Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells

A fusion protein of human interleukin-2 (hIL-2) and green fluorescent protein (GFP) was expressed in insect Sf-9 cells infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of hIL-2 from the fusion, and the model product hIL-2. Successful production of hIL-2 as a fusion protein (approximately 52,000 Da) with GFPuv was obtained. GFPuv enabled rapid monitoring and quantification of the hIL-2 by simply checking the fluorescence, obviating the need for Western blot and/or ELISA assays during infection and production stages. There was no increased 'metabolic burden' due to the presence of GFPuv in the fusion product. The additional histidine residues at the N-terminus enabled efficient one-step purification of the fusion protein using IMAC. Additional advantages of GFP as a fusion marker were seen, particularly during separation and purification in that hIL-2 containing fractions were identified simply by illumination with UV light. Our results demonstrated that GFP was an effective non-invasive on-line marker for the expression and purification of heterologous protein in the suspended insect cell/baculovirus expression system.     

Insect larval expression process is optimized by generating fusions with green fluorescent protein.

The insect larvae/baculovirus protein production process was dramatically simplified by expressing fusion proteins containing green fluorescent protein (GFP) and the product-of-interest. In this case, human interleukin-2 (hIL-2) and chloramphenicol acetyl-transferase (CAT) were model products. Specifically, our fusion construct was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), the UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of the product from the fusion, and the product, hIL-2 or CAT. Both the approximately 52 kDa GFPuv/hIL-2 and approximately 63 kDa GFPuv/CAT fusions were expressed in Trichoplusia ni larvae at 9.0 microg-hIL-2 and 24.1 microg-CAT per larva, respectively. The GFP enabled clear identification of the infection process, harvest time, and more importantly, the quantity of product protein. Because the GFP served as a marker, this technique obviates the need for in-process Western analyses (during expression, separation, and purification stages). As a purification marker, GFP facilitated rapid identification of product-containing elution fractions (Cha et al., 1999b), as well as product-containing waste fractions (e.g., cell pellet). Also, because the fluorescence intensity was linear with hIL-2 and CAT, we were able to select the highest-producing larvae. That is, three fold more product was found in the brightest larva compared to the average. Finally, because the GFP is attached to the product protein and the producing larvae can be selected, the infection and production processes can be made semi-continuous or continuous, replacing the current batch process. These advantages should help to enable commercialization of larvae as expression hosts.     

New fusion protein systems designed to give soluble expression in Escherichia coli.

Three native E. coli proteins-NusA, GrpE, and bacterioferritin (BFR)-were studied in fusion proteins expressed in E. coli for their ability to confer solubility on a target insoluble protein at the C-terminus of the fusion protein. These three proteins were chosen based on their favorable cytoplasmic solubility characteristics as predicted by a statistical solubility model for recombinant proteins in E. coli. Modeling predicted the probability of soluble fusion protein expression for the target insoluble protein human interleukin-3 (hIL-3) in the following order: NusA (most soluble), GrpE, BFR, and thioredoxin (least soluble). Expression experiments at 37 degrees C showed that the NusA/hIL-3 fusion protein was expressed almost completely in the soluble fraction, while GrpE/hIL-3 and BFR/hIL-3 exhibited partial solubility at 37 degrees C. Thioredoxin/hIL-3 was expressed almost completely in the insoluble fraction. Fusion proteins consisting of NusA and either bovine growth hormone or human interferon-gamma were also expressed in E. coli at 37 degrees C and again showed that the fusion protein was almost completely soluble. Starting with the NusA/hIL-3 fusion protein with an N-terminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography.     

Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: monitoring protein expression and solubility

We have constructed three plasmid vectors for the expression of green fluorescent protein (GFP) fusion proteins using the following motif: (His)(6)-GFP-EK-X, where X represents chloramphenicol acetyl-transferase (CAT), human interleukin-2 (hIL-2), and organophosphorous hydrolase (OPH), respectively, (His)(6) represents a histidine affinity ligand for purification, and EK represents an enterokinase cleavage site for recovering the protein-of-interest from the fusion. The CAT and OPH fusion products ( approximately 63 kDa GFP/CAT and approximately 70 kDa GFP/OPH) were expressed at 4.85 microg/mL (19.9 microg/mg-total protein) and 1.42 microg/mL (4.2 microg/mg-total protein) in the cell lysis supernatant, and, in both cases, enzymatic activity was retained while coupled to GFP. In the case of hIL-2 fusion ( approximately 52 kDa), however, the GFP fluorescence was significantly reduced and most of the fusion was retained in the cell pellet. Linear relationships between GFP fluorescence and CAT or OPH concentration, and with enzymatic activity of CAT or OPH, indicated, for the first time, that in vivo noninvasive quantification of proteins-of-interest, was made possible by simple measurement of GFP fluorescence intensity. The utility of GFP as a reporter was not realized without disadvantages however, in particular, an incremental metabolic cost of GFP was found. This could be offset by many benefits foreseen in expression and purification efficiencies.     

Engineering green fluorescent protein as a dual functional tag

A hexa-histidine (6 x His) sequence was inserted into a surface loop of the green fluorescent protein (GFP) to develop a dual functional GFP useful for both monitoring and purification of recombinant proteins. Two variants (GFP172 and GFP157), differentiated by the site of insertion of the 6xHis sequence, were developed and compared with a control variant (GFPHis) having the 6xHis sequence at its C-terminus. The variants were produced in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The purification efficiencies by IMAC for all variants were found to be comparable. Purified GFP172 and GFP157 variants retained approximately 60% of the fluorescence compared to that of GFPHis. The reduction in the fluorescence intensity associated with GFP172 and GFP157 was attributed to the lower percentage of fluorescent GFP molecules in these variants. Nonetheless, the rates of fluorescence acquisition were found to be similar for all functional variants. Protein misfolding at an elevated temperature (37 degrees C) was found to be less profound for GFP172 than for GFP157. The dual functional properties of GFP172 were tested with maltose binding protein (MBP) as the fusion partner. The MBP-GFP172 fusion protein remained fluorescent and was purified from E. coli lysate as well as from spiked tobacco leaf extracts in a single-step IMAC. For the latter, a recovery yield of approximately 75% was achieved and MBP-GFP172 was found to coelute with a degraded product of the fusion protein at a ratio of about 4:1. The primary advantage of the chimeric GFP tag having an internal hexa-histidine sequence is that such a tag allows maximum flexibility for protein or peptide fusions since both N- and C-terminal ends of the GFP are available for fusion.     

Overexpression and purification of an immunologically reactive His-BIV capsid fusion protein

The gene of the capsid protein of bovine immunodeficiency virus (BIV) was linked to a sequence encoding for six histidines and expressed as the (His)6 p26 capsid fusion protein. The fusion protein was strongly expressed as both soluble and insoluble forms after induction by isopropylthio-beta-d-galactoside. Purification was based on interaction of the hexa-histidine polypeptide with metal ions. Expression could represent 11% of the total protein in Escherichia coli, allowing more than 20 mg of highly purified protein to be obtained per liter of bacterial culture. The (His)6 p26 capsid fusion protein purified by immobilized metal affinity chromatography reacted specifically in Western blot with sera from cattle experimentally infected by BIV, as well as with two monoclonal antibodies directed against different epitopes of the Gag protein. The ease of expression, purification, and specificity of this fusion protein should permit a thorough study of prevalence of BIV infection in large-scale serological studies of field samples.     

