Purification and characterization of human interleukin-1 beta expressed in recombinant Escherichia coli

The high-level expression of human interleukin-1 beta in Escherichia coli is described. The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell. A method for the purification of the protein is given which is based on anion- and cation-exchange chromatographies. The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N- and C-terminal sequence analysis and analytical centrifugation. The protein is biologically active as demonstrated by two different in vitro assays, namely, the mononuclear cell factor (IL-1/MCF) assay and lymphocyte activating factor (IL-1/LAF) assay. The specific activities determined with the IL-1/MCF and IL-1/LAF assays, are 2 X 10(7) and 4 X 10(7) units mg-1, respectively.     

Purification and characterization of recombinant human interleukin-2 produced in Escherichia coli

Recombinant human interleukin-2 (rIL-2) produced in Escherichia coli was purified to apparent homogeneity by cation exchange chromatography and reverse phase high performance liquid chromatography. The amino acid composition, amino terminal amino acid sequence, and carboxyl terminal amino acid were consistent with those deduced from the cDNA sequence. Besides the molecular species with the amino terminal Ala, the purified preparation contained another species having an additional Met residue at the amino terminus corresponding to the initiation codon AUG. The molar absorption coefficient of rIL-2 was determined to be 9.58 X 10(3) M-1 cm-1 at 280nm in water. Ultracentrifugal analyses revealed that it existed as a monomeric form in 0.1 M NaCl. The apparent sedimentation coefficient (S20,w) was calculated to be 1.8 S.     

Purification and characterization of human interleukin-1 alpha produced in Escherichia coli

The production of human interleukin-1 alpha (IL-1 alpha) in Escherichia coli is described together with a method for its purification. The isolated protein was shown to be pure and physically homogeneous. The in vitro biological activity of IL-1 alpha was tested with the mononuclear-cell factor and the lymphocyte-activating factor assays. The specific activity determined with both assays was about 3 X 10(7) units mg-1 and is similar to that observed with recombinant human IL-1 beta. The purified protein was resolved by chromatofocusing into two species of isoelectric points 5.45 and 5.20 (75% and 25%, respectively, of the total protein). Both species had similar chemical properties and biological activities to the unfractionated protein. The charge difference between the species was attributed to the deamidation of a single Asn or Gln residue.     

Purification and characterization of human recombinant interleukin-1 beta

A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli. The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates. The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay. Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes. The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm. NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No initiator Met was observed. Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds. S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay. Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.     

Purification and characterization of recombinant human interleukin-29 expressed in Escherichia coli

Human interleukin (IL)-29 is the latest member of the class II cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-29, little is known of its functions in man. In the present study, an Escherichia coli expression system for the rapid expression of the human IL-29 gene was developed. It involved of cloning IL-29 gene into the pET-44 Ek/LIC vector, which allowed expression of IL-29 with a fusion tag consisting of the NusA protein, polyhistidine and S peptide (Nus-His-S-tag), and introducing a thrombin recognition site between the fusion tag and IL-29. The expressed fusion protein was purified by S-protein agarose affinity chromatography, and the fusion tag was removed from recombinant IL-29 by cleavage with thrombin. The purified IL-29 appeared a single band on SDS-PAGE, and the yield of IL-29 was 60 mg from 1 l of bacterial culture. N-terminal sequencing confirmed the identity of the purified protein. The recombinant IL-29 showed specific antiviral activity that was comparable to the commercially available IFN alfa-2b preparation.     

Purification of human interleukin-4 produced in Escherichia coli

An interleukin-4 (IL4)-encoding cDNA isolated from human splenocytes was used to construct an expression plasmid that directs a high-level synthesis of mature IL4 protein in Escherichia coli. The expression was under the control of the major leftward promoter, pL, of phage lambda and the phage Mu ribosome-binding site. The IL4 protein was present as insoluble inclusion bodies in the bacterial extract. The IL4 could be solubilized in 5 M MgCl2 and was purified to homogeneity by several chromatographic steps. The yield of protein from bacteria ranged between 3 and 5 mg of IL4 protein per gram of wet cells. The specific activity of the recombinant human IL4 was about the same as that of the natural product.     

