High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells.

Much is known about the physical properties of the Cu,Zn- and Mn-superoxide dismutases (SODs). However, the biochemical characteristics and pharmacological properties of extracellular (EC)-SOD have been severely limited due to difficulties in obtaining and purifying the enzyme. The EC-SOD cDNA was inserted into the Escherichia coli expression plasmid pET-28a(+) which contains the T7 promoter and transformed into the E. coli BL21(DE3). After induction with 1 mmol/L isopropyl beta-D-thiogalactoside, the recombinant human EC-SOD was highly expressed as inclusion bodies. SDS-PAGE analysis revealed that recombinant EC-SOD accumulated up to 26% of the total soluble protein of E. coli cells. The expression product was purified by a Ni(2+)-IDA-Sepharose 6B column. After the denaturing and refolding processes, the recombinant human EC-SOD retains the specific enzymatic activity of 920 U/mg of the purified product. The gene encoding human EC-SOD mature peptide was also inserted into the donor plasmid pFastBacHTb. After transposition, transfection, and amplification were performed, the recombinant baculoviruses infected the Tn-5B1-4 cells and EC-SOD was highly expressed in Tn-5B1-4 cells. SDS-PAGE and Western blot analysis revealed that the subunit molecular weight of the expression product is 28 kDa. Furthermore, recombinant human EC-SOD retains the enzymatic specific activity of 200 U/mg of the Tn-5B1-4 cell lysates.     

High-level expression of functional recombinant human coagulation factor VII in insect cells

Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human γ-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future.     

High-level expression of human glycosyltransferases in insect cells as biochemically active form

cDNAs, encoding human beta1,4-galactosyltransferase (hGalT I, EC 2.4.1.22), human Galbeta1,3(4)-GlcNAc alpha2,3-sialyltransferase (hST3GalIII, EC 2.4.99), and human Galbeta1,4-GlcNAc alpha2,6-sialyltransferase (hST6Gal I, EC 2.4.99.1), were cloned from human cell lines. In order to express these glycosyltransferases as secreted form in insect cells, cDNAs were inserted into a novel baculovirus transfer vector equipped with the mouse IgM signal peptide and IgG binding domain of the Staphylococcus aureus protein A as an N-terminal fusion partner. About 14 mg hGalT I, 8 mg hST3GalIII, and 6.4 mg hST6Gal I were purified from 1 liter of recombinant baculovirus infected insect cell culture media. The specific activities of recombinant hGalT I and hST6Gal I were determined as 0.65 and 1.6 U/mg protein, respectively. These results indicated that the recombinant hGalT I and hST6Gal I retained enzyme activities at similar level to those of the authentic one although they were fused with the IgG binding domain at the N-terminus. Taken together, the mouse IgM signal peptide and IgG binding domain of the protein A could be efficiently used as an N-terminus fusion partner for the over-expression of heterologous proteins in insect cells.     

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.     

High-level expression and purification of human xylosyltransferase I in High Five insect cells as biochemically active form

Human xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to consensus serine residues of proteoglycan core proteins. Expression of a soluble form of recombinant histidine-tagged XT-I (rXT-I-HIS) was accomplished at a high level with High Five/pCG255-1 insect cells in suspension culture. The recombinant protein was purified to homogeneity by a combination of heparin affinity chromatography and metal (Ni(2+)) chelate affinity chromatography. Using the modern technique of perfusion chromatography, a rapid procedure for purification of the rXT-I-HIS from insect cell culture supernatant was developed. The purified, biologically active enzyme was homogeneous on SDS-PAGE, was detected with anti-XT-I-antibodies, and had the expected tryptic fragment mass spectrum. N-terminal amino acid sequencing demonstrated that the N-terminal signal sequence of the expressed protein was quantitatively cleaved. The total yield of the enzyme after purification was 18% and resulted in a specific XT-I activity of 7.9mU/mg. The K(m) of the enzyme for recombinant [Val(36),Val(38)](delta1),[Gly(92),Ile(94)](delta2)bikunin was 0.8microM. About 5mg purified enzyme could be obtained from 1L cell culture supernatant. The availability of substantial quantities of active, homogeneous enzyme will be of help in future biochemical and biophysical characterization of XT-I and for the development of a immunological XT-I assay.     

