Secretory production of human leptin in Escherichia coli

Human leptin is a 16 kDa (146 amino acids) protein secreted from adipocytes and influences body weight homeostasis. In this study, human leptin was produced and secreted efficiently in Escherichia coli using a novel Bacillus sp. endoxylanase signal peptide. The endoxylanase signal sequence consisted of 28 amino acids (84 bp) was fused to the leptin structural gene. The fused gene was expressed using an inducible promoter (T7 or Trc) by adding 1 mM IPTG. Using T7 promoter in E. coli BL21(DE3), most of protein produced was in a premature form. Using the Trc promoter, which is weaker than T7, leptin was efficiently produced and secreted as a mature form (40% of total proteins) at 37 degrees C. However, most of leptin (about 90%) formed the inclusion bodies in the periplasmic space of E. coli. At 30 degrees C, ca. 90% of leptin was produced in a soluble form, but the total amount of leptin produced was 40% less than that obtained at 37 degrees C. When the periplasmic oxidoreductase of E. coli, DsbA, was co-expressed, 69% of the secreted leptin (26% of total proteins) was produced as soluble form at 37 degrees C without the decrease of the amount of leptin produced.     

Adipose differentiation related protein: expression, purification of recombinant protein in Escherichia coli and characterization of its fatty acid binding properties

Adipose differentiation related protein (ADRP) is a 53 kDa protein encoded by a cDNA originally cloned by differential hybridization from murine adipocytes. ADRP is induced during the early onset of the adipose differentiation program and is expressed at high level in mature adipocytes. We have demonstrated that ADRP stimulated the uptake of fatty acids thereby providing evidence for a functional role of ADRP in lipid metabolism. In the present paper, the murine ADRP has been expressed as a recombinant histidine-tagged protein in Escherichia coli, and purified from expressing cultures in order to examine its biochemical properties. We report here that the purified recombinant ADRP binds fatty acids and exhibits stoichiometric saturable binding of NBD-stearic acid with a K(d)=0.145+/-0.003 microM and a B(max)=0.99+/-0.05. Analysis of fluorescence emission spectra indicates that the polarity of the ADRP binding site is near epsilon approximately 23, close to that observed for fatty acid binding sites in other lipid binding proteins such as the liver fatty acid binding protein. The data presented here provide evidence that isolated ADRP purified in the experimental conditions described here can be used for functional studies.     

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization

Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.     

Design, production, and characterization of recombinant neocarzinostatin apoprotein in Escherichia coli

Neocarzinostatin (NCS) is the first discovered anti-tumor antibiotic having an enediyne-containing chromophore and an apoprotein with a 1:1 complex. An artificial gene library for NCS apoprotein (apo-NCS) production in Escherichia coli was designed and constructed on a phage-display vector, pJuFo. The recombinant phages expressing pre-apo-NCS protein were enriched with a mouse anti-apo-NCS monoclonal antibody, 1C7D4. The apo-NCS gene (encsA) for E. coli was successfully cloned, and then re-cloned into the pRSET A vector. After the his-tagged apo-NCS protein had been purified and cleaved with enterokinase, the binding properties of the recombinant protein as to ethidium bromide (EtBr) were studied by monitoring of total fluorescence intensity and fluorescence polarization with a BEACON 2000 system and GraphPad Prism software. A dissociation constant of 4.4 +/- 0.3 microM was obtained for recombinant apo-NCS in the fluorescence polarization study. This suggests that fluorescence polarization monitoring with EtBr as a chromophore mimic may be a simplified method for the characterization of recombinant apo-NCS binding to the NCS chromophore. When Phe78 on apo-NCS was substituted with Trp78 by site-directed mutagenesis using a two stage megaprimer polymerase chain reaction, the association of the apo-NCS mutant and EtBr observed on fluorescence polarization analysis was of the same degree as in the case of the wild type, although the calculated maximum change (DeltaIT(max)) in total fluorescence intensity decreased from 113.9 to 31.3. It was suggested that an environmental change of the bound EtBr molecule on F78W might have dramatically occurred as compared with in the case of wild type apo-NCS. This combination of monitoring of fluorescence polarization and total fluorescence intensity will be applicable for determination and prediction of the ligand state bound or associated with the target protein. The histone-specific proteolytic activity was also re-investigated using this recombinant apo-NCS preparation, and calf thymus histone H1, H2A, H2B, H3, and H4. The recombinant apo-NCS does not act as a histone protease because a noticeable difference was not observed between the incubation mixtures with and without apo-NCS under our experimental conditions.     

