor VII activation following complex formation with cell-surface tissue factor, which may play an important role in the initiation of extrinsic coagulation in normal hemostasis.     

Proteolytic alterations of factor Va bound to platelets

The coagulation protein Factor Va forms the receptor for the serine protease Factor Xa at the platelet surface. This membrane-bound complex of Factor Va and Factor Xa plus calcium constitutes the enzymatic complex prothrombinase, which effects the conversion of prothrombin to the clotting enzyme, thrombin. Studies were undertaken to investigate the proteolytic events accompanying the inactivation of platelet-bound Factor Va by activated protein C as well as the ability of Factor Xa to protect Factor Va from activated protein C inactivation. During the course of these studies, observations were made which indicated that Factor Va was also cleaved by both a platelet-associated protease, as well as Factor Xa. When Factor Va was incubated with washed platelets, electrophoresis and autoradiography of solubilized platelet pellets indicated that three Factor Va peptides were associated with the platelet: component D (Mr = 94,000), component E (Mr = 74,000), and a 90,000-dalton peptide (component D') which appeared with time as the result of a platelet-associated protease cleavage of component D. The Factor Va peptides bound to platelets were proteolytically inactivated by activated protein C, resulting in five peptide products, all of which remained associated with the platelet-membrane surface. Factor Va was protected from activated protein C proteolysis by complex formation with Factor Xa or active site-blocked Factor Xa. However, active Factor Xa cleaved platelet-bound Factor Va to peptide products which also remained associated with the platelet. Whereas activated protein C rapidly cleaved components D and D' with secondary cleavages occurring in component E, Factor Xa rapidly cleaved component E with secondary cleavages occurring in components D and D'. The Factor Xa-cleaved Factor Va is catalytically functional. To determine whether cleavage was necessary for function, prothrombin conversion reaction mixtures were monitored for thrombin formation and Factor Va cleavage with time in a defined phospholipid vesicle model system. The results indicated that Factor Xa cleavage of Factor Va is not essential for Factor Va activity but may promote its ability to function in the prothrombinase complex.     

Purification and characterization of recombinant antistasin: a leech-derived inhibitor of coagulation factor Xa

Antistasin (ATS) is a selective, tight-binding inhibitor of blood coagulation Factor Xa originally isolated from the salivary glands of the Mexican leech Haementeria officinalis. In order to provide sufficient quantities of ATS to further investigate the role of Factor Xa in blood coagulation, a recombinant version of ATS has been produced in an insect baculovirus host-vector system. In this study, we describe the purification and in vitro and in vivo characterization of a single recombinant antistasin (rATS) isoform. The purified protein constitutes a minor isoform relative to the more abundant ATS isoforms present in leech salivary gland extracts. In vitro, rATS inhibits purified human Factor Xa stoichiometrically, prolongs plasma-based clotting assays at nanomolar concentrations, and like native ATS, is cleaved at a single position by Factor Xa during the course of inhibition. An initial evaluation of the in vivo efficacy of rATS was addressed utilizing a rhesus monkey model of mild disseminated intravascular coagulation. rATS was shown to fully suppress thromboplastin-induced fibrinopeptide A generation in a dose-dependent fashion. The availability of rATS should provide a valuable tool for the critical evaluation of the specific role played by Factor Xa in coagulation.     

Expression and Purification of Recombinant Tick Anticoagulant Peptide (Y1W/D10R) Double Mutant Secreted bySaccharomyces cerevisiae

A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast α-mating factor pre-proleader peptide. Expression in yeast (Saccharomyces cerevisiae) resulted in the secretion of the TAP mutein into the culture medium. An HPLC-based assay was used to screen yeast strains to find those giving highest expression levels. Efficiency of cleavage at the junction of the leader-TAP mutein varied from strain to strain, and a rapid purification method followed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products. A purification scheme was developed which separated the TAP mutein from improperly processed peptides present in the medium. This scheme employed cation-exchange chromatography and reversed-phase HPLC. Scale-up of the process was successful and produced 100 mg of fully functional TAP mutein of >96% homogeneity from a 50-L yeast culture.     

