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  • 1
    Electronic Resource
    Electronic Resource
    Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae (2000)
    Springer
    Applied microbiology and biotechnology 53 (2000), S. 575-582 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract  In order to direct the persistent expression of recombinant human serum albumin (HSA) from the GAL10 promoter in the yeast Saccharomyces cerevisiae, we carried out periodic feeding of galactose during shake-flask cultures. Unexpectedly, the recombinant protein secreted was observed to undergo rapid degradation, which was apparently accelerated by carbon-source feeding. The extracellular degradation of HSA occurred even in the strain deficient in the major vacuolar proteases PrA and PrB, and in the strain lacking the acidic protease Yap3p (involved in the generation of HSA-truncated fragments). Interestingly, the degradation correlated closely with the acidification of extracellular pH and thus was significantly overcome either by buffering the culture medium above pH 5.0 or by adding amino acid-rich supplements to the culture medium, which could prevent the acidification of medium pH during cultivation. Addition of arginine or ammonium salt also substantially minimized the degradation of HSA, even without buffering. The extracellular degradation activity was not detected in the cell-free culture supernatant but was found to be associated with intact cells. The results of the present study strongly suggest that the HSA secreted in S. cerevisiae is highly susceptible to the pH-dependent proteolysis mediated by cell-bound protease(s) whose activity and expression are greatly affected by the composition of the medium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530051659
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  • 2
    Electronic Resource
    Electronic Resource
    Identification of three classes of hydrogenase in the genus, Desulfovibrio
    Amsterdam : Elsevier
    Biochemical and Biophysical Research Communications 149 (1987), S. 369-377 
    Details
    ISSN: 0006-291X
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Chemistry and Pharmacology , Physics
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/0006-291X(87)90376-7
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  • 3
    Electronic Resource
    Electronic Resource
    Simple technique for the simultaneous measurements of the four-probe resistivity and the thermoelectric power (1998)
    [S.l.] : American Institute of Physics (AIP)
    Review of Scientific Instruments 69 (1998), S. 3705-3706 
    Details
    ISSN: 1089-7623
    Source: AIP Digital Archive
    Topics: Physics , Electrical Engineering, Measurement and Control Technology
    Notes: We propose simple configurations to measure the thermoelectric power and the four-probe resistivity simultaneously for different types of samples.© 1998 American Institute of Physics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.1149164
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  • 4
    Electronic Resource
    Electronic Resource
    Thermoelectric power measurement under hydrostatic pressure using a self-clamped pressure cell (2002)
    [S.l.] : American Institute of Physics (AIP)
    Review of Scientific Instruments 73 (2002), S. 2999-3002 
    Details
    ISSN: 1089-7623
    Source: AIP Digital Archive
    Topics: Physics , Electrical Engineering, Measurement and Control Technology
    Notes: A thermoelectric power (TEP) measurement technique in a self-clamped pressure cell is presented. Thermal and electrical contacts were glued to heaters by Stycast epoxy, which enhances thermal integration. The pressure effect of Chromel–Constantan and Chromel–AuFe0.07% thermocouples are compared to Chromel–Alumel thermocouples, which are known to be pressure insensitive between 4.2 and 300 K. The investigated thermocouples are found to have a small pressure effect; ∼±4% at maximum in the measured temperature and pressure range. Any pressure effect on Au wires was also found to be very small from the pressure-dependent TEP measurement of YBCO superconductor below Tc. © 2002 American Institute of Physics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.1489076
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  • 5
    Electronic Resource
    Electronic Resource
    Low-frequency method for magnetothermopower and Nernst effect measurements on single crystal samples at low temperatures and high magnetic fields (2001)
    [S.l.] : American Institute of Physics (AIP)
    Review of Scientific Instruments 72 (2001), S. 2392-2397 
    Details
    ISSN: 1089-7623
    Source: AIP Digital Archive
    Topics: Physics , Electrical Engineering, Measurement and Control Technology
    Notes: We describe an alternating current method for the measurement of the longitudinal (Sxx) and transverse (Sxy, i.e., Nernst) thermopower of millimeter-size crystal samples at low temperatures (T〈1 K) and high magnetic fields (B∼30 T). A low-frequency (33 mHz) heating method is used to increase the resolution and to determine the temperature gradient reliably in high magnetic fields. Samples are mounted between two thermal blocks which are heated by a sinusoidal frequency f0 with a π/2 phase difference. The phase difference between two heater currents gives a temperature gradient at 2f0. The corresponding thermopower and Nernst effect signals are extracted by using a digital signal processing method due to the low frequency of the measurement. An important component of the method involves a superconducting link, YBa2Cu3O7+δ, which is mounted in parallel with sample to remove the background magnetothermopower of the lead wires. The method is demonstrated for the quasi-two-dimensional organic conductor α-(BEDT–TTF)2KHg(SCN)4, which exhibits a complex, magnetic field dependent ground state above 22.5 T at low temperatures. © 2001 American Institute of Physics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.1353192
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  • 6
    Electronic Resource
    Electronic Resource
    Chemical vapor deposition of copper with a new metalorganic source (1996)
    Woodbury, NY : American Institute of Physics (AIP)
    Applied Physics Letters 68 (1996), S. 1017-1019 
    Details
    ISSN: 1077-3118
    Source: AIP Digital Archive
    Topics: Physics
    Notes: Copper deposition based on a newly synthesized metalorganic compound shows promise for reproducible deposition which has been a major problem. The deposition can be carried out without adverse effect on the film qualities at the bubbler temperature of 65 °C. Preliminary deposition results reveal that the resistivity is 2.5 μΩ cm in the deposition temperature range between 175 and 200 °C. Unlike (hfac)Cu⋅VTMS, the resistivity increases with increasing deposition temperature due to loose packing of the grains at higher temperatures. © 1996 American Institute of Physics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.116214
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  • 7
    Electronic Resource
    Electronic Resource
