High Cell-Density Expression System: Yeast Cells in a Phalanx Efficiently Produce a Certain Range of “Difficult-to-Express” Secretory Recombinant Proteins

2014 ◽  
pp. 169-178
Author(s):  
Yasuaki Kawarasaki ◽  
Takeshi Kurose ◽  
Keisuke Ito
Keyword(s):  
Cell Density ◽  
Recombinant Proteins ◽  
High Cell Density ◽  
Expression System ◽  
Yeast Cells ◽  
High Cell

scholarly journals A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

2010 ◽  
Vol 9 (1) ◽  
pp. 11 ◽  
Author(s):  
Mirja Krause ◽  
Kaisa Ukkonen ◽  
Tatu Haataja ◽  
Maria Ruottinen ◽  
Tuomo Glumoff ◽  
...  
Keyword(s):  
Cell Density ◽  
Recombinant Proteins ◽  
High Cell Density ◽  
High Cell ◽  
Fed Batch ◽  
Cultivation Method

scholarly journals Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (12) ◽  
pp. 5166-5178 ◽  
Author(s):  
H Jakubowski ◽  
E Goldman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Density ◽  
De Novo ◽  
High Cell Density ◽  
Degradation Products ◽  
Yeast Cells ◽  
High Cell ◽  
Yeast Saccharomyces Cerevisiae ◽  
Type Locus ◽  
Low Cell Density

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.

Preparation of Very-High-Yield Recombinant Proteins Using Novel High-Cell-Density Bacterial Expression Methods

2010 ◽  
Vol 2010 (8) ◽  
pp. pdb.prot5475 ◽  
Author(s):  
Victoria Murray ◽  
Jianglei Chen ◽  
Yuefei Huang ◽  
Qianqian Li ◽  
Jianjun Wang
Keyword(s):  
Cell Density ◽  
Recombinant Proteins ◽  
High Cell Density ◽  
Bacterial Expression ◽  
High Yield ◽  
High Cell ◽  
Very High

High cell-density expression system: A novel method for extracellular production of difficult-to-express proteins

2012 ◽  
Vol 113 (2) ◽  
pp. 154-159 ◽  
Author(s):  
Koichi Kimata ◽  
Masaya Yamaguchi ◽  
Yuta Saito ◽  
Hiroki Hata ◽  
Kasumi Miyake ◽  
...  
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
Expression System ◽  
High Cell ◽  
Extracellular Production ◽  
System A ◽  
Novel Method

The use of elements of the E. coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations

1999 ◽  
Vol 75 (2-3) ◽  
pp. 241-250 ◽  
Author(s):  
Volker Schroeckh ◽  
Rolf Wenderoth ◽  
Marian Kujau ◽  
Uwe Knüpfer ◽  
Dieter Riesenberg
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
Recombinant Expression ◽  
Expression System ◽  
High Cell ◽  
E Coli ◽  
Recombinant Expression System

scholarly journals Role for Cell Density in Antifungal Drug Resistance in Candida albicans Biofilms

2007 ◽  
Vol 51 (7) ◽  
pp. 2454-2463 ◽  
Author(s):  
Palani Perumal ◽  
Satish Mekala ◽  
W. LaJean Chaffin
Keyword(s):  
Candida Albicans ◽  
Quorum Sensing ◽  
Cell Density ◽  
High Cell Density ◽  
Yeast Cells ◽  
Drug Efflux ◽  
High Cell ◽  
Phenotypic Resistance ◽  
Drug Efflux Pumps ◽  
Low Cell Density

ABSTRACT Biofilms of Candida albicans are less susceptible to many antifungal drugs than are planktonic yeast cells. We investigated the contribution of cell density to biofilm phenotypic resistance. Planktonic yeast cells in RPMI 1640 were susceptible to azole-class drugs, amphotericin B, and caspofungin at 1 × 103 cells/ml (standard conditions) using the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] assay. As reported by others, as the cell concentration increased to 1 × 108 cells/ml, resistance was observed with 10- to 20-fold-greater MICs. Biofilms that formed in microtiter plate wells, like high-density planktonic organisms, were resistant to drugs. When biofilms were resuspended before testing, phenotypic resistance remained, but organisms, when diluted to 1 × 103 cells/ml, were susceptible. Drug-containing medium recovered from high-cell-density tests inhibited low-cell-density organisms. A fluconazole-resistant strain showed greater resistance at high planktonic cell density, in biofilm, and in resuspended biofilm than did low-density planktonic or biofilm organisms. A strain lacking drug efflux pumps CDR1, CDR2, and MDR1, while susceptible at a low azole concentration, was resistant at high cell density and in biofilm. A strain lacking CHK1 that fails to respond to the quorum-sensing molecule farnesol had the same response as did the wild type. FK506, reported to abrogate tolerance to azole drugs at low cell density, had no effect on tolerance at high cell density and in biofilm. These observations suggested that cell density has a role in the phenotypic resistance of biofilm, that neither the drug efflux pumps tested nor quorum sensing through Chk1p contributes to resistance, and that azole drug tolerance at high cell density differs mechanistically from tolerance at low cell density.

scholarly journals Pichia pastoris: SEL RAGI UNTUK PRODUKSI PROTEIN REKOMBINAN

2018 ◽  
Vol 17 (2) ◽  
Author(s):  
Neng Herawati ◽  
Arizah Kusumawati ◽  
Adi Santoso
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Protein ◽  
Cell Density ◽  
Mammalian Cells ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
High Cell Density ◽  
Expression System ◽  
Scale Up ◽  
High Cell

Pichia pastoris is a group of methylotropic yeast known as a host of expression and protein production which is widely used for biopharmaceutical-based drug production. This yeast can grow fast with a high cell density. Its genetic stability, high cell density, and stress resistance make the development process and scale-up of P. pastoris can increase to a scale of 200,000 liters of culture. In contrast to the expensive and complex development of recombinant protein production in mammalian cells, the development of production in P. pastoris is relatively simple and cheaper. The advantage of P. pastoris as an expression system is that it is able to use methanol as a carbon source by inducing the expression of alcohol oxidase oxidase (AOX) enzyme. Promoter used by this enzyme is also used as a strong promoter for the expression of proteins that we want. Unlike in bacterial and mammalian systems, recombinant protein production in Pichia cells is not contaminated with endotoxins or viruses so it is safer and simplifies the downstream processes in bioproduction. The level of endogenous protein in the low supernatant allows Pichia to cultivate with a high volumetric productivity, therefore the process of protein production becomes very economical. This review provides an overview of several things that must be considered in utilizing P. pastoris as an expression system including the selection of vectors, strains, vector integration mechanisms into the genome, glycosylation processes, and applications in industry.

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