High-level expression and production of recombinant human interleukin-6 analogs.

We have constructed and analyzed different mutant forms of interleukin-6 (IL-6) expressed in Escherichia coli that can be divided into two groups. The first group contains four full-length IL-6 molecules that differ in the presence of cysteine residues involved in disulfide bridges. The second group contains 22 N-terminal amino acid deletions in addition to the differences in the cysteine residues. The different IL-6 muteins were extracted and their expression levels and solubility were compared. We found that the production levels of IL-6 can be dramatically improved by deleting the first 22 N-terminal amino acids of the molecule. We have also found that the production of IL-6 containing the four cysteine residues is lower than the production of the mutant molecules that lack one or both pairs of cysteines. The yield of soluble and properly refolded IL-6 was the highest when the disulfide bond between the cysteines at positions 74 and 84 was present in the mutein form, which also lacked the 22 N-terminal amino acids.     

High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli.

The collagen prolyl 4-hydroxylases (C-P4Hs), enzymes residing within the lumen of the endoplasmic reticulum, play a central role in the synthesis of all collagens. The vertebrate enzymes are alpha(2)beta(2) tetramers in which the two catalytic sites are located in the alpha subunits, and protein disulfide isomerase serves as the beta subunit. All attempts to assemble an active C-P4H tetramer from its subunits in in vitro cell-free systems have been unsuccessful, but assembly of a recombinant enzyme has been reported in several cell types by coexpression of the two types of subunit. An active type I C-P4H tetramer was obtained here by periplasmic expression in Escherichia coli strains BL21 and RB791. Further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human type I C-P4H tetramer. The specific activity of the C-P4H tetramer purified from the cytoplasmic expression was within the range of values reported for human type I C-P4H isolated as a nonrecombinant enzyme or produced in the endoplasmic reticulum of insect cells, but the expression level, about 25 mg/l in a fermenter, is about 5-10 times that obtained in insect cells. The enzyme expressed in E. coli differed from those present in vivo and those produced in other hosts in that it lacked the N glycosylation of its alpha subunits, which may be advantageous in crystallization experiments.     

Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.

The previous and following articles in this issue describe the recombinant synthesis of three mutant beta-globins (beta 1 Val----Ala, beta 1 Val----Met, and the addition mutation beta 1 + Met), their assembly with heme and natural alpha chains into alpha 2 beta 2 tetramers, and their X-ray crystallographic structures. Here we have measured the equilibrium and kinetic allosteric properties of these hemoglobins. Our objective has been to evaluate their utility as surrogates of normal hemoglobin from which further mutants can be made for structure-function studies. The thermodynamic linkages between cooperative oxygenation and dimer-tetramer assembly were determined from global regression analysis of multiple oxygenation isotherms measured over a range of hemoglobin concentration. Oxygen binding to the tetramers was found to be highly cooperative (maximum Hill slopes from 3.1 to 3.2), and similar patterns of O2-linked subunit assembly free energies indicated a common mode of cooperative switching at the alpha 1 beta 2 interface. The dimers were found to exhibit the same noncooperative O2 equilibrium binding properties as normal hemoglobin. The most obvious difference in oxygen equilibria between the mutant recombinant and normal hemoglobins was a slightly lowered O2 affinity. The kinetics of CO binding and O2 dissociation were measured by stopped-flow and flash photolysis techniques. Parallel studies were carried out with the mutant and normal hemoglobins in the presence and absence of organic phosphates to assess their allosteric response to phosphates. In the absence of organic phosphates, the CO-binding and O2 dissociation kinetic properties of the mutant dimers and tetramers were found to be nearly identical to those of normal hemoglobin. However, the effects of organic phosphates on CO-binding kinetic properties of the mutants were not uniform: the beta 1 + Met mutant was found to deviate somewhat from normalcy, while the beta 1 Val----Met mutant reproduced the native allosteric response. Further characterization of the allosteric properties of the beta 1 Val----Met mutant was made by measuring the pH dependence of its overall oxygen affinity by tonometry. Regulation of oxygen affinity by protons was found to be nearly identical to normal hemoglobin from pH 5.8 to 9.3 (0.52 +/- 0.07 protons released per oxygen bound at pH 7.4). The present study demonstrates that the equilibrium and kinetic functional properties of the recombinant beta 1 Val----Met mutant mimic reasonably well those of normal hemoglobin. We conclude that this mutant is well-suited to serve as a surrogate system of normal hemoglobin in the production of mutants for structure-function studies.     