Secretory production of human granulocyte colony-stimulating factor in Escherichia coli.

Human granulocyte colony-stimulating factor (hG-CSF) is a glycoprotein, consisting of 174 amino acids, which plays an important role in hematopoietic cell proliferation, differentiation of hemopoietic precursor cells, and activation of mature neutrophilic granulocytes. In this study, secretory production of hG-CSF in the periplasmic space of Escherichia coli using the Bacillus sp. endoxylanase signal peptide was examined. For the efficient expression of hG-CSF gene, the first five codons at the N-terminal were altered based on the E. coli high-frequency codon database. The hG-CSF gene fused to the endoxylanase signal sequence was expressed using an inducible trc promoter. However, recombinant E. coli cells were completely lysed after induction with 1 mM isopropyl-beta-D-thiogalactopyranoside. Insertion of a small oligopeptide (13 amino acids) containing the histidine hexamer and factor Xa cleavage site between the signal peptide and the mature hG-CSF protein allowed successful secretion of hG-CSF into the periplasm without cell lysis. Among the several E. coli strains examined, E. coli BL21(DE3) and E. coli MC4100 allowed production of hG-CSF to the highest levels (20-22% of total proteins) with the secretion efficiencies greater than 98%. The circular dichroism spectra showed that the conformation of purified hG-CSF is almost identical to native hG-CSF.     

Refolding and characterization of recombinant human GST-PD-1 fusion protein expressed in Escherichia coli

Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed onactivated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibitsproliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involvedin antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain wasamplified and cloned into expression plasmid pGEX-Sx-3. The fusion protein GST-PD-1 was effectivelyexpressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was per-formed to obtain bioactive soluble GST-PD-I. Fusion protein of above 95% purity was acquired by a conve-nient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependentin vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-PD-1 could competently block the interaction between PD-Ll and PD-l and increase the production of IL-2 and IFN-γ of phytohemagglutinin-activated T cells.     

Expression, purification, and characterization of a novel recombinant fusion protein, rhTPO/SCF, in Escherichia coli

Thrombopoietin (TPO) is the principal regulatory cytokine of megakaryopoiesis and thrombopoiesis and promotes all aspects of megakaryocyte development. Stem cell factor (SCF) is mainly a pleiotropic cytokine acting on hematopoiesis by promoting the survival and proliferation of hematopoietic stem cells and has a potent synergistic effect on megakaryopoiesis in the presence of TPO. Here, we report the construction, expression, and purification of a novel recombinant human thrombopoietin/stem cell factor (rhTPO/SCF) fusion protein, which consists of a truncated human thrombopoietin (1-157 a.a.) plus a truncated human stem cell factor (1-145 a.a.), linked by a peptide (GGGGSPGGSGGGGSGG). The TPO/SCF gene was cloned into the Escherichia coli expression vector pET28a and expressed in BL21(DE3) strain. The rhTPO/SCF constituted up to 6% of the total bacterial protein. Co-expression with E. coli chaperones, Trigger Factor (TF) and GroES/GroEL, and lowering cultivation temperature cooperatively improved the solubility of expressed rhTPO/SCF, resulting in about fourfold increase in the yield soluble rhTPO/SCF. The rhTPO/SCF was purified to homogeneity using anion exchange followed by metal affinity chromatography. Western blot analysis confirmed the identity of the purified protein. rhTPO/SCF stimulated a dose-dependent cell proliferation in both TF1 and Mo7e cell lines.     

