scholarly journals Secretion of Human Serum Albumin by Kluyveromyces lactis Overexpressing KlPDI1 and KlERO1

2005 ◽  
Vol 71 (8) ◽  
pp. 4359-4363 ◽  
Author(s):  
Tiziana Lodi ◽  
Barbara Neglia ◽  
Claudia Donnini
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Protein Conformation ◽  
Protein Production ◽  
Heterologous Protein ◽  
Kluyveromyces Lactis ◽  
Heterologous Proteins ◽  
Disulfide Bridges ◽  
Genes Encoding

ABSTRACT The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1, an orthologue of Saccharomyces cerevisiae ERO1, was cloned by functional complementation of the ts phenotype of an Scero1 mutant. The expression of KlERO1 was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either PDI1 or ERO1 led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of KlERO1 did not affect the production of human interleukin 1β, a protein that has no disulfide bridges. The results confirm that the ERO1 genes of S. cerevisiae and K. lactis are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of KlERO1 copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of KlERO1 and KlPDI1 is important for the production of high levels of heterologous proteins of this type.

scholarly journals Use of the KlADH4 Promoter for Ethanol-Dependent Production of Recombinant Human Serum Albumin inKluyveromyces lactis

1999 ◽  
Vol 65 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Michele Saliola ◽  
Cristina Mazzoni ◽  
Nicola Solimando ◽  
Alessandra Crisà ◽  
Claudio Falcone ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Kluyveromyces Lactis ◽  
Medium Composition ◽  
Heterologous Proteins ◽  
Batch Cultures ◽  
Alcohol Dehydrogenase Activity ◽  
Recombinant Human Serum Albumin ◽  
Yeast Genes

ABSTRACT KlADH4 is a gene of Kluyveromyces lactisencoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host toSaccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression inK. lactis of the bacterial β-glucuronidase and of the human serum albumin (HSA) genes under the control of theKlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation ofKlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UASE) which is sufficient for the induction ofKlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source.

scholarly journals Induction by Hypoxia of Heterologous-Protein Production with the KlPDC1 Promoter in Yeasts

2006 ◽  
Vol 73 (3) ◽  
pp. 922-929 ◽  
Author(s):  
Andrea Camattari ◽  
Michele M. Bianchi ◽  
Paola Branduardi ◽  
Danilo Porro ◽  
Luca Brambilla
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Gene Expression ◽  
Kluyveromyces Lactis ◽  
Expression System ◽  
Promoter Sequence ◽  
Heterologous Protein Production ◽  
Heterologous Proteins ◽  
Hypoxic Induction ◽  
Production Ratio

ABSTRACT The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1β was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get β-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.

Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability

2016 ◽  
Vol 192 ◽  
pp. 178-187 ◽  
Author(s):  
Xin Peng ◽  
Xiangchao Wang ◽  
Wei Qi ◽  
Rongxin Su ◽  
Zhimin He
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Protein Conformation ◽  
Rosmarinic Acid ◽  
Conformation Stability

Betulinic acid binding to human serum albumin: A study of protein conformation and binding affinity

2009 ◽  
Vol 94 (1) ◽  
pp. 8-12 ◽  
Author(s):  
Rajagopal Subramanyam ◽  
Anilkishor Gollapudi ◽  
Persis Bonigala ◽  
Madhurarekha Chinnaboina ◽  
Damu G. Amooru
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Binding Affinity ◽  
Betulinic Acid ◽  
Protein Conformation

scholarly journals Ligand-binding properties of proalbumin Christchurch

1980 ◽  
Vol 191 (1) ◽  
pp. 281-283 ◽  
Author(s):  
R G Reed ◽  
T Peters ◽  
S O Brennan ◽  
R W Carrell
Keyword(s):  
Fatty Acids ◽  
Human Serum Albumin ◽  
Ligand Binding ◽  
Serum Albumin ◽  
Human Serum ◽  
Protein Conformation ◽  
Binding Sites ◽  
Bromocresol Green ◽  
Binding Properties ◽  
Normal Albumin

Proalbumin Christchurch, a circulating variant of human serum albumin, is secreted from the liver without cleavage of the hexapeptide situated at the N-terminal end of the peptide chain of proalbumin. We compared ligand-binding properties of proalbumin Christchurch and of normal albumin A from the same individual in order to test the effect of the presence of the hexapeptide. The two albumin forms exhibited similar affinities for palmitate, bilirubin, 8-anilinonaphthalene-1-sulphonate and Bromocresol Green. The patterns of endogenous fatty acids bound to the two forms of albumin were slightly different, although the differences were probably not of physiological significance. From these studies it would appear that the propeptide of proalbumin does not alter the protein conformation in such a way as to alter binding sites for organic anions.

scholarly journals Spectroscopic investigations on the binding of an iodinated chlorin p6-copper complex to human serum albumin

2017 ◽  
Vol 16 (12) ◽  
pp. 1762-1770 ◽  
Author(s):  
P. Sarbadhikary ◽  
A. Dube
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Binding Site ◽  
Copper Complex ◽  
Protein Conformation ◽  
High Affinity ◽  
Spectroscopic Investigations ◽  
Chlorin P6

An iodinated chlorin p6 copper complex showed high affinity to bind human serum albumin, the binding site was predicted and it was demonstrated that binding did not affect protein conformation.

Lysine modification of human serum albumin and its effect on protein conformation and nalidixic acid binding

2021 ◽  
Vol 98 (3) ◽  
pp. 100031
Author(s):  
Cheau Yuaan Tan ◽  
Chun Shen Lim ◽  
Siew Mun Liew ◽  
Adyani Azizah Abd Halim ◽  
Saad Tayyab
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Nalidixic Acid ◽  
Protein Conformation ◽  
Lysine Modification

scholarly journals A nucleotide insertion and frameshift cause albumin Kénitra, an extended and O-glycosylated mutant of human serum albumin with two additional disulfide bridges

2001 ◽  
Vol 268 (2) ◽  
pp. 344-352 ◽  
Author(s):  
Lorenzo Minchiotti ◽  
Monica Campagnoli ◽  
Antonio Rossi ◽  
Maria E. Cosulich ◽  
Maria Monti ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Disulfide Bridges ◽  
Nucleotide Insertion

Human serum albumin as protecting agent of silver nanoparticles: role of the protein conformation and amine groups in the nanoparticle stabilization

2013 ◽  
Vol 15 (1) ◽  
Author(s):  
Emilio I. Alarcon ◽  
Carlos J. Bueno-Alejo ◽  
Christopher W. Noel ◽  
Kevin G. Stamplecoskie ◽  
Natalia L. Pacioni ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Protein Conformation ◽  
Nanoparticle Stabilization ◽  
Amine Groups
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