High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells

A scalable transfection procedure using polyethylenimine (PEI) is described for the human embryonic kidney 293 cell line grown in suspension. Green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) were used as reporter genes to monitor transfection efficiency and productivity. Up to 75% of GFP-positive cells were obtained using linear or branched 25 kDa PEI. The 293 cell line and two genetic variants, either expressing the SV40 large T-antigen (293T) or the Epstein–Barr virus (EBV) EBNA1 protein (293E), were tested for protein expression. The highest expression level was obtained with 293E cells using the EBV oriP-containing plasmid pCEP4. We designed the pTT vector, an oriP-based vector having an improved cytomegalovirus expression cassette. Using this vector, 10- and 3-fold increases in SEAP expression was obtained in 293E cells compared with pcDNA3.1 and pCEP4 vectors, respectively. The presence of serum had a positive effect on gene transfer and expression. Transfection of suspension-growing cells was more efficient with linear PEI and was not affected by the presence of medium conditioned for 24 h. Using the pTT vector, >20 mg/l of purified His-tagged SEAP was recovered from a 3.5 l bioreactor. Intracellular proteins were also produced at levels as high as 50 mg/l, representing up to 20% of total cell proteins.     

High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector

An expression cassette for continuous high-level expression of secreted glycoproteins by transformed lepidopteran insect cells has been developed as an alternative to baculovirus and mammalian cell expression systems. The expression cassette utilizes the promoter of the silkmoth cytoplasmic actin gene to drive expression from foreign gene sequences, and also contains the ie-1 transactivator gene and the HR3 enhancer region of BmNPV to stimulate gene expression. Using an antibiotic-resistance selection scheme, we have cloned a Bm5 (silkmoth) cell line overexpressing the secreted glycoprotein juvenile hormone esterase (JHE-KK) at levels of 190 mg/L in batch suspension cultures. A baculovirus (AcNPV) expressing the same gene under the control of the p10 promoter of AcNPV produced only 4 mg/L active JHE in static cultures of infected Sf21 cells. A cloned Bm5 cell line overexpressing a soluble isoform of the alpha-subunit of the granulocyte-macrophage colony stimulating factor receptor (solGMRalpha) was also generated and produced five times more solGMRalpha in static cultures than a cloned BHK cell line obtained by transformation with a recombinant expression cassette utilizing the human cytomegalovirus (CMV) enhancer-promoter system. Finally, we show that recombinant protein expression levels in transformed Bm5 cells remain high in serum-free media, that expression is stable even in the absence of antibiotic selection, and that lepidopteran cells other than Bm5 may be used equally efficiently with this new expression cassette for producing recombinant proteins.     

High-level production and isolation of human recombinant alpha 1-proteinase inhibitor in yeast

The cDNA coding for mature human alpha 1-proteinase inhibitor (alpha 1-PI) has been inserted into a variety of yeast expression vectors. Yeast cells transformed with these plasmids were then assayed for the production of mature, unglycosylated alpha 1-PI. The production level is optimal when the recombinant plasmid carries the TDH promoter, the complete 2mu and the leu2D selection marker. Biologically active recombinant alpha 1-PI can be purified either analytically, by affinity chromatography using a monoclonal antibody, or on a large scale, by a procedure involving precipitation of high-Mr yeast material with polyethylene glycol 3300 followed by successive chromatography on DEAE-agarose, Zn-chelate agarose, kappa-chain agarose, heparin-agarose and aminohexyl-agarose.     

Mammalian glycosyltransferase expression allows sialoglycoprotein production by baculovirus-infected insect cells.