Expanding the landscape of recombinant protein production in Escherichia coli

Over the years, several vectors and host strains have been constructed to improve the overexpression of recombinant proteins in Escherichia coli. More recently, attention has focused on the co-expression of genes in E. coli, either by means of a single vector or by cotransformation with multiple compatible plasmids. Co-expression was initially designed to generate protein complexes in vivo, and later served to extend the use of E. coli as a platform for the production of heterologous proteins. This review shows how the co-expression of genes in E. coli is challenging the production of protein complexes and proteins bearing post-translational modifications or unnatural amino acids. In addition, the importance of co-expression to achieve efficient secretion of recombinant proteins in E. coli is discussed, with recent insights into the use of co-expression to overproduce membrane proteins.     

Expression, purification, and characterization of recombinant human interleukin 24 in Escherichia coli

Interleukin-24 (IL-24) can induce apoptosis of a broad range of tumor cells, and this function of IL-24 is independent of classic tumor suppressor genes, such as p53, Rb and p16. Here, we report the expression, purification and preparation of a recombinant IL-24 protein (rIL-24) without post-translational modifications, which may selectively induce apoptosis of tumor cells in vitro. We found that non-fusion rIL-24 was not able to be expressed by vectors pET11c, 28a, and 22b in Escherichia coli. To obtain recombinant non-fusion IL-24 protein, the encoding region for IL-24 was cloned between KpnI and BamHI in pET32a. The Trx (Thioredoxin)/IL-24 fusion proteins were expressed in the form of inclusion bodies in E. coli host strain BL21 (DE21). The expression level was more than 30% of total cell lysate. Inclusion bodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in a buffer containing 2M urea at pH 9.0. After nickel ion metal affinity chromatography, gel filtration chromatography, and renaturation, the refolded fusion proteins with a purity of >96% were obtained. Trx/IL-24 proteins were digested by enterokinase (EK) to both Trx and rIL-24 fragments which then were separated by cation exchange chromatography. Cell proliferation experiments proved that the rIL-24 (98% purity) retains its cancer-selective apoptosis-inducing properties. This result suggested that the rIL-24 may have cancer therapeutic applications.     

Combined effect of protein fusion and signal sequence greatly enhances the production of recombinant human GM-CSF in Escherichia coli

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been successfully produced in Escherichia coli, the reported levels are extremely poor. In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. This combination had a dramatic effect on expression levels, which increased from 0.8 μg/mL in the control to 40 μg/mL. When a larger fusion partner, such as the Maltose-binding protein (MBP-tag), was used the expression levels increased further to 69.5 μg/mL, which along with the MBP-tag represented approx 12% of the total cellular protein.     

Expression, purification, and characterization of recombinant human flotillin-1 in Escherichia coli

Human flotillin-1 (reggie-2), a major hydrophobic protein of biomembrane microdomain lipid rafts, was cloned and expressed in Escherichia coli with four different fusion tags (hexahistidine, glutathione S-transferase, NusA, and thioredoxin) to increase the yield. The best expressed flotillin-1 with thioredoxin tag was solubilized from inclusion bodies, first purified by immobilized metal affinity column under denaturing condition and direct refolded on column by decreasing urea gradient method. The thioredoxin tag was cleaved by thrombin, and the flotillin-1 protein was further purified by anion exchanger and gel filtration column. The purified protein was verified by denaturing gel electrophoresis and Western blot. The typical yield was 3.4 mg with purity above 98% from 1L culture medium. Using pull-down assay, the interaction of both the recombinant flotillin-1 and the native flotillin-1 from human erythrocyte membranes with c-Cbl-associated protein or neuroglobin was confirmed, which demonstrated that the recombinant proteins were functional active. This is the first report describing expression, purification, and characterization of active recombinant raft specific protein in large quantity and highly purity, which would facilitate further research such as X-ray crystallography.     

Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli strain

Several protease negative mutant strains including HM114, HM126, and HM130 as well as their parent strain KS272 were compared for their growth and secretory production of a model fusion protein, protein A-beta-lactamase. HM114, a strain deficient in two cell envelope proteases, grew slightly faster and produced more fusion protein than the other strains deficient in more proteases. HM114 was grown to a cell dry weight of 47.86 g/L in 29 h using pH-stat, fed-batch cultivation. The beta-lactamase activity was 11.25 x 10(4) U/L, which was 30% higher than that obtained with its parent strain KS272. Up to 96% of protein A-beta-lactamase fusion protein could be recovered by a simple cold osmotic shock method. The specific beta-lactamase activity obtained with HM114 after fractionation was 4.5 times higher than that obtained with KS272.     