Purification and characterization of recombinant single-chain urokinase produced in Escherichia coli

Recombinant single-chain urokinase (rUK1) has been purified from Escherichia coli. The purification utilizes a refractile body purification, followed by batch DE-52 cellulose extraction, hydroxylapatite chromatography, and S-200 chromatography. Two-chain rUK (rUK2) is separated from rUK1 on benzamidine--Sepharose. The purification eliminates proteases early in the procedure so the rUK1 will not be cleaved to rUK2. The rUK1 has been characterized by amino-terminal analysis as well as carboxy-terminal analysis after cleavage by plasmin.     

Purification and characterization of the immunostimulatory properties of recombinant human interleukin-1 beta

In this study we have used a new method for human recombinant IL-1 beta (rIL-1 beta) purification and investigated its immunostimulatory biological activity. The IL-1 beta gene was cloned using a novel mRNA preparation from activated human blood monocytes. The purification protocol consists of extraction and two chromatographic steps using the new Soloza cation exchange resin. The purified protein was characterized electrophoretically, by amino acid analysis and reverse phase chromatography. The protein migrated on SDS-PAGE with a molecular weight of 18.200 but demonstrated the minor presence of aggregates (dimers and trimers). Specific activity of purified rIL-1 beta in comitogenic assay on mouse thymocytes was 10(8) U/mg protein. rIL-1 beta increased in a dose dependent manner proliferation of Con A-stimulated murine thymocytes, splenocytes, PHA-stimulated human peripheral blood lymphocytes and transformed B-cell lines. Comitogenic activity depended on the degree of lymphocyte preactivation and was similar to that of natural human IL-1 beta. rIL-1 beta enhanced IL-2 production by murine spleen cells and EL-4 cell line and IL-2 receptor expression by human peripheral blood mononuclear cells. It induced PGE2 release from human blood monocytes but had no effect on human neutrophil chemotaxis, phagocytosis and respiratory burst.     

Purification and characterization of recombinant bovine growth hormone produced in Escherichia coli

Using the pUBJ10 plasmid containing the modified bovine growth hormone (bGH) cDNA, large production has been attempted in E. coli BL21 strain. The bGH was highly expressed upto the level of 35% of total cell proteins by IPTG induction and temperature shift to 40°C. The recombinant bGH (rbGH) was isolated from inclusion bodies by solubilization in 10 M urea and followed by DEAE-TOYOPEARL 650C ion exchange and Sephadex G-100 column chromatography. The pUBJ10-derived bGH was eluted at 25.28 min similar to the standard bGH (at 25.18 min) by reverse-phase HPLC. The analysis of N-terminal amino acid showed that the mature bGH has glutamic acid as a first amino acid in agreement with DNA sequencing data. The biological activity was indirectly measured by radioreceptor assay and compared with a pituitary-derived bGH.     

On-column refolding purification and characterization of recombinant human interferon-lambda1 produced in Escherichia coli

Interferon-lambda1 (IFN-lambda1) is a member of the recently discovered type III IFNs (IFN-lambda), which possesses antiviral, antitumor, and immunomodulatory activities. In this study, the recombinant human IFN-lambda1 containing a hexahistidine tag was expressed in Escherichia coli. IFN-lambda1 was overexpressed under the control of T7 promoter and most of the protein existed in the form of inclusion bodies. The expressed insoluble protein was solubilized with urea, purified and refolded by one-step immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purified IFN-lambda1 appeared as a single band on SDS-PAGE and the purity was more than 95%. The yield was 86 mg IFN-lambda1 from 1L of bacterial culture. Western blotting and N-terminal sequencing confirmed the identity of the purified protein. The purified IFN-lambda1 exhibited specific antiviral activity as demonstrated by a cytopathic effect reduction assay. Thus, this on-column refolding method provides an efficient way to obtain an active IFN-lambda1 with high yield and high purity.     