Incorporation of factor Va into prothrombinase is required for coordinated cleavage of prothrombin by factor Xa

Prothrombin is activated to thrombin by two sequential factor Xa-catalyzed cleavages, at Arg271 followed by cleavage at Arg320. Factor Va, along with phospholipid and Ca2+, enhances the rate of the process by 300,000-fold, reverses the order of cleavages, and directs the process through the meizothrombin pathway, characterized by initial cleavage at Arg320. Previous work indicated reduced rates of prothrombin activation with recombinant mutant factor Va defective in factor Xa binding (E323F/Y324F and E330M/V331I, designated factor VaFF/MI). The present studies were undertaken to determine whether loss of activity can be attributed to selective loss of efficiency at one or both of the two prothrombin-activating cleavage sites. Kinetic constants for the overall activation of prothrombin by prothrombinase assembled with saturating concentrations of recombinant mutant factor Va were calculated, prothrombin activation was assessed by SDS-PAGE, and rate constants for both cleavages were analyzed from the time course of the concentration of meizothrombin. Prothrombinase assembled with factor VaFF/MI had decreased k(cat) for prothrombin activation with Km remaining unaffected. Prothrombinase assembled with saturating concentrations of factor VaFF/MI showed significantly lower rate for cleavage of plasma-derived prothrombin at Arg320 than prothrombinase assembled with saturating concentrations of wild type factor Va. These results were corroborated by analysis of cleavage of recombinant prothrombin mutants rMz-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A), which can be cleaved only at Arg320 or Arg271, respectively. Time courses of these mutants indicated that mutations in the factor Xa binding site of factor Va reduce rates for both bonds. These data indicate that the interaction of factor Xa with the heavy chain of factor Va strongly influences the catalytic activity of the enzyme resulting in increased rates for both prothrombin-activating cleavages.     

The factor V activation paradox

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V activation are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation. alpha-Thrombin, derived from factor Xa cleavage of phospholipid-bound prothrombin via the prethrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation.     

Isolation of heparin-binding growth factors from bovine, porcine and canine hearts

Fresh bovine, porcine and canine hearts were homogenized and mitogens for mesoderm-derived cells were purified in three different steps. Extraction by two different ammonium sulfate precipitations was followed by cation-exchange chromatography and by heparin-Sepharose affinity chromatography. A heparin-Sepharose fraction from heart (eluted at 1.1 M NaCl) increased mitotic activity in serum-deprived cultures of porcine aortic endothelial and smooth muscle cells, and in human fibroblasts. This mitogenic activity is potentiated by heparin and inhibited by gamma-interferon. The heart mitogenic fraction showed one double peak on HPLC at A215 and one polypeptide band on SDS/PAGE. These peaks and bands were identical to those obtained from bovine brain. The heart acidic fibroblast growth factor (aFGF) showed a positive signal in Western blots using antibodies raised against brain aFGF. Gas-phase amino acid sequencing established that the mitogens were identical to aFGF and the N-terminally truncated aFGF. Extraction in the presence of a protease inhibitor (pepstatin A) produced a higher-molecular mass form of aFGF with a blocked amino terminus. Another mitogen, eluted at 1.6 M NaCl from heparin-Sepharose, reacted with polyclonal antiserum against human recombinant basic fibroblast growth factor (bFGF) and showed a 66% (12 from 18 amino acids determined by gas-phase sequencing) similarity with bFGF. This polypeptide increased the mitotic activity of the same cell lines but was more potent than aFGF.     

