Recombinant protein production in insect cell cultures infected with a temperature-sensitive baculovirus

Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Grace's). In temperature-shift experiments (cell growth at 33°C followed by virus replication at 27°C 3–4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27°C for both cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant temperature. It appeared that infected cells were severely damaged at 33°C (6°C above the optimal 27°C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (141 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27°C) to produce recombinant protein (β-galactosidase). A cell density as high as 1×10⁷ cells.ml⁻¹, and a β-gal concentration of up to 104,000 unit.ml⁻¹ were achieved.     

Kinetics of protein release from yeast using enzymatic lysis for selective product recovery

The kinetics of release of four intracellular enzymes from different yeast cell locations using the Differential Product Release (DPR) method has been investigated. The method uses a combination of physical, chemical and biological agents such as lytic enzymes, an osmotic support and a spheroplast stabilizer. Using the DPR technique a wall enzyme, invertase, was released with a very high specific activity in the first step from a breadmaking strain ofS. cerevisiae. Maximum release could be obtained in this step when the incubation time was extended from 60 min to 100 min. Two cytosol enzymes, α-D-glucosidase and alcohol dehydrogenase were released in the second step. Fumarase was released in the third step almost instantaneously after disruption of the mitochondria which reduces considerably, by ca. 1 hour, the total incubation time of DPR. This paper investigates the kinetics of enzyme release during the 3 steps of DPR.     

Alignment of protein sequences using secondary structure: a modified dynamic programming method

A method for comparison of protein sequences based on their primary and secondary structure is described. Protein sequences are annotated with predicted secondary structures (using a modified Chou and Fasman method). Two lettered code sequences are generated (Xx, where X is the amino acid and x is its annotated secondary structure). Sequences are compared with a dynamic programming method (STRALIGN) that includes a similarity matrix for both the amino acids and secondary structures. The similarity value for each paired two-lettered code is a linear combination of similarity values for the paired amino acids and their annotated secondary structures. The method has been applied to eight globin proteins (28 pairs) for which the X-ray structure is known. For protein pairs with high primary sequence similarity (greater than 45%), STRALIGN alignment is identical to that obtained by a dynamic programming method using only primary sequence information. However, alignment of protein pairs with lower primary sequence similarity improves significantly with the addition of secondary structure annotation. Alignment of the pair with the least primary sequence similarity of 16% was improved from 0 to 37% 'correct' alignment using this method. In addition, STRALIGN was successfully applied to seven pairs of distantly related cytochrome c proteins, and three pairs of distantly related picornavirus proteins.     

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture

An insecticidal protein gene from Bacillus thuringiensis var. aizawal was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteloytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resist-ant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CFI ceils but not dipteran (Aedes albopictus) calls. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawal toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran ceil membranes, which may be the receptor for this toxin.     

Evaluation of Escherichia coli cell disruption and inclusion body release using nucleic acid binding fluorochromes and flow cytometry

Many types of commercially valuable recombinant proteins produced by fermentation are expressed at high levels in Escherichia coli. Often, high-level expression in the host results in the formation of insoluble inclusion bodies. The release of these intracellular inclusion bodies from E. coli following cell disruption is a requirement for further downstream recovery. The ability to discern between intact unruptured cells and granules released from broken cells can provide valuable information for improving recovery yields in downstream purification. This paper describes a rapid and sensitive cytometry-based method that allows the simultaneous measurement of intact heat-killed E. coli and inclusion bodies using staining with nucleic acid binding fluorochromes.     

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture

An insecticidal protein gene from Bacillus thuringiensis var. aizawai was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteolytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resistant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CF1 cells but not dipteran (Aedes albopictus) cells. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawai toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran cell membranes, which may be the receptor for this toxin.     