Functional characterization of human recombinant apolipoprotein AIV produced in Escherichia coli

Apolipoprotein AIV (apoAIV), a protein which is known to activate the enzyme lecithin: cholesterol acyltransferase, to bind to apoAI/AII receptor sites and also to promote cholesterol efflux from adipose cells, may play an important role in reverse cholesterol transport. In this report, the high-level production of soluble recombinant mature human apoAIV (isoform 1) in Escherichia coli is described. The recombinant protein was purified by avoiding lipid extraction or denaturation. The apoAIV preparation was analysed by its reactivity with antibodies raised against human apoAIV, SDS-gel electrophoresis, isoelectric focusing and N-terminal sequencing. The purified recombinant protein retains an extra methionine at the N-terminus. Purified recombinant and natural apoAIV proteins were indistinguishable with regard to their denaturation properties, thermo-stability or their fluorescence emission properties in the presence of various quantities of a quenching agent. Complexes of ApoAIV with L-alpha-dimyristoyl-glycerophosphocholine (Myr2GroPCho), glycerophosphocholine (GroPCho), or L-alpha-1-palmitoyl-2-oleoylglycerophosphocholine (PamOleGroPCho) prepared from plasmatic and from recombinant apoAIV proteins have similar densities as revealed by analytical centrifugation. They also share the same cofactor properties for the lecithin:cholesterol acyltransferase reaction. Recombinant apoAIV complex with Myr2GroPCho was also able to bind to the same apoAI/AII receptor sites and to promote cholesterol efflux to an equal extent from adipose cells. It is concluded that the recombinant protein is functionally identical to the plasmatic apoAIV and may therefore be very useful in helping to elucidate the physiological role of apoAIV.     

Biochemical characterization of recombinant human phenylalanine hydroxylase produced in Escherichia coli

A full-length human phenylalanine hydroxylase cDNA has been recombined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express phenylalanine hydroxylase immunoreactive protein and pterin-dependent conversion of phenylalanine to tyrosine. Recombinant human phenylalanine hydroxylase produced in E. coli has been partially purified, and biochemical studies have been performed comparing the activity and kinetics of the recombinant enzyme with native phenylalanine hydroxylase from human liver. The optimal reaction conditions, kinetic constants, and sensitivity to inhibition by aromatic amino acids are the same for recombinant phenylalanine hydroxylase and native phenylalanine hydroxylase. These data indicate that the recombinant human phenylalanine hydroxylase is an authentic and complete phenylalanine hydroxylase enzyme and that the characteristic aspects of phenylalanine hydroxylase enzymatic activity are determined by a single gene product and can be constituted in the absence of any specific accessory functions of the eukaryotic cell. The availability of recombinant human phenylalanine hydroxylase produced in E. coli will expedite physical and chemical characterization of human phenylalanine hydroxylase which has been hindered in the past by inavailability of the native enzyme for study.     

重组大肠杆菌右旋糖酐蔗糖酶的纯化及其性质

[目的]研究工程菌E.coli BL21(DE3)/pET28-dexYG产右旋糖酐蔗糖酶的纯化和酶学性质.[方法]工程菌经过IPTG诱导后生产含His-tag融合蛋白的右旋糖酐蔗糖酶,通过硫酸铵沉淀、Ni-NTA亲和层析纯化,得到纯度较高的酶蛋白,并对纯酶进行了酶学性质及动力学研究.[结果]经过SDS-PAGE测得该酶的分子量约为170 kDa,与理论推测值基本相同.以蔗糖为底物,酶促反应的最适温度为25～30℃,最适pH值为5.4,动力学常数Km值为10.43 mmol/L;酶活在pH 5.0～8.0较为稳定,在室温(25 ℃)保藏4天仍有59%的酶活力,4℃保存7周酶活力仅下降一半,但在35℃以上失活很快;Ca2 对催化作用有较大的促进,Mg2 有微弱的促进作用,K 对催化反应无影响,Cu2 的抑制作用最强.其他试剂对重组酶的活性有不同程度的影响,其中SDS抑制作用很强.[结论]研究为重组右旋糖酐蔗糖酶纯酶的获取、得到稳定性好、活性高的酶反应体系及利用该酶进行催化反应和工业化应用提供了重要参数.     