High yield expression and purification of isotopically labelled human endothelin-1 for use in NMR studies

Human endothelin-1 (ET-1) is a potent vasocontractile 21-residue peptide hormone with significant pharmacological importance. An efficient and straightforward expression strategy that enables cost-effective incorporation of stable isotopes is not available thus far. In this report, we describe a cost-effective expression system in Escherichia coli for the production of ET-1 enriched with (15)N and (13)C isotopes. Employing thioredoxin as carrier protein, specific and nearly quantitative cleavage of ET-1 from the fusion was mediated by Factor Xa, and purification to homogeneity (final purity of >95%) was achieved by RP-HPLC. Purified recombinant ET-1 was found to be indistinguishable from the synthetic counterpart as determined by mass spectrometry and NMR spectroscopy. Our expression strategy offers the potential for production of isotopically labeled ET-1 in large (mg) quantities for the purpose of heteronuclear NMR experiments. Moreover, the method devised should be applicable for recombinant expression of small peptides in general.     

Expression of a Synthetic Gene Encoding the Anticoagulant-Antimetastatic Protein Ghilanten by the Methylotropic YeastPichia pastoris

Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leechHaementeria ghilianii.In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments. A yeast expression plasmid, pPIC9HG-2, was constructed by making an in-frame fusion of the ghilanten-coding sequences with the region encoding the pre-pro α-mating factor signal sequence for secretion. The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter.Pichia pastorisyeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5′-AOX1locus via homologous recombination. Both strains yielded His⁺transformants that secreted a potent anticoagulant activity into the medium. Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone. The protease-deficient strain, SMD 1168, secreted about a twofold higher level ofr-ghilanten than KM 71. Significant clonal variation in the expression ofr-ghilanten was found among the His⁺transformants. A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales.r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography. Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions.     

Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase

The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(d) approximately 1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K(d) = 260-500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.     

Identification and functional analysis of truncated human glutamic acid decarboxylase 65

Human brain glutamate decarboxylase 65 (hGAD65) was found to exist as full-length and truncated forms when the glutathione S-transferase-tagged hGAD65 fusion protein was subjected to factor Xa cleavage. The truncated form is produced by cleavage at arginine 69 based on N-terminal amino acid sequence analysis, and has a molecular weight of 58 kD. It is resistant to further factor Xa cleavage or mild trypsin treatment and is more active and more stable than the full-length form. Both the full-length and truncated forms of GAD are also observed in brain preparations in the presence of protease inhibitors. Furthermore, full-length GAD could be converted to the truncated form by endogenous proteases, suggesting that the conversion of full-length to truncated GAD mediated by endogenous protease may represent an important mechanism in the regulation of GABA biosynthesis in the brain.     

重组人肝刺激物在原核细胞中的表达与纯化

利用基因重组技术 ,构建成人肝刺激因子 (hHSS)和谷胱甘肽转移酶 (GST)融合表达载体 ,转化大肠杆菌BL 2 1(DE3 ) ,以His·Tag亲和层析纯化表达产物 ,FactorXa切割分离hHSS单体 ,并检测其生物学活性。结果显示 ,在pET 4 2a表达体系中hHSS以可溶性蛋白和包涵体两种形式存在 ,GST hHSS表达量占菌体可溶性蛋白的3 0 % ;FactorXa切割GST与hHSS之间肽腱 ,得到 3 3和 15kD两条蛋白带 ,经Western杂交证实 3 3kD条带为GST ,而 15kD条带的分子量与hHSS基因序列推测蛋白结果相符。经His·Tag再次纯化可获得hHSS单体 ,初步证实重组hHSS具有促进肝癌细胞增殖活性     

A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides

An expression/purification system was developed using artificial oil bodies (AOB) as carriers for producing recombinant proteins. A target protein, green fluorescent protein (GFP), was firstly expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a linker sequence susceptible to factor Xa cleavage. Artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble recombinant protein, oleosin-Xa-GFP. After centrifugation, the oleosin-fused GFP was exclusively found on the surface of artificial oil bodies presumably with correct folding to emit fluorescence under excitation. Proteolytic cleavage with factor Xa separated soluble GFP from oleosin embedded in the artificial oil bodies; thus after re-centrifugation, GFP of high yield and purity was harvested simply by concentrating the ultimate supernatant.