Microbial cell disruption methods for efficient release of enzyme L-asparaginase

Abstract

The efficacy of a simple laboratory method for cell disruption based on the glass bead stirring, sonication, osmotic shock, freezing and grinding, or use of solvents and detergents was assessed in this study, via measurements of the release of total protein and L-asparaginase activity. Three different microbial sources of L-asparaginase were used: Escherichia coli BL21 (DE3), Leucosporidium muscorum, and Aspergillus terreus (CCT 7693). This study adjusted and identified the best procedure for each kind of microorganism. Sonication and glass bead stirring led to obtaining filamentous fungus cell-free extracts containing high concentrations of soluble proteins and specific activity; however, sonication was the best since it obtained 4.61?±?0.12?IU?mg^?1 after 3?min of operation time. Mechanical methods were also the most effective for yeast cell disruption, but sonication was the technique which yielded a higher efficiency releasing 7.3 IUtotal compared to glass bead stirring releasing 2.7 IUtotal at the same operation time. For bacterium, sonication proved to be the best procedure due to getting the highest specific activity (9.01?IU?mg^?1) and total enzyme activity (61.7?IU). The data presented lead to conclude that the mechanical methods appeared to be the most effective for the disintegration of the all microbial cells studies. This is the first report related to the experimental comparison of L-ASNase extraction procedures from different microorganisms, which can also be used for extracting periplasm located enzymes from other organisms.     

An alternative Candida spp. cell wall disruption method using a basic sorbitol lysis buffer and glass beads

This report describes a modified, cost-effective method of cell wall disruption for the yeast Candida spp., which employs the use of glass beads in a simple sorbitol lysis buffer. This method can be used in conjunction with a commercial RNA or genomic DNA isolation method to obtain high-quality RNA or DNA.     

An alternative Candida spp. cell wall disruption method using a basic sorbitol lysis buffer and glass beads

This report describes a modified, cost-effective method of cell wall disruption for the yeast Candida spp., which employs the use of glass beads in a simple sorbitol lysis buffer. This method can be used in conjunction with a commercial RNA or genomic DNA isolation method to obtain high-quality RNA or DNA.     

Expression of epoxide hydrolase in insect cells: a focus on the infected cell

Insect cell culture and the baculovirus vector expression system have emerged to be a promising production technique for heterologous proteins. In this article, expression characteristics for membrane-bound epoxide hydrolase are examined. A generic process is presented whereby cells are grown in serum-free media supplemented with serum and then resuspended in serum-free media to simplify purification after infection. The infected cells retain significant metabolic activity during the postinfection stage. Thus, maintaining nutrient supply during the postinfection period is critical, and a low stirring rate will result in oxygen depletion and shift the metabolism of the infected cells toward lactate production which then lowers product yield. This is the first report indicating that glucose is supplied from sucrose decomposition and then metabolized for viral DNA and recombinant protein production in recombinant baculovirus insect expression system. (c) 1993 John Wiley & Sons, Inc.     

A modified method for determining protein binding capacity of plant polyphenolics using radiolabelled protein

A modified radiochemical protein binding method for determining the protein binding capacity of plant polyphenolics (tannins) is described. Purified tannin or unfractionated plant extracts were immobilised on filter paper discs and incubated with the 125I-labelled bovine serum albumin. Protein bound to the disc was proportional to the amount of tannin applied to the disc, although at high concentrations of polyphenolics the discs became saturated and the relationship was no longer applicable. The method was validated using purified procyanidin from Sorghum grain and has been applied to crude polyphenolic extracts from maple, white oak, black oak, walnut and tulip poplar leaves. Specific chemical assays for the determination of proanthocyanidins (acid butanol method) and hydrolysable tannins (modified potassium iodate method) were employed to validate the new protein binding method with the complex plant extracts.     

Preparation of N-acylated proteins modified with fatty acids having a specific chain length using an insect cell-free protein synthesis system

To establish a strategy to generate N-acylated proteins modified with fatty acids having a specific chain length, tGelsolin-streptag, an epitope-tagged model protein having an N-myristoylation motif, was synthesized using an insect cell-free protein synthesis system in the presence of acyl-CoA with various fatty acid chain lengths. It was found that the fatty acid species attached to the N-termini fully depended on the acyl-CoA species added to the reaction mixture. N-Acylated proteins with fatty acid chain lengths of 8, 10, 12, and 14 were generated successfully.     

