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  • 1
    Electronic Resource
    Electronic Resource
    Chaperone and foldase coexpression in the baculovirus-insect cell expression system (1996)
    Springer
    Cytotechnology 20 (1996), S. 149-159 
    Details
    ISSN: 1573-0778
    Keywords: aggregation ; BiP ; folding ; protein disulfide isomerase ; protein production ; secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Conclusions The BEVS has become widely utilized for production of recombinant proteins. However, protein aggregation and inefficient processing often limit yields, especially for secreted and membrane proteins. Since many proteins of pharmaceutical interest require similar posttranslational processing steps, engineering the folding, assembly, and secretion pathway may enhance the production of a wide variety of valuable complex proteins. Efforts should be undertaken to coexpress the relevant chaperones or foldases at low levels in concert with the final product to ensure the ideal folding and assembly environment. In the future, expression of oligosaccharide modifying enzymes and secretion factors may further improve secretion rates of assembled proteins and provide heterologous proteins with altered glycoforms. Also significant is the use of BEVS as an in vivo eucaryotic laboratory to study the fundamental roles of differnt chaperones, foldases, and secretion factors. The coexpression of chaperones and foldases will complement other approaches such as the development of alternative insect cell lines, promoters, and signal peptides to optimize the baculovirus-insect cell expression system for generating high yields of valuable proteins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00350396
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  • 2
    Electronic Resource
    Electronic Resource
    II. Electrostatic effect in the aggregation of heat-denatured RNase A and implications for protein additive design (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 281-285 
    Details
    ISSN: 0006-3592
    Keywords: protein aggregation ; RNase A ; protein formulation ; protein additives ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the previous study (part I), heat-denatured RNase A aggregation was shown to depend on the solution pH. Interestingly, at pH 3.0, the protein did not aggregate even when exposed to 75°C for 24 h. In this study, electrostatic repulsion was shown to be responsible for the absence of aggregates at that pH. While RNase A aggregation was prevented at the extremely acidic pH, this is not an environment conducive to maintaining protein function in general. Therefore, attempts were made to confer electrostatic repulsion near neutral pH. In this study, heat-denatured RNase A was mixed with charged polymers at pH 7.8 in an attempt to provide the protein with excess surface cations or anions. At 75°C, SDS and dextran sulfate were successful in preventing RNase A aggregation, whereas their cationic, nonionic, and zwitterionic analogs did not do so. We believe that the SO3- groups present in both additives transformed the protein into polyanionic species, and this may have provided a sufficient level of electrostatic repulsion at pH 7.8 and 75°C to prevent aggregation from proceeding. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:281-285, 1998.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Bcl-2 inhibits apoptosis and extends recombinant protein production in cells infected with Sindbis viral vectors (1996)
    Springer
    Cytotechnology 22 (1996), S. 169-178 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; bcl-2 ; cell culture ; chloramphenicol acetyltransferase ; recombinant protein ; Sindbis virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Viruses carrying foreign genes are often used for the production of recombinant proteins in mammalian cells and other eukaryotic expression systems. Though high levels of gene expression are possible using viral vectors, the host cell generally responds to the infection by inducing apoptotic cell death within several days, abruptly ending protein production. It has recently been demonstrated, however, that apoptosis can be suppressed in virally infected cells using anti-apoptotic genes, such as bcl-2. In this study, stably transfected rat carcinomal cell lines, AT3-bcl2 and AT3-neo, were infected with a Sindbis virus carrying the gene for chloramphenicol acetyltransferase (CAT) in an effort to determine the effect of bcl-2 on cell viability and recombinant protein production. Infected AT3-bcl2 cells consistently maintained viabilities close to 100% and a growth rate equivalent to that of uninfected cells (0.040 h-1). In contrast, the Sindbis viral vector induced apoptosis in the AT3-neo cells, which were all dead by three days post-infection. Though infected AT3-neo cells generated higher levels of heterologous protein, over 1000 mUnits per well, CAT activity fell to zero by two days post-infection. In contrast, chloramphenicol acetyltransferase was present in AT3-bcl2 cells for almost a week, reaching a maximum level of 580 mUnits per well. In addition, recombinant protein production in AT3-bcl2 cells was extended and amplified by the regular addition of virus to the culture medium, a process which resulted in expression for the duration of the cell culture process.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353936
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  • 4
    Electronic Resource
    Electronic Resource
    Modeling assembly, aggregation, and chaperoning of immunoglobulin G production in insect cells (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 106-116 
    Details
    ISSN: 0006-3592
