Improved cell disruption of Pichia pastoris utilizing aminopropyl magnesium phyllosilicate (AMP) clay

Kim, Sunil; Wu, Yanrui; Kim, Ka-Lyun; Kim, Geun-Joong; Shin, Hyun‐Jae

doi:10.1007/s11274-013-1262-z

Cited by 7 publications

(3 citation statements)

References 12 publications

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“…(5) aminoclays exhibit antibacterial and antifungal activities under experimental conditions [20] , [21] , which may lower the possibility of bacterial contamination during the transformation procedure. Finally, (6) a further DNA extraction step can be performed in the presence of 0.2 wt% of aminoclay, which facilitates destabilization of cell walls during the bead-beating process [40] .…”

Section: Discussionmentioning

confidence: 99%

A Simple and Non-Invasive Method for Nuclear Transformation of Intact-walled Chlamydomonas reinhardtii

et al. 2014

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Genetic engineering in microalgae is gaining attraction but nuclear transformation methods available so far are either inefficient or require special equipment. In this study, we employ positively charged nanoparticles, 3-aminopropyl-functionalized magnesium phyllosilicate (aminoclay, approximate unit cell composition of [H2N(CH2)3]8Si8Mg6O12(OH)4), for nuclear transformation into eukaryotic microalgae. TEM and EDX analysis of the process of transformation reveals that aminoclay coats negatively-charged DNA biomolecules and forms a self-assembled hybrid nanostructure. Subsequently, when this nanostructure is mixed with microalgal cells and plated onto selective agar plates with high friction force, cell wall is disrupted facilitating delivery of plasmid DNA into the cell and ultimately to the nucleus. This method is not only simple, inexpensive, and non-toxic to cells but also provides efficient transformation (5.03×102 transformants/µg DNA), second only to electroporation which needs advanced instrumentation. We present optimized parameters for efficient transformation including pre-treatment, friction force, concentration of foreign DNA/aminoclay, and plasticity of agar plates. It is also confirmed the successful integration and stable expression of foreign gene in Chlamydomonas reinhardtii through molecular methods.

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Section: Discussionmentioning

confidence: 99%

A Simple and Non-Invasive Method for Nuclear Transformation of Intact-walled Chlamydomonas reinhardtii

et al. 2014

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“…1). This might be due to difficulties in lysis of Pichia cells that possess very complex and rigid cell walls [35]. In particular, a prolonged induction period prevented cell breaking, so the gain of total crude extracts was extremely difficult after 3 days of induction.…”

Section: Discussionmentioning

confidence: 99%

Expression of recombinant full‐length plant phytochromes assembled with phytochromobilin in Pichia pastoris

et al. 2014

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a b s t r a c tWe have successfully developed a system to produce full-length plant phytochrome assembled with phytochromobilin in Pichia pastoris by co-expressing apophytochromes and chromophore biosynthetic genes, heme oxygenase (HY1) and phytochromobilin synthase (HY2) from Arabidopsis. Affinity-purified phytochrome proteins from Pichia cells displayed zinc fluorescence indicating chromophore attachment. Spectroscopic analyses showed absorbance maximum peaks identical to in vitro reconstituted phytochromobilin-assembled phytochromes, suggesting that the co-expression system is effective to generate holo-phytochromes. Moreover, mitochondria localization of the phytochromobilin biosynthetic genes increased the efficiency of holophytochrome biosynthesis. Therefore, this system provides an excellent source of holophytochromes, including oat phytochrome A and Arabidopsis phytochrome B.

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“…It has previously been reported that protein yields above 20 mg ml −1 are required for productive CFPS (Fujiwara & Doi, 2016). As P. pastoris is difficult to lyse (Kim, Wu, Kim, Kim, & Shin, 2013;Stowers & Boczko, 2007), we investigated a variety of methods to determine their suitability for extract preparation. The optimal method would lead to a low number of unlysed, viable cells, without requiring unnecessary dilution (i.e.…”

Section: Optimal Conditions For Harvest and Lysismentioning

confidence: 99%

Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform

Polizzi

2019

Biotech & Bioengineering

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Cell‐free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell‐free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three‐fold increase in recombinant protein yield compared with those from the wild‐type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg ml −1. Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.

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Improved cell disruption of Pichia pastoris utilizing aminopropyl magnesium phyllosilicate (AMP) clay

Cited by 7 publications

References 12 publications

A Simple and Non-Invasive Method for Nuclear Transformation of Intact-walled Chlamydomonas reinhardtii

A Simple and Non-Invasive Method for Nuclear Transformation of Intact-walled Chlamydomonas reinhardtii

Expression of recombinant full‐length plant phytochromes assembled with phytochromobilin in Pichia pastoris

Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform

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