A simple method for bakers' yeast cell disruption using a three-phase fluidized bed equipped with an agitator

A three-phase fluidized bed equipped with a turbine agitator was utilized as a simple device for disrupting bakers' yeast cells (Saccharomyces cerevisiae). The degree of yeast cell disruption was evaluated based on the number of broken cells and its validity was confirmed by the total amount of crude soluble proteins released and by microscopic observation. It was found that the equipment could yield 90% of yeast cell disruption. With the presence of glass beads, the degree of cell disruption became higher as agitating speed is increased. The disruption enhancement would be attributed to the grinding effect resulting from the interaction between yeast cells and glass beads. One-thousand micrometers of glass beads yielded a higher degree of disruption than larger ones. An increase in liquid flow rate hindered the degree of disruption because of shorter contact time although the shear rates in the yeast suspension would become more rigorous.     

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.     

Determination of the cell surface hydrophobicity of oral bacteria using a modified hydrocarbon adherence method

Abstract Hydrophobic interactions between bacterial cell surfaces and colonisable substrates have been implicated in the mechanisms of bacterial adherence. However, current methods of assessing bacterial hydrophobicity as a function of adherence to liquid hydrocarbons (especially hexadecane) do not always produce accurate or reproducible results. Therefore, the present technique was developed using xylene. The hydrophobic surface properties of fresh and type strains of Bacteriodes gingivalis, Bacteriodes intermedius, Capnocytophaga spp., Streptococcus salivarius and Streptococcus sanguis suspended either in saliva ions buffer (SIB) or in saliva diluted in SIB were measured. In SIB the test strains were predominantly hydrophobic. The addition of saliva caused a significant reduction ( P < 0.05) in hydrophobicity compared to SIB alone, with 80% of the strains tested. Since oral bacteria will be suspended in saliva in vivo, it is concluded that bacteria in the oral cavity may be less hydrophobic than previous studies have suggested.     

Transfection of insect cell lines using polyethylenimine

Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85–90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.

Neural repair in an insect: cell recruitment and deployment following selective glial disruption

Summary In Periplaneta americana, SEM of abdominal nervous connectives revealed a rapid accumulation of haemocytes on the surface of the neural lamella within 24 h of selective disruption of the underlying neuroglia by ethidium bromide. After 4 days the neural lamella was effectively clear of adhering haemocytes, but showed characteristic blisters, which, it is postulated, represented the points of entry of the cells from the haemocoel into the underlying tissues. A notable subsequent feature was a substantial increase in the number of cells within repairing connectives. Initially, there was a marked asymmetry in their distribution, with significantly higher numbers of cells anterior to, and within, the lesion area. It seems likely that this polarity resulted from differential cell division within the connectives. The initial asymmetry disappeared after seven days. However, increased perineurial cell numbers were maintained in the lesion area after one month and were still apparent two months after selective glial disruption. There was no equivalent increase in cell numbers in the lesion zone of cultured cords or, in vivo, after injection of the DNA-scission drug, bleomycin, treatments which preclude haemocyte involvement. It is suggested that in the absence of haemocytes and with suppression of proliferation by endogenous cells, repair is achieved by redeployment or growth of adjacent, undamaged glia.     

On-line monitoring of respiration in recombinant-baculovirus infected and uninfected insect cell bioreactor cultures

Respiration rates in Spodoptera frugiperda (Sf-9) cell bioreactor cultures were successfully measured on-line using two methods: The O(2) uptake rate (OUR) was determined using gas phase pO(2) values imposed by a dissolved oxygen controller and the CO(2) evolution rate (CER) was measured using an infrared detector. The measurement methods were accurate, reliable, and relatively inexpensive. The CER was routinely determined in bioreactor cultures used for the production of several recombinant proteins. Simple linear relationships between viable cell densities and both OUR and CER in exponentially growing cultures were used to predict viable cell density. Respiration measurements were also used to follow the progress of baculoviral infections in Sf-9 cultures. Infection led to increases in volumetric and per-cell respiration rates. The relationships between respiration and several other culture parameters, including viable cell density, cell protein, cell volume, glucose consumption, lactate production, viral titer, and recombinant beta-galactosidase accumulation, were examined. The extent of the increase in CER following infection and the time postinfection at which maximum CER was attained were negatively correlated with the multiplicity of infection (MOI) at multiplicities below the level required to infect all the cells in a culture. Delays in the respiration peak related to the MOI employed were correlated with delays in the peak in recombinant protein accumulation. DO levels in the range 5-100% did not exert any major effects on viable cell densities, CER, or product titer in cultures infected with a baculovirus expressing recombinant beta-galactosidase. (c) 1996 John Wiley & Sons, Inc.