    Keywords: BiP ; immunoglobulin ; aggregation ; mathematical model ; chaperones ; baculovirus-insect cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A model for immunoglobulin G (IgG) production in the baculovirus-insect cell system was developed that incorporates polypeptide synthesis, oligomer assembly, protein aggregation, and protein secretion. In addition, the capacity of a chaperone to protect heavy and light chain polypeptides from protein aggregation was considered by including in vitro chaperone-peptide binding and dissociation kinetic constants from the literature. Model predictions were then compared to experiments in which the chaperone immunoglobulin heavy chain binding protein, BiP, was coexpressed by coinfecting insect cells with BiP-containing baculovirus. The model predicted a nearly twofold increase in intracellular and secreted IgG that was similar to the behavior observed experimentally after approximately 3 days of coexpressing heterologous IgG and BiP. However, immunoglobulin aggregation was still significant in both the model simulation and experiments, so the model was then used to predict the effect of strategies for improving IgG production even further. Increasing expression of the chaperone BiP by 10-fold over current experimental levels provided a 2.5-fold increase in secreted IgG production over IgG assembly without BiP. Alternatively, the expression of BiP earlier in the baculovirus infection cycle achieved a twofold increase in protein secretion without requiring excessive BiP production. The potential effect of cochaperones on BiP activity was considered by varying the BiP binding and release constants. The utilization of lower binding and release kinetic constants led to a severalfold increase in IgG secretion because the polypeptides were protected from aggregation for greater periods. An optimized strategy for chaperone action would include the rapid peptide binding of a BiP-ATP conformation along with the slow peptide release of a BiP-ligand conformation. However, even with an optimized chaperoning system, limitations in the secretion kinetics can result in the accumulation of intracellular IgG. Thus, the entire secretory pathway must be considered when enhanced secretion of heterologous proteins is desired. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 106-116, 1997.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    I. Study of protein aggregation due to heat denaturation: A structural approach using circular dichroism spectroscopy, nuclear magnetic resonance, and static light scattering (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 273-280 
    Details
    ISSN: 0006-3592
    Keywords: heat denaturation ; protein aggregation ; RNase A ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of this study was to investigate the relationship between oxidized RNase A protein structure and the occurrence of protein aggregation using several spectroscopic techniques. Circular dichroism spectroscopy (CD) measurements taken at small temperature intervals were used to determine the protein's melting temperature, Tm, of approximately 65°C in deionized water. A more detailed examination of the protein structure was undertaken at several temperatures around Tm using near- and far-UV CD and one-dimensional nuclear magnetic resonance (NMR) measurements. These measurements revealed the presence of folded structures at 55°C and below, while denatured structures appeared at 65°C and above. Concurrent static light scattering (SLS) measurements, employed to detect the presence of RNase A aggregates, showed that RNase A aggregation was observed at 65°C and above, when much of the protein was denatured. Subsequent NMR time-course data demonstrated that aggregates forming at 75°C and pH 7.8 were indeed derived from heat-denatured protein. However, aggregation was also detected at 55°C when the spectroscopic data suggested the protein was present predominantly in the folded configuration. In contrast, heat denaturation did not lead to RNase A aggregation in a very acidic environment. We attribute this phenomenon to the effect of charge-charge repulsion between the highly protonated RNase A molecules in very acidic pH. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:273-280, 1998.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Overexpression of a cytosolic chaperone to improve solubility and secretion of a recombinant IgG protein in insect cells (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 196-203 
    Details
    ISSN: 0006-3592
    Keywords: baculovirus ; chaperone ; hsp70 ; insect cell ; immunoglobulin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The secretion of heterologous IgG proteins in the baculovirus-insect cell expression system is accompanied by substantial insoluble immunoglobulin in the infected cells. The accumulation of these insoluble forms suggests a limitation in the processing and secretory pathway of the infected cells. As a result, cytosolic hsp70 chaperones, which are known to associate and prevent aggregation of polypeptides in vitro, have been coexpressed in the infected cells. The hsp70 protein coprecipitated with the immunoglobulin to indicate the formation of a specific hsp70-immunoglobulin complex in vivo. Immunoblot and pulse chase studies indicated that coexpression of hsp70 increased intracellular immunoglobulin solubility. Metabolic labeling experiments revealed that hsp70 increased secreted immunoglobulin levels after several days infection as compared to infection with control baculoviruses. Pulse chase studies indicated that hsp70 increases the solubility of immunoglobulin precursors that are then processed and assembled into the complete antibody oligomer. A comparison of the action of cytosolic hsp70 chaperone to the endoplasmic reticulum chaperone BiP suggests sequential action in which hsp70 increases the solubility of preprocessed immunoglobulin, while BiP enhances the solubility of processed immunoglobulin chains. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 196-203, 1998.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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