N-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines

Summary The capacity of two Trichoplusia ni (TN-368 and BTI-Tn-5bl-4) and a Spodoptera frugiperda (IPLB-SF-21A) cell lines to glycosylate recombinant, baculovirus-encoded, secreted, placental alkaline phosphatase was compared. The alkaline phosphatase from serum-containing, cell culture medium was purified by phosphate affinity column chromatography. The N-linked oligosaccharides were released from the purified protein with PNGase F and analyzed by fluorophore-assisted carbohydrate electrophoresis. The majority of oligosaccharide structures produced by the three cell lines contained two or three mannose residues, with and without core fucosylation, but there were structures containing up to seven mannose residues. The oligosaccharides that were qualitatively or quantitatively different between the cell lines were sequenced with glycosidase digestions. The S. frugiperda cells produced more fucosylated oligosaccharides than either of the T. ni cell lines. The smallest oligosaccharide produced by S. frugiperda cells was branched trimannose. In contrast, both T. ni cell lines produced predominantly dimannose and linear trimannose structures devoid of α 1–3-linked mannose.     

Comparative recombinant protein production of eight insect cell lines

Summary A recombinantAutographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived fromSpodoptera frugiperda, Trichoplusia ni, Mamestra brassicae andEstigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, theT. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than theS. frugiperda-derived Sf9 or Sf21 cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.     

Modelling the growth and protein production by insect cells following infection by a recombinant baculovirus in suspension culture

A mathematical model has been developed to describe the growth and infection of insect cells by recombinant baculoviruses. The model parameters were determined from a series of independent experiments involving batch suspension culture. The profiles generated by the model for cell growth, virus production and protein production agree with those observed in experiments. Presently, the model simulates only systems where cells are not growth-limited. The model is useful in aiding the design and optimization of large-scale systems for production of biological insecticides as well as recombinant proteins and in delineating those areas which are limiting the process and require further, more fundamental, investigation.     

Comparison of oligosaccharide processing among various insect cell lines expressing a secreted glycoprotein

Summary The processing of the N-linked oligosaccharide modifying a secreted alkaline phosphatase glycoprotein (SEAP) expressed with a recombinantAutographa californica nuclear polyhedrosis virus was evaluated in insect cell lines established fromSpodoptera frugiperda, Trichoplusia ni, andMamestra brassicae. Studies with Endoglycosidase H (Endo H), which removes high-mannose oligosaccharides, revealed that 79% of the intracellular SEAP produced in theM. brassicae-derived MB0503 cell line was Endo H resistant. The commonly usedS. frugiperda Sf21 and Sf9 cell lines produced 44 and 21% Endo H-resistant intracellular SEAP, respectively. Detection of oligosaccharide moieties with lectins, which selectively recognize terminal sugars, identified only mannose residues on SEAP expressed in the six insect cell lines. However, the oligosaccharide moiety of SEAP expressed in a Chinese hamster ovary cell line contained sialic acid. Therefore, when expressed in mammalian cells, the oligosaccharide present on SEAP is processed into complex oligosaccharide, but in insect cells it is of the high-mannose type. Studies with inhibitors of the initial oligosaccharide processing steps demonstrated that all six cell lines possessed glycosidase I/II and mannosidase I activity and that glycosylation was required for secretion.     

The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell-baculovirus expression system.

The effect of dissolved oxygen concentration on human secreted alkaline phosphatase (SEAP) glycosylation by the insect cell-baculovirus expression system was investigated in a well-controlled bioreactor. Oligomannose-type N-linked glycans (i.e., Man2 to Man6 and Man3F) were present in SEAP produced by Spodoptera frusiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines. The relative amounts of the most highly processed glycans (i.e., Man3F and Man2 in the SEAP from Sf-9 and Tn-5B1-4 cells, respectively) were significantly higher at 50% of air saturation than at either 10% or 190% of air saturation. That is, glycan processing was inhibited at both low and high dissolved oxygen concentrations.     

Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: Large scale production of the E1 protein

The two envelope glycoproteins of rubella virus (RV), El of 58 kDa and E2 of 42–47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10−1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.     

The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.     

Production of recombinant endostatin from stably transformed Trichoplusia ni BTI Tn 5B1-4 cells

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Trichoplusia ni BTI Tn 5B1-4 (Tn 5B1-4) cells. Recombinant endostatin expressed in the stably transformed Tn 5B1-4 cells was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED₅₀) for recombinant endostatin was approximately 0.35 g ml^–1. In a T-flask, the stably transformed Tn 5B1-4 cells produced 14.3 mg recombinant endostatin l^–1 at 6 days of cultivation.     

Catalase effect on cell death for the improvement of recombinant protein production in baculovirus-insect cell system

Baculovirus expression vector system (BEVS) in host insect cells is a powerful technology to produce recombinant proteins, as well as virus-like particles (VLP). However, BEVS is based on baculovirus infection, which limits the recombinant protein production by inducing insect cell death. Herein a new strategy to enhance cell life span and to increase recombinant protein production was developed. As baculovirus infection induces cellular oxidative stress, the ability of several antioxidants to inhibit cell death was tested during infection. The production of rotavirus structural proteins was used as model to analyse this new strategy. We found that only catalase is able to partially prevent cell death triggered by baculovirus infection and to inhibit lipid peroxidation. An increase in recombinant protein production was coupled with the partial cell death inhibition. In summary, the addition of catalase is a promising strategy to improve recombinant protein production in BEVS, by delaying insect cell death.     

A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells

The use of Seleno‐methionine (SeMet) incorporated protein crystals for single or multi‐wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X‐ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet‐incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60–90% compared with native protein expression.     

Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells

The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing -galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C₁₂FDG) — was used to monitor -galactosidase production. Specific -galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units -galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.Abbreviations ONPG ortho-phenyl 2--D-galactopyranoside - OUR oxygen uptake rate (-mol O₂/liter/hour) - q_glucose specific glucose uptake rate (mg glucose/10⁶cell/hour) - q_glutamine specific glutamine uptake rate (mg glutamine/10⁶cell/hour) - qO₂ specific oxygen uptake rate (-mol O₂/10⁶cell/hour) - MOI virus multiplicity of infection (viral plaque forming units/cell)     

Crystal structure of the cofactor-binding domain of the human phase II drug-metabolism enzyme UDP-glucuronosyltransferase 2B7

Human UDP-glucuronosyltransferases (UGT) are the dominant phase II conjugative drug metabolism enzymes that also play a central role in processing a range of endobiotic compounds. UGTs catalyze the covalent addition of glucuronic acid sugar moieties to a host of therapeutics and environmental toxins, as well as to a variety of endogenous steroids and other signaling molecules. We report the 1.8-A resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT isoform 2B7 (UGT2B7), which catalyzes the conjugative elimination of opioid, antiviral, and anticancer drugs. This is the first crystal structure of any region of a mammalian UGT drug metabolism enzyme. Designated UGT2B7 mutants at residues predicted to interact with the UDP-glucuronic acid cofactor exhibited significantly impaired catalytic activity, with maximum effects observed for amino acids closest to the glucuronic acid sugar transferred to the acceptor molecule. Homology modeling of UGT2B7 with related plant flavonoid glucosyltransferases indicates human UGTs share a common catalytic mechanism. Point mutations at predicted catalytic residues in UGT2B7 abrogated activity, strongly suggesting human UGTs also utilize a serine hydrolase-like catalytic mechanism to facilitate glucuronic acid transfer.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the α-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.