Quantification of metabolic limitations during recombinant protein production in Escherichia coli

Escherichia coli is one of the major microorganisms for recombinant protein production because it has been best characterized in terms of molecular genetics and physiology, and because of the availability of various expression vectors and strains. The synthesis of proteins is one of the most energy consuming processes in the cell, with the result that cellular energy supply may become critical. Indeed, the so called metabolic burden of recombinant protein synthesis was reported to cause alterations in the operation of the host's central carbon metabolism.To quantify these alterations in E. coli metabolism in dependence of the rate of recombinant protein production, ¹³C-tracer-based metabolic flux analysis in differently induced cultures was used. To avoid dilution of the ¹³C-tracer signal by the culture history, the recombinant protein produced was used as a flux probe, i.e., as a read out of intracellular flux distributions. In detail, an increase in the generation rate rising from 36 mmol_ATP g_CDW⁻¹ h⁻¹ for the reference strain to 45 mmol_ATP g_CDW⁻¹ h⁻¹ for the highest yielding strain was observed during batch cultivation. Notably, the flux through the TCA cycle was rather constant at 2.5 ± 0.1 mmol g_CDW⁻¹ h⁻¹, hence was independent of the induced strength for gene expression. E. coli compensated for the additional energy demand of recombinant protein synthesis by reducing the biomass formation to almost 60%, resulting in excess NADPH. Speculative, this excess NADPH was converted to NADH via the soluble transhydrogenase and subsequently used for ATP generation in the electron transport chain. In this study, the metabolic burden was quantified by the biomass yield on ATP, which constantly decreased from 11.7 g_CDW mmol_ATP⁻¹ for the reference strain to 4.9 g_CDW mmol_ATP⁻¹ for the highest yielding strain. The insights into the operation of the metabolism of E. coli during recombinant protein production might guide the optimization of microbial hosts and fermentation conditions.     

Expression, purification, and characterization of recombinant human beta-amyloid42 peptide in Escherichia coli

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive function. Evidence indicates that abnormal processing and extracellular deposition of the beta-amyloid42 peptide, the longer form of proteolytic derivative of the transmembrane glycoprotein-amyloid precursor protein (APP), is a key step in the pathogenesis of AD. Since it is convenient and economical to obtain such a peptide biologically, in this study, we report for the first time a method to express in E. coli and purify beta-amyloid42 using glutathione-S-transferase (GST) fusion system. beta-Amyloid42 gene was inserted into a vector pGEX-4T-1 to construct a GST-fusion protein. The fusion protein GST-beta-amyloid42, expressed in BL21 (DE3) strain, was purified with GSH-affinity chromatography followed by thrombin cleavage. The digested product was further purified with an additional GSH-affinity and a Benzamidine chromatography step. After cleavage and purification, the beta-amyloid42 moiety showed the expected size of 4.5 kDa on Tricine-SDS-PAGE, and was further confirmed by Western blot. Moreover, the fibrillar recombinant beta-amyloid42 exhibited great aggregation activity and showed neurotoxicity on neuron cells in vitro. These results suggest that our method will be useful in obtaining a large quantity of recombinant beta-amyloid42 peptide for further physiological and biochemical studies.     

High-level recombinant protein production by overexpression of Mlc in Escherichia coli

Escherichia coli excretes acetate during aerobic growth on LB broth containing glucose and growth ceases before depletion of glucose because of the low pH caused by the accumulation of acetate. It has been known that the acetate accumulation is reduced even when E. coli is grown in the presence of high concentration of glucose if Mlc is overexpressed. The intracellular concentration of Mlc is very low in E. coli because of autoregulation and a low efficiency of mlc translation. We constructed various mutants that can express higher levels of Mlc using site-directed mutagenesis and one of the Mlc-overproducing mutant showed reduced glucose consumption rate and low production of acetate. The mutant showed higher foreign gene expression level than that of its parental strain in the presence of glucose. These results suggest that the Mlc overproducing E. coli strain having an improved ability of glucose utilization can be a better host for high-level production of useful recombinant proteins.     

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.     

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 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.