The baculovirus-insect cell expression system is widely used to produce recombinant mammalian glycoproteins, but the glycosylated end products are rarely authentic. This is because insect cells are typically unable to produce glycoprotein glycans containing terminal sialic acid residues. In this study, we examined the influence of two mammalian glycosyltransferases on N-glycoprotein sialylation by the baculovirus-insect cell system. This was accomplished by using a novel baculovirus vector designed to express a mammalian alpha2,6-sialyltransferase early in infection and a new insect cell line stably transformed to constitutively express a mammalian beta1,4-galactosyltransferase. Various biochemical assays showed that a foreign glycoprotein was sialylated by this virus-host combination, but not by a control virus-host combination, which lacked the mammalian glycosyltransferase genes. Thus, this study demonstrates that the baculovirus-insect cell expression system can be metabolically engineered for N-glycoprotein sialylation by the addition of two mammalian glycosyltransferase genes.     

High-level production of human leptin by fed-batch cultivation of recombinant Escherichia coli and its purification.

Human leptin is a 16-kDa (146-amino-acid) protein that is secreted from adipocytes and influences body weight homeostasis. In order to obtain high-level production of leptin, the human obese gene coding for leptin was expressed in Escherichia coli BL21(DE3) under the strong inducible T7 promoter. The recombinant leptin was produced as inclusion bodies in E. coli, and the recombinant leptin content was as high as 54% of the total protein content. For production of recombinant human leptin in large amounts, pH-stat fed-batch cultures were grown. Expression of leptin was induced at three different cell optical densities at 600 nm (OD600), 30, 90, and 140. When cells were induced at an OD600 of 90, the amount of leptin produced was 9.7 g/liter (37% of the total protein). After simple purification steps consisting of inclusion body isolation, denaturation and refolding, and anion-exchange chromatography, 144.9 mg of leptin that was more than 90% pure was obtained from a 50-ml culture, and the recovery yield was 41.1%.     

High-level expression of human interferon alpha-2b in transgenic carrot (Daucus carota L.) plants

In this study, we report the obtaining of carrot plants expressing human interferon alpha-2b via Agrobacterium-mediated transformation using two vector constructs containing the sequence coding for interferon gene fused with Nicotiana plumbagenifolia calreticulin apoplast targeting signal driven by 35S CaMV promoter and root-specific Mll promoter. The human interferon alpha-2b gene was correctly translated in carrot plants according to Western blot analysis. The recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus replication in established piglet testicular cells. The results demonstrated the higher activity of interferon accumulated in carrot plants for young leaves (up to 50.7 × 10³ IU/g FW) compared to the mature ones probably due to the degradation-susceptible nature of this protein. The taproot-expressing system could have also provided the sufficient protein amounts (up to 16.5 × 10³ IU/g FW) and could possibly be used for generating interferon alpha-2b protein in planta for preventing and curing infectious diseases.     

Optimization of the production of virus-like particles in insect cells

In this work the maximal operational hydrodynamic conditions (agitation and aeration rate) that cause no adverse effect in Sf-9 cells growth in SF900II serum-free medium were determined. Shear stresses higher than 1 N m-2 and aeration rates higher than 0.04 vvm affect cell growth and when these conditions increase to 1.5 N m-2 and 0.11 vvm, cell growth is completely inhibited with significant cell morphology changes and a strong decrease in viability. Although the pO2 did not show a significant effect upon cell growth in the range from 10 to 50%, cell infection and specific productivity were dramatically affected. The production was optimal at a pO2 of 25% with decreases higher than 50% being observed when the pO2 decreased to 10 or increased to 50%. The maximum product quality, i.e., the percentage of product in the form of high molecular weight particles, is not coincident with maximum product titer. Although the highest Pr55gag particle titer was obtained at 96 hours post infection (hpi) and at pO2 of 25%, the best product quality (defined by gel filtration chromatography and Western immunoblot) was obtained at 48 hpi, independently of the pO2 used. The effect of overcritical conditions upon productivity was also studied. As obtained for cell growth, cell infection is affected by shear stresses above 1 N m-2 and by aeration rates higher than 0.04 vvm, with decreases in Pr55gag particle titer higher than 70%, even when the overcritical values are still far from the limit at which cell death occurs. The results obtained and the optimization strategy used allowed the maximization of the oxygen supply without damaging the cells, with important consequences on the scale-up of a production process involving this insect cell/baculovirus expression system.