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  • 1
    Electronic Resource
    Electronic Resource
    The importance of Arg40 and 45 in the mitogenic activity and structural stability of basic fibroblast growth factor: Effects of acidic amino acid substitutions (1995)
    Springer
    The protein journal 14 (1995), S. 263-274 
    Details
    ISSN: 1573-4943
    Keywords: bFGF ; heparin ; sucrose octasulfate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract High-affinity binding of basic fibroblast growth factor (bFGF) to the tyrosine kinase receptor requires cell-surface heparan sulfate proteoglycan or exogenous addition of heparin. The crystal structure of bFGF shows Arg40 and 45 on the surface opposite to the heparin-binding region, suggesting that these charged residues may be involved in the receptor binding. Therefore, these amino acids were mutated to aspartic acid separately or simultaneously, and also a simultaneous mutation to glutamic acid was introduced. These mutants displayed a mitogenic activity decreased greater than tenfold compared to the wild-type protein. Addition of heparin had no effect on the activity, while these mutants showed heparin-binding characteristics resembling those of the native sequence protein. The mutants exhibited decreased stability compared to the native sequence protein. Gradual changes in conformation were observed by circular dichroic and infrared spectroscopy. Heparin chromatography also showed the presence of denatured form for these mutants. However, in the presence of multivalent anions such as citrate, sucrose octasulfate, and heparin, the conformation of the mutants resembled that of the wild-type protein, as revealed by X-ray crystallography and circular dichroism spectra of the mutant with a Arg40 → Asp substitution.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01886783
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    A New Strategy for Enhancing the Stability of Lyophilized Protein: The Effect of the Reconstitution Medium on Keratinocyte Growth Factor (1995)
    Springer
    Pharmaceutical research 12 (1995), S. 1447-1452 
    Details
    ISSN: 1573-904X
    Keywords: proteins ; aggregation ; reconstitution ; lyophilization ; additives ; stability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Protein stabilization during lyophilization has previously focused on optimization of the formulation as well as the freezing and dehydration process parameters. However, the effect of the reconstitution medium has been largely neglected. We have investigated its effect on aggregate formation using recombinant keratinocyte growth factor (KGF). Methods. The protein was lyophilized under suboptimal conditions to induce aggregation and precipitation upon reconstitution with water. A series of additives were examined by UV spectrophotometry and size exclusion chromatography (SEC-HPLC) for their effects on decreasing the degree of KGF aggregation and precipitation by the increase in recovery of soluble monomer. Results. Several additives resulted in a significant reduction of aggregation, including sulfated polysaccharides, surfactants, polyphosphates, and amino acids. A similar effect was achieved by adjusting the ionic strength of the reconstitution medium. SEC-HPLC indicated that the amount of soluble monomer was also increased by these additives suggesting that the recovery of the soluble protein correlates with the native, monomeric protein. Conclusions. These results suggest that optimization of reconstitution conditions will be a useful methodology for increasing the recovery of soluble, active proteins and that for KGF, the recovery of the soluble protein correlates with the native, monomeric form.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1016219000963
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    The Effect of the Reconstitution Medium on Aggregation of Lyophilized Recombinant Interleukin-2 and Ribonuclease A (1996)
    Springer
    Pharmaceutical research 13 (1996), S. 643-646 
    Details
    ISSN: 1573-904X
    Keywords: proteins ; aggregation ; reconstitution ; lyophilization ; additives ; stability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1016074811306
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    Paper (German National Licenses)
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  • 4
    Electronic Resource
    Electronic Resource
    Optimization of Lyophilization Conditions for Recombinant Human Interleukin-2 by Dried-State Conformational Analysis Using Fourier-Transform Infrared Spectroscopy (1995)
    Springer
    Pharmaceutical research 12 (1995), S. 1250-1259 
    Details
    ISSN: 1573-904X
    Keywords: lyophilization ; interleukin-2 ; protein conformation ; infrared spectroscopy ; stability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Examination of the dried-state conformation of interleukin-2 (IL-2) was used to determine the pH conditions and stabilizers that provide optimal storage stability for the lyophilized product. Methods. Fourier-transform infrared spectroscopy and accelerated stability studies which examined solubility, aggregate formation, and covalent cross-linking were used. Results. Varying the pH in the absence of excipients resulted in dramatic differences in the dried state conformation of IL-2. At pH 7, IL-2 unfolds extensively upon lyophilization while at pH below 5 it remains essentially native. Additional unfolding was observed upon incubation at elevated temperatures. A strong direct correlation between the retention of the native (aqueous) structure during freeze-drying and enhanced stability is demonstrated. IL-2 prepared at pH 5 is approximately an order of magnitude more stable than at pH 7 with regard to formation of soluble and insoluble aggregates. A similar pH profile was observed in the presence of excipients, although the excipients alter the overall stability profile. Additional accelerated stability studies examined the stabilizers necessary for optimal stability. Conclusions. Excipients with the capacity to substitute for water upon dehydration better preserve the native structure resulting in enhanced stability. Those that have high glass transition temperatures provide the highest level of stability during storage, although they do not prevent dehydration induced unfolding.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1016296801447
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    Paper (German National Licenses)
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