重组水蛭素相关肽Hi-lys的表达与纯化(英文)

为开发一种新的有临床应用价值的抗血栓药物,根据水蛭素保持抗凝活性的2 0肽片段,设计并构建了水蛭素相关肽(Hi lys)与天冬酰胺酶C端的融合表达系统.为方便目的肽与融合伙伴的分离,增加了富含带电序列的8肽(KRKRKKSR)及酸敏感的天冬氨酰 脯氨酸(Asp Pro)位点,获得了表达质粒pED P8 Hi lys.将其转化E .coliBL 2 1,玉米浆培养基(kanr)培养,乳糖诱导获得融合蛋白(AnsB C P8 Hi lys)的高效表达.通过细菌裂解、包涵体洗涤、尿素溶解、乙醇沉淀、酸水解和DEAE 纤维素5 2柱层析纯化获得目的肽Hi lys ,用凝血酶测定法测得其抗凝活性为5 0ATU mg .     

Tandem multimer expression of angiotensin I-converting enzyme inhibitory peptide in Escherichia coli

It is common for small tandem peptide multimer genes to be indirectly inserted into expression vectors and fused with a protein tag. In this study, a multimer of the tandem angiotensin I-converting enzyme inhibitory peptide (ACE-IP) gene was directly transferred to a commercially available vector and the designed gene was expressed as a repeated peptide in Escherichia coli BL21(DE3)pLysS. The process further developed in our study was the construction of six-repeated ACE-IP synthetic genes and their direct insertion. Protein expression in inclusion bodies was confirmed by SDS-PAGE and Western blot. Acid hydrolysis of inclusion bodies produced single-unit peptides through cleavage of the aspartyl-prolyl bonds. This cleaved recombinant peptide (rACE-IP) was purified using immuno-affinity chromatography followed by reversed phase-HPLC. 105–115 mg of the lyophilized recombinant peptide was obtained from 1 L E. coli culture. In vitro biological activity of rACE-IP was indistinguishable from that of the natural peptide produced by hydrolysis in artificial gastric juice or by acidic hydrolysis. The rACE-IP prepared by recombinant DNA technology and solid-phase synthesis methods showed a similar IC₅₀. This strategy could be used for the expression of important peptides, which have N-terminal proline (P) and C-terminal aspartic acid residues (D) for commercial applications, e.g. functional foods and drinks.     

Expression kinetics and plasmid stability of recombinant E. coli encoding urease-derived peptide with bioinsecticide activity

The nucleotide sequence encoding for an insecticidal peptide derived from the Canavalia ensiformis urease gene jbureII (AF 468788), was cloned and expressed in the pET101/Escherichia coli expression system. Bacterial cultivation in shaker with lactose as inducer produced 1.26 μg of recombinant peptide/mg protein, after 8 h of growth. The plasmid stability and the expression of the recombinant peptide were studied in bioreactor. Expression of the recombinant peptide was strongly affected by pH of cultures, with a decrease of more than 50% when acidification was freely allowed. Likewise, peptide production and plasmid stability were shown to be affected by aeration and agitation speed, both decreasing for higher values of oxygen mass transfer rates. Despite these difficulties, in bioreactor cultures carried out with controlled pH, low oxygen mass transfer rates and using lactose as inducer, we were able to achieve a total peptide production of 7.14 μg/mg protein, which represents approximately 2% of total cell protein. Bioassays were carried out using the purified peptide on insect models. The peptide fed to Dysdercus peruvianus nymphs produced 100% mortality after 11 days, deaths starting with a lag phase of 3–4 days, confirming that the bioreactor-produced peptide retained its biological activity.     

Expression and preparation of recombinant hepcidin in Escherichia coli

Hepcidin is a low-molecular-weight, highly disulfide bonded peptide relevant to small intestine iron absorption and body iron homeostasis. In this work, hepcidin was expressed in Escherichia coli as a 10.5 kDa fusion protein (His-hepcidin) with a N-terminal hexahistidine tag. The expressed His-hepcidin existed in the form of inclusion bodies and was purified by IMAC under denaturation condition. Since the fusion partner for hepcidin did not contain other cysteine residues, the formation of disulfide bonds was performed before the His-tag was removed. Then, the oxidized His-hepcidin monomer was separated from protein multimers through gel filtration. Following monomer refolding, hepcidin was cleaved from fusion protein by enterokinase and purified with reverse-phase chromatography. The recombinant hepcidin exhibited obvious antibacterial activity against Bacillus subtilis.     