Purification and characterization of human IL-10/Fc fusion protein expressed in Pichia pastoris

Interleukin (IL)-10 is an anti-inflammatory cytokine that could be potentially applied for clinical therapy. However, its short circulating half-life in the serum limits its clinical applications. In this study, we designed a fusion protein containing human IL-10 and an IgG Fc fragment (hIL-10/Fc), and expressed it in Pichia pastoris. This hIL-10/Fc fusion protein was purified from the culture supernatant using MabSelect affinity chromatography and size-exclusion chromatography. The hIL-10/Fc yield was about 5mg/L in shake flasks, with purity exceeding 95%. In addition, the hIL-10/Fc fusion protein suppressed the phytohemagglutinin-induced IFN-γ production in human peripheral blood mononuclear cells. Pharmacokinetic study also revealed that hIL-10/Fc has a prolonged circulating half-life of about 30h in rats. More importantly, the hIL-10/Fc fusion protein displayed highly specific biological activity, which was slightly higher than that of the commercial recombinant human IL-10 (rhIL-10). Therefore, P. pastoris is useful in the large-scale production of hIL-10/Fc fusion protein for both research and therapeutic applications.     

A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase

 Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5′ end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence. Received: 27 April 1999 / Received last revision: 18 October 1999 / Accepted: 1 November 1999     

Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein

The entire coding region of open reading frame 2 (ORF2) of porcine circovirus 2 (PCV2) was linked to the 3'-end of the maltose-binding protein (MBP)-His(8)-tag gene. The fusion protein was expressed as soluble form after induction by isopropylthio-beta-d-galactoside. MBP-His(8)-ORF2 was purified to homogeneity by immobilized metal affinity chromatography based on the interaction of the polyhistidine-tag with metal ions. Expression could represent 1% of the total protein in Escherichia coli, allowing approximately 1 mg of highly purified protein to be obtained per liter of bacterial culture. The fusion protein was recognized in Western blot by anti-PCV2 polyclonal antibody and swine sera with PCV2 infection. In addition, rabbit polyclonal antibody raised against purified MBP-His(8)-ORF2 fusion protein reacted with the ORF2 protein in immunoprecipitation. The availability of this fusion protein should permit a thorough study of prevalence of PCV2 infection in large-scale serological studies of field samples.     

A novel fusion partner for enhanced secretion of recombinant proteins in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.     

A self-cleavable sortase fusion for one-step purification of free recombinant proteins

A new protein fusion system has been developed to generate free recombinant protein in a single affinity chromatographic step. The key component in the fusion is the catalytic core of sortase A from Staphylococcus aureus (SrtAc), which recognizes and cleaves the Thr-Gly bond at an LPXTG sequence with moderate activity. The fusion here consists of an N-terminal His6 tag, SrtAc, and an LPETG linker followed by protein of interest at the C-terminus. The fusion protein is expressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography (IMAC). The immobilized fusion then undergoes on-column SrtAc-mediated cleavage at the LPETG site in the presence of Ca2+ and/or triglycine. The target protein with an extra N-terminal glycine is released from the fusion while the N-terminal portion remains bound to the column. Because the cleavage enzyme SrtAc is co-expressed as a fusion with the target protein, the purification system eliminates exogenous proteolysis. This purification approach is simple, robust, inexpensive, time saving, and allows purification of free recombinant protein via one-step chromatography.     

Facile monitoring of avian reovirus σB expression and purification processes by tagged green fluorescent protein

Avian reovirus capsid protein σB was genetically fused with a histidine (His₆) tag and a UV-optimized green fluorescent protein (GFPuv) and expressed in Sf-9 cells. The fluorescence of GFPuv allowed for easy identification of protein localization and revealed that the fusion protein was quite stable in the cell culture. The fluorescence intensity (FI) exhibited a linear relationship (r² = 0.93) with the recombinant protein yield and therefore allowed for on-line tracking of the expression profile, which revealed an extremely high maximum yield of 70 μg per 10⁶ cells. The recombinant protein was purified via immobilized metal affinity chromatography (IMAC) and a high purity (85%) was achieved in one step. During the purification, the fluorescence again enabled qualitative and quantitative monitoring of when and how much the desired product was eluted. The GFP-tagging strategy eliminated the need for cumbersome and time-consuming assays (e.g. Western blot or ELISA) for product analysis, thus GFP is an effective non-invasive on-line marker for the expression and purification of recombinant proteins in the baculovirus expression system.     

