N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.      

Protein production from recombinant protein bodies

In this study, we describe a process for protein expression and purification from plants and insect cells based on the accumulation of recombinant proteins in protein bodies. This technology is using Zera^®, which sequence has the capacity to trigger in vivo the formation of dense, non-secretory storage protein body-like organelles derived from the endoplasmic reticulum (ER). With this method, recombinant human growth hormone (hGH) was expressed and purified from protein bodies accumulated in plants (Nicotiana benthamiana) and in insect cells (Spodoptera frugiperda). We found that recombinant Zera-hGH are stored in large quantity inside those proteins bodies and can be easily recovered during a one-step process from plant and insect cell biomass. After solubilization of recombinant protein bodies and cleavage of Zera tag from the fusion protein, active hGH was finally purified by a single chromatography step. These results indicate that recombinant proteins derived from Zera-fusion could provide both an efficient protein production system and eased purification downstream process.     

Preparation of recombinant RNase single-chain antibody fusion proteins

This article describes the construction, expression, and purification of RNase single-chain antibody fusion proteins. To construct a fusion protein, the gene for each moiety, the RNase and the binding ligand, is modified separately to contain complementary DNA encoding a 13 amino acid spacer that separates the RNase from the binding moiety. Appropriate restriction enzyme sites for cloning into the vector are also added. The modified DNA is combined and fused using the PCR technique of splicing by overlap extension (1). The resulting DNA construct is expressed in inclusion bodies in BL21(DE3) bacteria that are specifically engineered for the expression of toxic proteins (2). After isolation and purification of the inclusion bodies, the fusion protein is solubilized, denatured, and renatured. The renatured RNase fusion protein mixture is purified to homogeneity by two chromatography steps. The first column, a CM-Sephadex C-50 or a heparin Sepharose column, eliminates the majority of contaminating proteins while the second column, an affinity column (Ni²⁺-NTA agarose), results in the final purification of the RNase fusion protein.     

Purification of the type II insulin-like growth factor receptor from rat placenta

The membrane receptor for insulin-like growth factor II (IGF II) has been purified to near homogeneity from rat placenta by chromatography of crude plasma membranes solubilized in Triton X-100 on agarose-immobilized IGF II. Elution of the IGF II receptor from the matrix at pH 5.0 in the presence of 1.5 M NaCl resulted in a receptor purification of 1100-fold from isolated plasma membranes, or 340-fold from the Triton extract with an average yield of about 50% in five separate purifications. Analysis of 125I-IGF II binding to the solubilized receptor in the Triton extract and in purified form by the method of Scatchard demonstrated no change in receptor affinity (Kd = 0.72 nM). Sodium dodecyl sulfate electrophoresis of the purified receptor showed one major band at Mr = 250,000 with only minor contamination. Affinity labeling of the receptor in isolated placenta membranes and in purified form using 125I-IGF II and the cross-linking agent disuccinimidyl suberate resulted in covalent labeling of only the Mr = 250,000 band. Such labeling was abolished by unlabeled IGF II but was unaffected by insulin, consistent with the previously reported specificity of IGF II receptor (Massague, J., and Czech, M.P. (1982) J. Biol. Chem. 257, 5038-5045). These results establish a one step affinity method for the purification of the type II IGF receptor that is rapid and highly efficient.     

Comparison of the efficiencies of different affinity tags in the purification of a recombinant secretory protein expressed in silkworm larval hemolymph

Silkworms are useful bioreactors for heterologous protein expression when used in conjunction with the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. However, purification from silkworm hemolymph is difficult since it contains various kinds of proteins. In this study, we investigated an effective single-step method for the purification of affinity-tagged single-chain antibody variable region fragment (scFv) from silkworm larval hemolymph. A 5-fold higher expression level was obtained when scFv was fused with the His tag than when it was fused with the Strep II or GST tags. However, the His tag was inadequate for single-step purification since it led to the nonspecific binding of contaminants. The purification recoveries of GST-, Strep II-, and His-tagged scFvs were 91.8%, 43.7%, and 27.2%, respectively. The specific amount of single-step purified GST-tagged scFv was 2.2∼2.7 fold higher than the amounts of the His- and Strep II-tagged constructs. The purities of Strep II- and GST-tagged scFvs in the eluent were 98.4% and 83.0%, respectively. Thus, both the short peptide Strep II and GST protein are suitable fusion tags for the affinity purification of proteins from silkworm larvae.     

Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand

The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)6 for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-2 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker; we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.     

Production of scFv-displaying BmNPV in silkworm larvae and its efficient purification

Baculovirus-display technology utilizing the gp64 envelope protein has been developed. A simple and efficient process to separate the virus from the majority of the protein contaminants may be needed for the future demand of pure and functional baculovirus vectors ideal for vaccine- and gene-delivery applications. In the present study, using Bombyx mori (silkworm) larvae as a host, scFv (single-chain variable fragment)-surface displaying recombinant baculovirus production and its purification from silkworm larval haemolymph by SEC (size-exclusion chromatography) were demonstrated. The amounts of scFv were 4-8 μg/ml in the haemolymph. The scFv-gp64 fusion protein was confirmed to be incorporated into the cell membrane and the BmNPV (B. mori nucleopolyhedrovirus) surface by immunofluorescence microscopy and Western blotting. rBmNPV (recombinant BmNPV) was purified to higher purity by SEC using Sephacryl S-1000 column chromatography than by sucrose-density-gradient centrifugation. The recovery of purified rBmNPV was 22.2%, and the virus purity in the SEC fraction was increased 269-fold compared with its purity in haemolymph. Judging from the results of ELISA, approx. 0.9% of the total baculovirus-particle proteins were occupied by scFv on their surface. A BmNPV-based silkworm-larval system is suitable for large-scale production of baculovirus-surface-displayed proteins or peptides in comparison with a cell-culture system. The present study will be useful for future BmNPV-application studies for gene delivery and vaccine trials.     

Purification of dual-tagged intact recombinant proteins

Large-scale purification of recombinant proteins has been used extensively to assist numerous protein studies, including investigation of function, substrate identification and protein-protein interaction of low abundance proteins. Genetic fusion of affinity tags to these proteins has also been widely used for ease of purification by affinity chromatography. However, this technique sometimes yields unstable and degraded protein products limiting its application. In this study, we show a facile and straightforward method of dual-tagged recombinant protein purification that eliminates contamination by degraded protein products. A 6His-containing BamHI-HindIII fragment from pQE12 was ligated into the pGEX-KG BamHI-HindIII fragment and the protein of interest (p25(nck5a), which is highly susceptible to proteolytic degradation when expressed and purified from bacteria) was cloned into the BamHI site without a termination codon. The resulting plasmid construct, designated as pGST-p25(nck5a)-6His, with GST at the N-terminal and 6His at the C-terminal was expressed in Escherichia coli DH5alpha and purified using a two-step procedure. We show that using Ni(2+)-NTA chromatography as a first purification step and GSH-agarose chromatography as a second step, rather than vice-versa, yields a highly purified intact protein that is free of any contaminating degraded protein product. The purified fusion protein is soluble and fully active.     

Process development for production of recombinant human insulin-like growth factor-I in Escherichia coli

Fed-batch cultures were carried out to overproduce human insulin-like growth factor I (IGF-I) in Escherichia coli. The effects of carbon sources (glucose or glycerol) and induction time on cell growth and IGF-I production were investigated in more detail. Glycerol was a better carbon source than glucose for IGF-I production in fed-batch culture. Induction at the mid-exponential phase with glycerol as a carbon source in the pH-stat fed-batch culture was optimal for IGF-I production. Under this condition, 2.8 g L⁻¹ of fusion IGF-I was produced as inclusion bodies. We have also developed downstream processing for preparative scale purification of IGF-I from the fusion protein produced by the fed-batch culture using glycerol as a carbon source. After the fusion protein expressed was solubilized in 8 M urea and cleaved with hydroxylamine, the released IGF-I was purified by cation exchange chromatography, refolding and preparative scale reverse phase HPLC (rp-HPLC) to give recombinant IGF-I of >98% purity. The biological activities of the purified IGF-I were measured and found to be identical to those of commercial IGF-I. Journal of Industrial Microbiology & Biotechnology (2000) 24, 94–99. Received 13 January 1999/ Accepted in revised form 02 October 1999     

Characterization of an extended form of recombinant human insulin-like growth factor II.

