Cloning, expression, and purification of the N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae in Pichia pastoris

Goossens, Katty; Greve, Henri De; Willaert, Ronnie

doi:10.1016/j.pep.2012.12.001

Cited by 3 publications

(3 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…5) because of glycosylation to different degrees. This is a common phenomenon; similar results were obtained for another FAD-GDH and other proteins expressed in P. pastoris [7,16,26]. In general, glycosylation may not affect the enzymatic activity [18], but the degree of glycosylation did affect the charge and redox hydrogel catalytic efficiency of GOX [31].…”

Section: Discussionsupporting

confidence: 77%

Efficient Expression, Purification, and Characterization of a Novel FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus in Pichia pastoris

Yang¹,

Huang²,

Wang³

et al. 2014

Journal of Microbiology and Biotechnology

View full text Add to dashboard Cite

Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) can utilize a variety of external electron acceptors and also has stricter substrate specificity than any other glucose oxidoreductases, which makes it the ideal diagnostic enzyme in the field of glucose biosensors. A gene coding for a hypothetical protein, similar to glucose oxidase and derived from Aspergillus terreus NIH2624, was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 260,000 U/l in the culture supernatant after fed-batch cultivation for 84 h. After a three-step purification protocol that included isopropanol precipitation, affinity chromatography, and a second isopropanol precipitation, recombinant FAD-GDH was purified with a recovery of 65%. This is the first time that isopropanol precipitation has been used to concentrate a fermentation supernatant and exchange buffers after affinity chromatography purification. The purified FAD-GDH exhibited a broad and diffuse band between 83 and 150 kDa. The recombinant FAD-GDH was stable across a wide pH range (3.5 to 9.0) with maximum activity at pH 7.5 and 55°C. In addition, it displayed very high thermal stability, with a half-life of 82 min at 60°C. These characteristics indicate that FAD-GDH will be useful in the field of glucose biosensors.

show abstract

Section: Discussionsupporting

confidence: 77%

Efficient Expression, Purification, and Characterization of a Novel FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus in Pichia pastoris

Yang¹,

Huang²,

Wang³

et al. 2014

Journal of Microbiology and Biotechnology

View full text Add to dashboard Cite

show abstract

“…In this study, we used the simplest and most cost-effective SLiCE identified: the extract prepared from the E. coli RecA − laboratory strain JM109 with the 3% Triton X-100 buffer, and compared its cloning efficiency with a commercially available seamless DNA cloning kit. The Clontech In-Fusion HD Cloning Kit, which is widely used for seamless DNA cloning, was selected as being representative of commercial kits [17] , [18] , [19] , [21] , [23] , [24] . In this comparative experiment, the cloning abilities of both seamless DNA cloning methods were evaluated under various molar ratios of insert DNA fragments to vector DNA by two parameters: colony formation rate and cloning efficiency.…”

Section: Resultsmentioning

confidence: 99%

“…By using the Triton X-100 buffer, the SLiCE-method becomes an ultra-low cost homemade seamless DNA cloning method [12] . On the other hand, commercially available kits for seamless DNA cloning have been widely used [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] . Although commercial kits are associated with a high cost per reaction, they are generally accepted to be easy to use and efficient.…”

Section: Introductionmentioning

confidence: 99%

A simple and ultra-low cost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit

Okegawa

Motohashi

2015

Biochemistry and Biophysics Reports

View full text Add to dashboard Cite

The seamless ligation cloning extract (SLiCE) method is a novel seamless DNA cloning tool that utilizes homologous recombination activities in Escherichia coli cell lysates to assemble DNA fragments into a vector. Several laboratory E. coli strains can be used as a source for the SLiCE extract; therefore, the SLiCE-method is highly cost-effective.The SLiCE has sufficient cloning ability to support conventional DNA cloning, and can simultaneously incorporate two unpurified DNA fragments into vector. Recently, many seamless DNA cloning kits have become commercially available; these are generally very convenient, but expensive. In this study, we evaluated the cloning efficiencies between a simple and highly cost-effective SLiCE-method and a commercial kit under various molar ratios of insert DNA fragments to vector DNA. This assessment identified that the SLiCE from a laboratory E. coli strain yielded 30−85% of the colony formation rate of a commercially available seamless DNA cloning kit. The cloning efficiencies of both methods were highly effective, exhibiting over 80% success rate under all conditions examined. These results suggest that SLiCE from a laboratory E. coli strain can efficiently function as an effective alternative to commercially available seamless DNA cloning kits.

show abstract

Flocculating Protein Flo1p from Saccharomyces cerevisiae W303-1A

Wang

Zhang

2018

Appl Biochem Microbiol

View full text Add to dashboard Cite

Cloning, expression, and purification of the N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae in Pichia pastoris

Cited by 3 publications

References 41 publications

Efficient Expression, Purification, and Characterization of a Novel FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus in Pichia pastoris

Efficient Expression, Purification, and Characterization of a Novel FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus in Pichia pastoris

A simple and ultra-low cost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit

Flocculating Protein Flo1p from Saccharomyces cerevisiae W303-1A

Contact Info

Product

Resources

About