Integrated bioprocessing in Saccharomyces cerevisiae using green fluorescent protein as a fusion partner

In this study, we examine the use of green fluorescent protein (GFP) for monitoring a hexokinase (HXK)-GFP fusion protein in Saccharomyces cerevisiae for various events including expression, degradation, purification, and localization. The fusion, HXK-EK-GFP-6 x His, was constructed where the histidine tag (6 x His) would allow for convenient affinity purification, and the enterokinase (EK) cleavage site would be used for separation of HXK from GFP after affinity purification. Our results showed that both HXK and GFP remained active in the fusion and, more importantly, that there was a linear correlation between HXK activity and GFP fluorescence. Enterokinase cleavage studies revealed that both GFP fluorescence intensity and HXK activity remained unchanged after separation of the fusion proteins, which indicated that fusion of GFP did not cause structural alteration of HXK and thus did not affect the enzymatic activity of HXK. We also found that degradation of the fusion protein occurred, and that degradation was limited to HXK with GFP remaining intact in the fusion. Confocal microscopy studies showed that while GFP was distributed evenly in the yeast cytosol, HXK-GFP fusion followed the correct localization of HXK, which resulted in a di-localization of both cytosol and the nucleus. GFP proved to be a useful fusion partner that may lead to the possibility of integrating the bioprocesses by quantitatively following the entire process visually.     

高活性重组hG-CSF的制备

我们构建了新的硫氧还蛋白（Ｔｈｉｏｒｅｄｏｘｉｎ）融合表达载体ｐＥＴＴｒｘＬ和ｐＥＴＴｒｘ－ＨｉｓＬ,它们可使功能蛋白在大肠杆菌胞质中以可溶性形式高效表达。利用此表达系统成功地获得的ｈＧ－ＣＳＦ－硫氧还蛋白融合蛋白的高效可溶性表达,表达水平达总细胞可溶蛋白的４１％以上。所表达的ｈＧ－ＣＳＦ－硫氧还蛋白融合蛋白可通过Ｃｕ２＋－ＩＤＡＳｅｐｈａｒｏｓｅＦＦ固相金属螯合层析柱,方便地从细胞破碎可溶上清中直接纯化。所获得的融合蛋白具有ｈＧ－ＣＳＦ特异的生物活性,其比活性达到０．５－１．３３×１０７ｕ／ｍｇ融合蛋白。这样表达的ｈＧ－ＣＳＦ融合蛋白能被ＩｇＡ蛋白酶特异地切割,将ｈＧ－ＣＳＦ从融合蛋白上切下获得与天然蛋白一级结构完全一致的重组ｈＧ－ＣＳＦ 。     

Expression of the murine leukemia virus protease in fusion with maltose-binding protein in Escherichia coli

The protease of murine leukemia virus (MLV) was cloned into pMal-c2 vector, expressed in fusion with maltose-binding protein (MBP), and purified to homogeneity after Factor Xa cleavage of the chimeric protein. Substantial degradation of the fusion protein was observed during expression, which severely diminished the yield. The degree of degradation of the fusion protein was even more pronounced when a single-chain form of the MLV protease was cloned after the gene coding for MBP. To increase the yield, a hexahistidine tag with an additional Factor Xa cleavage site was cloned after the protease and nickel chelate affinity chromatography was used as the first purification step. The modified procedure resulted in substantially higher yield as compared to the original procedure. The degradation of hexahistidine-tagged active site mutant MLV protease was very low and comparable to that obtained with hexahistidine-tagged MBP, but purified MLV protease alone was not able to degrade purified MBP, suggesting that during expression the active MLV protease may activate bacterial proteases which appear to be responsible for the degradation of the fusion proteins.     

In vivo monitoring of intracellular expression of human interleukin-2 using green fluorescent protein fusion partner in Pichia pastoris

The use of Pichia pastoris for protein production was simplified by creation of fusion proteins containing green fluorescent protein (GFP) and the product of interest. Human interleukin-2 (hIL-2) was used as a model product: GFP enabled clear identification of fusion protein expression and, more importantly, the quantification of hIL-2. Although GFP fusions for bioprocess monitoring have been demonstrated in other hosts, this is its first use in P. pastoris.     