To investigate the biological role of variants of human insulin-like growth factor II (IGF-II), an extended form designated IGF-IIE21, with a molecular mass of 9.8 kDa, was produced in Escherichia coli as a stable and soluble secreted fusion protein. After site-specific cleavage of the affinity purified fusion protein, followed by purification using ion exchange and reversed phase chromatography, it could be demonstrated that IGF-IIE21 and IGF-II have similar or identical activities according to radioimmunoassay and radioreceptor assay. However, IGF-IIE21 showed only 1% growth promotion activity as compared with IGF-II in a clonal expansion assay using human K562 cells which lacks IGF-I receptors. These results suggest that this extended variant of IGF-II can bind to the receptor but has limited growth promoting activity.     

Refolding and purification of recombinant OsNifU1A domain II that was expressed by Escherichia coli

OsNifU1A is a NifU-like rice (Oryza sativa) protein, discovered recently. Its amino acid sequence is very homologous to the sequence of cyanobacterial CnfU and to the sequences of NifU C-terminal domains. Based on its sequence, OsNifU1A is probably a modular structure consisting of two CnfU-like domains, with domain I (formed by residues Leu73 to Gly153) and domain II (formed by residues Leu154 to Ser226). Domain I have a conserved Cys-X-X-Cys motif, which may function as an iron-sulfur cluster assembly scaffold. Domain II lacks a Cys-X-X-Cys motif and therefore, cannot function analogously. Other NifU-like proteins, with sequences homologous to OsNifU1A domain II, have been identified during plant genomic projects; however, the biological roles of these domains remain unknown. We successfully constructed an Escherichia coli expression system for OsNifU1A domain II that enabled us to synthesize and purify milligram quantities of protein for use in structural and functional studies. Using the Gateway system, we built DNA sequences corresponding to two OsNifU1A domain II fusion proteins. One construct has a (His)6 sequence upstream of the OsNifU1A domain II sequence; the other has an upstream thioredoxin-(His)6 sequence. Recombinant OsNifU1A domain II fusion proteins were extracted from E. coli inclusion bodies by dissolving them in 6 M guanidine-HCl. About 36% of the total (His)6/OsNifU1A domain II fusion protein initially present remained soluble after guanidine-HCl was completely removed by step-wise dialysis; whereas, recovery of soluble Trx-(His)6 fusion protein was about 60% of the total cell lysate. About 2 mg of 15N-labeled OsNifU1A domain II was purified for NMR spectral studies. Examination of the OsNifU1A domain II 1H-15N HSQC NMR spectrum indicated that the purified protein was monomeric and correctly folded. Therefore, we established an efficient procedure for synthesis and purification of 15N-labeled OsNifU1A domain II in quantities sufficient for heteronuclear NMR solution structure studies.     

Production of a polyclonal antibody against recombinant goldfish prolactin and demonstration of its usefulness in a non-competitive antigen-capture ELISA

The cDNA encoding the goldfish (Carassius auratus) prolactin was expressed in Escherichia coli using the pRSETA expression vector. The recombinant goldfish prolactin (gfPRL) produced was a fusion protein containing a hexahistidyl sequence, which facilitated its purification on a Ni²⁺ column. The fusion protein was overexpressed in the bacteria as inclusion bodies and was successfully purified under denaturing conditions by one-step affinity chromatography. Repeated immunization of rabbits against the purified recombinant gfPRL allowed the production of a high-titer polyclonal antiserum. The IgG fraction of the antiserum was isolated on an immobilized Protein A–agarose column. The antibody recognized recombinant gfPRL, but not recombinant goldfish growth hormone (gfGH) or goldfish somatolactin (gfSL) on Western analyses. The purified antibody was able to recognize gfPRL, but not gfGH or gfSL, in a non-competitive antigen-capture ELISA. The assay was applied in monitoring the purification of native PRL from goldfish pituitaries.     