Expression, purification, and characterisation of nesiritide using an E. coli expression system

Nesiritide, the recombinant human brain natriuretic peptide, is involved in the regulation of cardiovascular homeostasis and has been approved for treatment of patients with congestive heart failure. We prepared a synthetic cDNA construct of Nesiritide to generate a fusion protein with an affinity handle and 41 amino acid peptide of β-galactosidase. The fusion protein was expressed mainly in the inclusion bodies and accounted for approximately 20% of total cellular protein. After purification by Ni-IDA affinity chromatography and renaturation, the fusion protein was cleaved with purified recombinant enterokinase. Nesiritide was purified by pH precipitation/ion exchange chromatography followed by source phenyl chromatography to obtain protein with > 99% purity (determined by RPHPLC) and a mass of 3,464 Daltons. The potency (ED₅₀) of the purified protein was equivalent to that of Natrecor (Innovator formulation). Analytical methods were developed to identify oxidised, reduced and other related impurities. The expression strategy described in this work allows the convenient generation of high yield Nesiritide and enabled ease of purification.     

A simple method for the purification of an antimicrobial peptide in recombinant Escherichia coli

A magainin derivative, designated MSI-344, was produced in Escherichia coli as fusion protein, by utilizing a truncated amidophsphoribosyltransferase of E. coli as a fusion partner. Bacterial cells transformed with the gene encoding the fusion protein were grown to a high cell density and induced with isopropyl-1-thio-b-D-galatoside (IPTG) to initiate product expression. The fusion protein was accumulated into cytoplasmic inclusion body and recombinant MSI-344 was released from the fusion partner by hydroxylamine treatment. Following cleavage of the fusion protein with hydroxylamine, the released MSI-344 was purified to homogeneity by cationic exchange chromatography. The final purity was at least 95% by reversed-phase high performance liquid chromatography (RP-HPLC). Purified recombinant MSI-344 was found to be indistinguishable from the synthetic peptide determined by amino acid sequences and antimicrobial activity assay.     

Recombinant production and purification of novel antisense antimicrobial peptide in Escherichia coli

A fusion protein was genetically engineered that contains an antimicrobial peptide, designated P2, at its carboxy terminus and bovine prochymosin at its amino terminus. Bovine prochymosin was chosen as the fusion partner because of its complete insolubility in Escherichia coli, a property utilized to protect the cells from the toxic effects of the antimicrobial peptide. This fusion protein was purified by centrifugation as an insoluble inclusion body. A methionine linker between prochymosin and the P2 peptide enabled P2 to be released by digestion with cyanogen bromide. Cation exchange HPLC followed by reversed-phase HPLC were used to purify the P2 peptide. The recombinant P2 peptide's molecular mass was confirmed by mass spectrometry to within 0.1% of the theoretical value (2480.9 Da), and the antimicrobial activity of the purified recombinant P2 against E. coli D31 was determined to be identical to that of the chemically synthesized peptide (minimal inhibitory concentration of 5 mg/mL). Although the yield of the fusion protein after expression by the cells was high (16% of the total cell protein), the percentage recovery of the P2 peptide in the inclusion bodies was relatively low, which appears to be due to losses in the cyanogen bromide digestion step.     

Purification of recombinant antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen using the expression system pH6EX3 followed by metal chelating affinity chromatography.

A novel plasmid expression vector (pH6EX3) that directs the synthesis of a fusion protein with a histidine hexapeptide at its N-terminus and a foreign protein at its C-terminus was constructed. The fusion gene is controlled by a strong tac promoter, leading to high-level expression of recombinant protein in several bacterial strains; the protein is deposited mainly as an insoluble mass in inclusion bodies. The fusion protein can be purified from the insoluble cell fraction by one-step affinity chromatography based on the selective interaction between the histidine hexapeptide and a metal chelating matrix charged with Ni2+ ions. The principle of this new system was tested by expressing and purifying antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein autoantigen. With the use of column chromatography and pH gradient elution, about 25 micrograms recombinant protein/ml of bacterial culture was obtained.     

Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein ( approximately 33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP ( approximately 2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.     