Quantitative monitoring for secreted production of human interleukin-2 in stable insect Drosophila S2 cells using a green fluorescent protein fusion partner

Human interleukin-2 (hIL-2) production in Escherichia coli and insect cell/baculovirus expression systems can be inefficient. Here we investigated secreted production of hIL-2 fused with green fluorescent protein (GFP) as a versatile fusion partner in optimized stably transfected insect Drosophila melanogaster S2 cells. This nonlytic S2 insect cell expression system employs a plasmid vector and allows for secretion of functional human proteins. We report that, following stable transfection and induction, S2 cells secreted hIL-2 as a fusion protein (approximately 2.3 microg/mL yield), with a secretion efficiency of approximately 90%. Regression analysis indicated a single linear relationship existed between GFP fluorescence and hIL-2 mass in both whole cell and secreted medium samples, indicating that in vivo monitoring and quantification of target foreign protein expression and even secretion is possible using this system. The simple comparative measurement of GFP fluorescence also allowed monitoring of secretion efficiency during periods of high GFP/hIL-2 expression.     

Construction and characterization of a bi-functional EGFP/sBAFF fusion protein

A fusion between gene encoding fluoresce-enhanced green fluorescent protein variant (EGFP) and soluble domain of human B-cell-activating factor of the TNF family (sBAFF) was constructed and expressed in Escherichia coli. The EGFP/sBAFF had an apparent molecular weight of 45 kDa and was detected with anti-hsBAFF and anti-His(6) monoclonal antibodies. After being purified by immobilized metal affinity chromatography (IMAC), the fusion protein retained similar fluorescence spectra to those of EGFP. Biological activity assays showed the EGFP/sBAFF as well as sBAFF could co-stimulated human B lymphocyte proliferation in vitro. In addition, EGFP/sBAFF has shown specific binding to BAFF receptors positive-cells and the stained cells could be analyzed with flow cytometry. Thus, the fusion protein represents a readily obtainable source of biologically active sBAFF that may prove useful in further studies on BAFF and its receptors.     

Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin

An antimicrobial peptide, piscidin, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The peptide was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The expression and purification process of piscidin encountered several problems such as the lysis of the bacterial cell upon induction of the peptide production, the unwanted cleavage of the fusion protein inside the bacterial cell, and high tendency to aggregate in the aqueous environment. Such problems were alleviated by employing ubiquitin as a fusion partner for piscidin, growing the cells at a lower temperature, and changing the order of the purification steps. The yields of the fusion protein and the peptide were around 15 and 1.5 mg per liter of LB or minimal medium, respectively. The recombinant expression and purification of piscidin will enable its structural and dynamic studies using multidimensional NMR spectroscopy.     

Expression, refolding, purification, and bioactivity of recombinant bifunctional protein, hIL-2/GM-CSF

Interleukin-2 (IL-2) can stimulate T cell proliferation and differentiation when binding to its receptor on T cells. It produces a marked effect by enhancing the cytotoxicity of CD8+ T cells and natural killer cells. Granulocyte-macrophage colony stimulating factor (GM-CSF) is associated with many cells proliferation, such as dendritic cells, macrophages. Here, we report the construction, expression and purification of a bifunctional protein, hIL-2/GM-CSF, which may facilitate interaction between T cells and the antigen presentation cells and improve the efficiency of antigen presentation. We found that the use of chemicals and temperature shift is a peculiar system for induction of the Escherichia coli transformed with an IPTG-regulated hIL-2/GM-CSF expression vector in this research. After renaturation, anion exchange chromatography, metal affinity chromatography, and strict endotoxin-free cation exchange chromatography, the fusion protein devoid of endotoxin showed high purity. Cell proliferation experiments proved that this bifunctional protein retains both hIL-2 and GM-CSF biological activities. These results will facilitate the numerous subsequent studies on this bifunctional molecule.