Purification of a 31,000-dalton insulin-like growth factor binding protein from human amniotic fluid. Isolation of two forms with different biologic actions

Human amniotic fluid has been shown to contain a protein that binds insulin-like growth factor I and II (IGF-I and IGF-II). Partially purified preparations of this protein have been reported to inhibit the biologic actions of the IGFs. In these studies our laboratory has used a modified purification procedure to obtain a homogeneous preparation of this protein as determined by polyacrylamide gel electrophoresis and amino acid sequence analysis. During purification the ion exchange chromatography step resulted in two peaks of material with IGF binding activity termed peaks B and C. Each peak was purified separately to homogeneity. Both peaks were estimated to be 31,000 daltons by polyacrylamide gel electrophoresis and their amino acid compositions were nearly identical. Amino acid sequence analysis showed that both peaks had identical N-terminal sequences through the first 28 residues. Neither protein had detectable carbohydrate side chains and each had a similar affinity for radiolabeled IGF-I (1.7-2.2 x 10(10) liters/mol). In contrast, these two forms had marked differences in bioactivity. Concentrations of peak C material between 2 and 20 ng/ml inhibited IGF-I stimulation of [3H]thymidine incorporation into smooth muscle cell DNA. In contrast, when peak B (100 ng/ml) was incubated with IGF-I there was a 4.4-fold enhancement of stimulation of DNA synthesis. Additionally, pure peak B was shown to adhere to cell surfaces, whereas peak C was not adherent. The non-adherent peak C inhibited IGF-I binding to its receptor and to adherent peak B. We conclude that human amniotic fluid contains two forms of IGF binding protein that have very similar physiochemical characteristics but markedly different biologic actions. Since both have similar if not identical amino acid compositions, N-terminal sequences, and do not contain carbohydrate, we conclude that they differ in some other as yet undefined post-translational modification.     

快速纯化重组腺联病毒的受体结合捕捉方法

【目的】建立用于重组腺联病毒(AAV)纯化的受体结合捕捉方法。【方法】将AAV受体的多囊肾病(PKD)结构域1和2与类弹性蛋白多肽(ELP)在重组大肠杆菌中进行融合表达,利用相变循环(ITC)纯化ELP-PKD融合蛋白;分别用昆虫和AAV-293细胞制备rAAV-GFP,与ELP-PKD融合蛋白共孵育后进行ITC,从沉淀复合物提取病毒DNA进行PCR检测;在优化条件下利用ELP-PKD蛋白结合捕捉rAAV-GFP,利用电子显微镜观察、免疫转印和细胞感染试验进行rAAV鉴定。【结果】ELP-PKD融合蛋白获得正确、可溶性表达,ITC纯化的蛋白纯度大于90%;ELP-PKD蛋白能特异结合rAAV-GFP,结合具有p H、温度和时间依赖性,受体结合捕捉方法可在1h内完成,从两种细胞纯化rAAV-GFP的回收率分别为58%和56%;rAAV-GFP洗脱具有p H和温度依赖性,洗脱rAAV-GFP的回收率分别为46%和44%;纯化rAAV-GFP具有AAV的典型形态和结构蛋白。【结论】建立的ELP-PKD结合捕捉法可用于不同细胞源rAAV的快速纯化。     

One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purity, as determined by SDS-PAGE analysis, was greater than 80%. The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca(2+) was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.     