Purification and characterization of human recombinant precursor interleukin 1 beta

We have purified the 31-kDa precursor of human interleukin 1 beta (proIL1 beta) from recombinant Escherichia coli expressing the protein. The recombinant precursor was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, Western blot, and for biological and receptor binding activity. The protein migrates at the expected molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration columns. The specific activity of the recombinant precursor is less than 10(2) units/mg in the EL4 thymoma assay compared with 5 x 10(8) units/mg for the recombinant 17-kDa mature protein. The inactivity of the precursor is attributable to the inability of the protein to bind the IL1 receptor on EL4 cells as shown by receptor competition studies using 125I-labeled 17-kDa IL1 beta. Inactivity of the IL1 beta precursor is not due to degradation of the protein in either the bioactivity or receptor binding assays. The inactive IL1 beta precursor is converted to an active form following proteolysis with chymotrypsin which generates a carboxyl-terminal fragment of 17 kDa that is 6 orders of magnitude more active than the starting IL1 beta precursor. Removal of the first 114 amino acids from proIL1 beta generates a fully active molecule. In contrast, removal of the first 77 amino acids by treatment with trypsin only partially restores activity. The resultant 22-kDa protein exhibits a 600-fold increase in both biological and receptor binding activity, demonstrating a direct correlation between the ability of sequences within the pro-region to inhibit biological activity and inhibit binding to the IL1 receptor. Far-UV circular dichroism spectroscopy indicates that proIL1 beta is similar in secondary structure to mature IL1 beta; both proteins are nonhelical beta sheet proteins.     

Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli.

The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays. Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines.     

Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli.

Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase.     

Purification and functional characterization of human 11beta hydroxylase expressed in Escherichia coli

The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES/GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS/PAGE. Upon mass spectrometry, the mass-to-charge ratio (m/z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its substrates at position 11-beta. Moreover, the determined NADPH coupling percentage for the hCYP11B1 catalyzed reactions using either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approximately 75% in both cases. Biacore and stopped-flow measurements indicate that hCYP11B1 possesses more than one binding site for its redox partner adrenodoxin, possibly resulting in the formation of more than one productive complexes. In addition, we performed CD measurements to obtain information about the structure of hCYP11B1.     

Purification and properties of recombinant rat catalase produced in Escherichia coli

Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.     

Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli.

Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.     

Purification and characterization of progenipoietins produced in Escherichia. coli

The progenipoietins (ProGPs) are a family of genetically engineered chimeric proteins that contain receptor agonist activity for both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. These unique proteins have previously been shown to induce the proliferation of multiple cell lineages. The characterization of two progenipoietins, ProGP-1 and ProGP-4, refolded and purified from an Escherichia coli expression system is described. These ProGP molecules differ in the orientation of the two receptor agonists and, in addition, ProGP-4 contains a fetal liver tyrosine kinase-3 receptor agonist that has been circularly permuted to modulate its activity. Static light scattering analyses demonstrated that both ProGP molecules exist as dimers, most likely through non-covalent interaction of the fetal liver tyrosine kinase-3 receptor agonist domains. ProGP-1 and ProGP-4 have comparable secondary structures, as analyzed by circular dichroism; however, their tertiary structures, as measured by intrinsic fluorescence, were demonstrated to be different. Differential scanning calorimetry demonstrated that the thermal stability of these two proteins was indistinguishable. Interestingly, these dual agonist proteins yielded only a single melting temperature value that was intermediate between that of their individual receptor agonist components, indicating that these chimeric molecules behave as a single domain protein during thermal denaturation. This study describes the purification and physico-chemical properties of this class of proteins generated using an E. coli expression system.