Design and expression of peptide antibiotic hPAB-beta as tandem multimers in Escherichia coli

Peptide antibiotics are small peptides encoded by organism genomic DNA. They are recognized to play important roles in the innate host defense of most living organisms. The growing resistance of bacteria to conventional antibiotics and the need for discovery of new antibiotics have stimulated great interest in the development of peptide antibiotics as human therapeutics. However, preparation of peptide antibiotics at a large scale is a great challenge in developing these commercial products. In this study, tandem repeat multimers of peptide antibiotic hPAB-beta were designed and the recombinant plasmids containing one to eight copies of hPAB-beta gene were generated. Eight genetic engineered bacteria harboring pQE-hPAB-beta1-8 recombinant were able to express the repetitive hPAB-beta multimers of interest in inclusion bodies, respectively. The expressed proteins could reach 2.6-28% of the total proteins. The hPAB-beta trimer construct was selected out for the subsequent study based on its higher expression level (27.8%), which yields in wet cell weights (3.15+/-0.45 g/l) and the fusion protein inclusion bodies was able to completely dissolve in 8 M urea. The tandem trimers could easily be captured by Ni-NTA affinity chromatography and cleaved into monomers by hydroxylamine. Then, the monomer hPAB-beta of interest was purified to 95% homogeneity by reverse phase chromatography and gel filtration. The final yield of purified recombinant monomer hPAB-beta was 680+/-12 mg/100 g wet cells. The minimum inhibitory concentrations (MICs) of the purified recombinant hPAB-beta against type or clinical strains of microorganisms were about 31-250 microg/ml and these results showed that the recombinant hPAB-beta could retain its bioactivity.     

Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli

Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.     

Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli.

Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.     

一种抗菌肽和aFGF融合蛋白的构建和表达

利用PCR技术扩增出带有凝血酶Xa因子切割位点的天蚕素蜂毒素杂合肽和aFGF的融合基因,插入大肠杆菌表达载体pET-3c中,构建出表达质粒pET-aF-CM,并转化至大肠杆菌BL21（DE3）中,氨苄青霉素抗性筛选重组转化子。IPTG诱导4h后,以包涵体形式表达的融合蛋白约占菌体总蛋白的17%。将包涵体溶解后透析复性,并利用肝素亲和层析纯化,得到电泳纯的融合蛋白。Western blot分析表明,该蛋白能与aFGF抗体产生免疫反应。MTT法检测显示,融合蛋白具有促3T3Bal/b细胞分裂活性,其比活为1.471×106IU/mg。利用凝血酶Xa因子裂解融合蛋白,可以获得抗菌肽和含凝血酶Xa因子裂解序列的aFGF蛋白。分子筛回收含杂合抗菌肽,抑菌活性检测表明其对大肠杆菌K12D31具有明显抑菌活性。微量稀释法检测结果表明,回收的抗菌肽对大肠杆菌DH5α、大肠杆菌K12D31、沙门氏菌、金黄色葡萄球菌、枯草芽孢杆菌和绿脓杆菌的MIC分别达6.25μg/ml、10μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml和5μg/ml。     

Cloning, expression, and refolding of a secretory protein ESAT-6 of Mycobacterium tuberculosis

A DNA encoding the 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis was inserted into a bacterial expression vector of pQE30 resulting in a 6x His-esat-6 fusion gene construction. This plasmid was transformed into Escherichia coli strain M15 and effectively expressed. The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea or 6M guanidine-hydrochloride at pH 7.4, and the recombinant protein was purified by Ni-NTA column. The purified fusion protein was refolded by dialysis with a gradient of decreasing concentration of urea or guanidine hydrochloride or by the size exclusion protein refolding system. The yield of refolded protein obtained from urea dialysis was 20 times higher than that from guanidine-hydrochloride. Sixty-six percent of recombinant ESAT-6 was successfully refolded as monomer protein by urea gradient dialysis, while 69% of recombinant ESAT-6 was successfully refolded as monomer protein by using Sephadex G-200 size exclusion column. These results indicate that urea is more suitable than guanidine-hydrochloride in extracting and refolding the protein. Between the urea gradient dialysis and the size exclusion protein refolding system, the yield of the monomer protein was almost the same, but the size exclusion protein refolding system needs less time and reagents.     

Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics

The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC₅₀ values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.     

Cloning, expression, and purification of a highly immunogenic recombinant gonadotropin-releasing hormone (GnRH) chimeric peptide

To design an anti-gonadotropin-releasing hormone (GnRH) vaccine capable of eliciting strong immunogenicity, a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide called GnRH3-hinge-MVP contained three linear repeats of GnRH (GnRH3), a fragment of the human IgG1 hinge region, and a T-cell epitope of measles virus protein (MVP). The expression plasmid contained the GnRH3-hinge-MVP construct ligated to its fusion partner (AnsB-C) via an unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in an inclusion body in Escherichia coli under IPTG or lactose induction and the target peptide was easily purified using washing of urea and ethanol precipitation. The target chimeric peptide was isolated from the fusion partner following acid hydrolysis and purified using DEAE-Sephacel chromatography. The purified GnRH3-hinge-MVP was determined to be highly homogeneous by IEF analysis and the N-terminal sequencing. Further, immunization of female mice with the recombinant chimeric peptide resulted in generation of high-titer antibodies specific for GnRH. The results showed that GnRH3-hinge-MVP could be considered as a candidate anti-GnRH vaccine.     

Expression,purification, phosphorylation and characterization of recombinant human statherin

This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria–Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47 mg per liter of cell culture, while 112 mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP–HPLC corresponded to 0.6 mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase.     

Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein (~33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP (~2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.     

A recombinant human G-CSF/GM-CSF fusion protein from E. coli showing colony stimulating activity on human bone marrow cells

Granulocyte-Macrophage colony stimulating factor (GM-CSF) and Granulocyte colony stimulating factor (G-CSF) are cytokines involved in the differentiation of bone marrow progenitor cells into myeloid cells. They also activate mature myeloid cells to mediate a variety of antimicrobial activities and inflammatory responses. Recombinant GM-CSF and G-CSF proteins have been used to treat various diseases including cancer and hematopoietic diseases and to isolate peripheral blood progenitor cells for bone marrow transplantation. A plasmid construct expressing recombinant human G-CSF/GM-CSF fusion protein has now been prepared by linking the human G-CSF and GM-CSF coding regions and the recombinant fusion protein has been successfully expressed in E. coli. The recombinant human G-CSF/GM-CSF fusion protein was extracted and purified from the cellular inclusion and refolded into the biologically active form to show colony stimulating activity. The recombinant fusion protein exhibited colony stimulating activity on human bone marrow cell cultures, indicating that the linkage of GM-CSF and G-CSF by a linker peptide may not interrupt activities of the cytokines in the fusion protein. The colony forming unit of the fusion protein was also higher than those of the cultures treated with the same molar numbers of the recombinant human GM-CSF and G-CSF separately, which suggests that the fusion protein presumably retains both G-CSF and GM-CSF activities.     

Production and purification of refolded recombinant human IL-7 from inclusion bodies

A recombinant form of human rhIL-7 was overexpressed in Escherichia coli HMS174 (DE3) pLysS under the control of a T7 promoter. The resulting insoluble inclusion bodies were separated from cellular debris by cross-flow filtration and solubilized by homogenization with 6 M guanidine HCl. Attempts at refolding rhIL-7 from solubilized inclusion bodies without prior purification of monomeric, denatured rhIL-7 were not successful. Denatured, monomeric rhIL-7 was therefore initially purified by size-exclusion chromatography using Prep-Grade Pharmacia Superdex 200. Correctly folded rhIL-7 monomer was generated by statically refolding the denatured protein at a final protein concentration of 80-100 microg/ml in 100 mM Tris, 2mM EDTA, 500 mM L-arginine, pH 9.0, buffer with 0.55 g/l oxidized glutathione at 2-8 degrees C for at least 48 h. The refolded rhIL-7 was subsequently purified by low-pressure liquid chromatography, using a combination of hydrophobic interaction, cation-exchange, and size-exclusion chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue, high-pressure size-exclusion chromatography (SEC-HPLC), and reverse-phase HPLC. The endotoxin level was <0.05 EU/mg. The final purified product was biologically active in a validated IL-7 dependent pre-B-cell bioassay. In anticipation of human clinical trials, this material is currently being evaluated for safety and efficacy in non-human primate toxicology studies.