Construction, overexpression, and purification of Arthrobacter globiformis amine oxidase-Strep-tag II fusion protein

The copper-containing amine oxidase from Arthrobacter globiformis has been expressed and purified as a fusion protein with a C-terminal Strep-tag II peptide. This tag facilitates the rapid purification of the enzyme on a large scale using the StrepTactin POROS medium. For example, we have demonstrated that 50 mg of protein can be obtained in 2 days from 2 L of Escherichia coli. The purified fusion protein displays turnover and spectroscopic properties that are essentially identical to those of the wild-type enzyme. Given the location of the C-terminus in four amine oxidase crystal structures, this strategy should be quite general for the rapid purification of amine oxidases from multiple sources.     

High-yield production of recombinant endothelin-1.

A fusion protein (pETB-42P), which encodes the 42-amino acid leader peptide and the 38-amino acid peptide of human big endothelin (ET)-1, was synthesized in Escherichia coli, isolated as inclusion bodies, and purified by DEAE-chromatography. Trypsin digestion of the purified pETB-42P gave big ET-1(1-37) in a yield of 70%; then pepsin digestion of the purified big ET-1(1-37) gave ET-1(1-21) in a yield of 74% (overall yield: 52%). Sequential trypsin and pepsin digestions of the purified fusion protein in the same reaction vessel also allowed recovery of ET-1 in a yield of 60%. One milligram of ET-1 or 2.0 mg of big ET-1(1-37) was obtained from 1.8 liters of culture broth. Recombinant ET-1 thus obtained was identical to authentic ET-1 in terms of amino acid sequence and vasoconstrictor potency, and recombinant big ET-1(1-37) had almost the same in vitro and in vivo biological activities as big ET-1(1-38).     

Hydrophobin (HFBI): A potential fusion partner for one-step purification of recombinant proteins from insect cells

Hydrophobins play an important role in binding and assembly of fungal surface structures as well as in medium-air interactions. These, hydrophobic properties provide interesting possibilities when purification of macromolecules is concerned. In aqueous micellar two-phase systems, based on surfactants, the water soluble hydrophobins are concentrated inside micellar structures and, thus, distributed to defined aqueous phases. This, one-step purification is attractive particularly when large-scale production of recombinant proteins is concerned. In the present study the hydrophobin HFBI of Trichoderma reesei was expressed as an N-terminal fusion with chicken avidin in baculovirus infected insect cells. The intracellular distribution of the recombinant fusion construct was analyzed by confocal microscopy and the protein subsequently purified from cytoplasmic extracts in an aqueous micellar two-phase system by using a non-ionic surfactant. The results show that hydrophobin and an avidin fusion thereof were efficiently expressed in insect cells and that these hydrophobic proteins could be efficiently purified from these cells in one-step by adopting an aqueous micellar two-phase system.     

Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase.

Encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which allowed easy purification of the fusion protein by affinity chromatography on immobilized glutathione. Inclusion of a thrombin cleavage site between the GST carrier and the viral enzyme facilitated the release of purified mature EMC virus RNA polymerase from the GST carrier by proteolysis with thrombin. The purified recombinant enzyme has a molecular mass of about 52 kDa and is recognized by polyclonal immune serum raised against a peptide sequence corresponding to the C-terminal region of the protein. The recombinant enzyme comigrates with immunoprecipitated EMC virus RNA polymerase from infected mouse L929 cell extracts when run in parallel lanes on a sodium dodecyl sulfate-polyacrylamide gel. The enzyme exhibits rifampin-resistant, poly(A)-dependent poly(U) polymerase activity and RNA polymerase activity, which are both oligo(U) dependent. Template-size products are synthesized in in vitro reactions with EMC virus genomic RNA or globin mRNA. The availability of recombinant EMC virus RNA polymerase in a purified form will allow biochemical analysis of its role in the replication of the virus as well as structure-function studies of this unique class of enzyme.     

The use of recombinant fusion proteases in the affinity purification of recombinant proteins

In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient, and efficient method of affinity purification that can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant "fusion protease" cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step, which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.