    Efficient production of intact human parathyroid hormone in a Saccharomyces cerevisiae mutant deficient in yeast aspartic protease 3 (YAP3) (1998)
    Springer
    Applied microbiology and biotechnology 50 (1998), S. 187-192 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract When human parathyroid hormone (hPTH) is expressed as a secretory product in yeast, the main problem is the aberrant proteolytic cleavage that reduces the yield of intact protein. To overcome this problem, we developed an hPTH expression system using a host strain in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. After 48 h of culture, most of the hPTH secreted by the yap3 disruptant remained intact, whereas more than 90% of the hPTH secreted by the wild-type strain was cleaved. When the authentic hPTH was incubated in each of the culture supernatants of untransformed yap3 disruptant and wild-type strain, the proteolysis proceeded much more slowly in the culture supernatant of yap3 disruptant than in that of the wild type. The extent of hPTH proteolysis was also significantly reduced by the addition of pepstatin A, a specific aspartic protease inhibitor. The results suggest that YAP3 is involved in the internal cleavage of hPTH expressed in yeast. The correct processing of the intact hPTH secreted in the yap3 disruptant demonstrates that the yeast mutant lacking the YAP3 activity is a suitable host for the high-level expression of intact hPTH.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530051275
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  • 8
    Electronic Resource
    Electronic Resource
    Optimization of the expression system using galactose-inducible promoter for the production of anticoagulant hirudin in Saccharomyces cerevisiae (1994)
    Springer
    Applied microbiology and biotechnology 42 (1994), S. 587-594 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have tried to optimize the galactose-inducible gene expression system for the overproduction of the potent thrombin-specific inhibitor, hirudin, in a genetically engineered yeast, Saccharomyces cerevisiae. The expression and secretion of hirudin were directed by the galactose-inducible promoter, GAL10, and the mating factor α pre-pro leader sequence. The initial hirudin expression level in shake-flask culture was 2.3 mg 1−1. Modification of the expression vector and optimization of culture conditions, including the induction conditions, improved the level of hirudin gene expression and secretion into the culture supernatant more than 20-fold (50 mg 1−1) in a 4-1 scale batch cultivation. The expression and secretion level of hirudin seemed to be partially dependent on cell growth when galactose was used as a carbon source. Overexpression of the transcriptional activator, GAL4, appeared to have only negative effects on the expression of the hirudin gene and lacZ directed by the GAL10 promoter in the strain used in this study, unlike the previously reported examples. The complex medium containing yeast extract used for the increase of the cell mass and hirudin level did not show any detrimental effect on plasmid stability and did not complicate the downstream purification of hirudin from the culture supernatant. Moreover, the complex medium could greatly improve the hirudin productivity and reduce the degradation of hirudin produced in the culture supernatants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00173925
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  • 9
    Electronic Resource
    Electronic Resource
    Optimization of the expression system using galactose-inducible promoter for the production of anticoagulant hirudin in Saccharomyces cerevisiae (1994)
    Springer
    Applied microbiology and biotechnology 42 (1994), S. 587-594 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have tried to optimize the galactose-inducible gene expression system for the overproduction of the potent thrombin-specific inhibitor, hirudin, in a genetically engineered yeast, Saccharomyces cerevisiae. The expression and secretion of hirudin were directed by the galactose-inducible promoter, GAL10, and the mating factor α pre-pro leader sequence. The initial hirudin expression level in shake-flask culture was 2.3 mg l–1. Modification of the expression vector and optimization of culture conditions, including the induction conditions, improved the level of hirudin gene expression and secretion into the culture supernatant more than 20-fold (50 mg l–1) in a 4-l scale batch cultivation. The expression and secretion level of hirudin seemed to be partially dependent on cell growth when galactose was used as a carbon source. Overexpression of the transcriptional activator, GAL4, appeared to have only negative effects on the expression of the hirudin gene and lacZ directed by the GAL10 promoter in the strain used in this study, unlike the previously reported examples. The complex medium containing yeast extract used for the increase of the cell mass and hirudin level did not show any detrimental effect on plasmid stability and did not complicate the downstream purification of hirudin from the culture supernatant. Moreover, the complex medium could greatly improve the hirudin productivity and reduce the degradation of hirudin produced in the culture supernatants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530050298
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  • 10
    Electronic Resource
    Electronic Resource
    A dominant selection system designed for copy-number-controlled gene integration in Hansenula polymorpha DL-1 (1999)
    Springer
    Applied microbiology and biotechnology 51 (1999), S. 800-807 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract To facilitate the selection of multiple gene integrants in Hansenula polymorpha, a rapid and copy-number-controlled selection system was developed using a vector containing a telomeric autonomous replication sequence and the bacterial aminoglycoside 3-phosphotransferase (APH) gene. Direct use of the unmodified APH gene as a dominant selectable marker resulted in the extremely slow growth of transformants and the frequent selection of spontaneous resistance. For the proper performance of the APHgene, a set of deleted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters of H. polymorpha were fused to the APH gene. The fusion construct with the 578-bp GAPDH promoter conferred G418 resistance sufficient to allow rapid growth of transformants, and thus facilitated the selection of transformants with up to 15 tandem copies of the vector. To increase further the integration copy number within the gene-dose-dependent range, the GAPDHpromoter was serially deleted down to the −61 nucleotide. With this weak expression cassette, the integration copy number could easily be controlled between 1 and 50. Tandemly integrated copies of plasmids near the end of the chromosome were mitotically stable over l50 generations. The dosage-dependent selection system of this study would provide a powerful tool for the development of H. polymorpha as an industrial strain to produce recombinant proteins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530051465
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