Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele. Results: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth. Conclusions: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii

Systematic Mutational Analysis of the Intracellular Regions of Yeast Gap1 Permease

The yeast general amino acid permease Gap1 is a convenient model for studying the intracellular trafficking of membrane proteins. Present at the plasma membrane when the nitrogen source is poor, it undergoes ubiquitin-dependent endocytosis and degradation upon addition of a good nitrogen source, e.g. ammonium. It comprises 12 transmembrane domains (TM) flanked by cytosol-facing N- and C-terminal tails (NT, CT). The NT of Gap1 contains the acceptor lysines for ubiquitylation and its CT includes a sequence essential to exit from the endoplasmic reticulum (ER).Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Search for the Structure of a Sulfur-Induced Reconstruction on Cu(111)

We have carried out an extensive DFT-based search for the structure of the (√43 × √43)R ± 7.5° reconstruction of S on Cu(111), which exhibits a honeycombtype structure in scanning tunneling microscopy (STM). We apply two criteria in this search: The structure must have a reasonably low chemical potential, and it must provide a good match with STM data, both our own and the data published by Wahlström et al. Phys. Rev. B 1999, 60, 10699. The best model has 12 S adatoms and 9 Cu adatoms per unit cell. Local defects within the Cu9S12 framework, consisting of one missing or one extra Cu adatom per unit cell, would be difficult to detect with STM and would not be energetically costly. There is no obvious correlation between this model and the structure of bulk CuS. If the √43 reconstruction is viewed in terms of local building blocks, then CuS3 and CuS2 clusters, linked by shared S atoms, provides the best description.Reprinted (adapted) with permission from Journal of Physical Chemistry C 118 (2014): 29218, doi: 10.1021/jp505351g. Copyright 2014 American Chemical Society.</p

Women and power: a theoretical approach using the example of copreneurial businesses

Despite the gradual recognition of strategic issues related to the integration of women into the economy, female entrepreneurship continues to receive little attention. Family business research attributes this situation to a lack of recognition given to the (decisive) role of women in these organizations. However, there is one type of family governance that formally acknowledges the man/woman combination: the copreneurial company. Copreneurs are couples who run a business together. This theoretical article highlights the role of women in the copreneurial context by distinguishing between formal and informal power- the latter being primarily held by women, but which is no less influential. The distribution of power reduces opposition costs between partners and the social costs of non-compliance, and improves the clarity of the entrepreneurial structure. Moreover, it increases satisfaction and a feeling of equality between the partners. These results can be generalized and shed light on the role of women in other entrepreneurial and social contexts. This article is published as part as part of a collection on the role of women in management and business

Genomic and functional analyses of mycobacterium tuberculosis strains implicate ald in D-cycloserine resistance.

CAPRISA, 2016.Abstract available in PDF file

Demixing processes in AgPd superlattices

The present scanning tunneling microscopy (STM) study describes the growth of silver-palladium heterostructures at room temperature, with ab initio simulations of ordered AgPd phases supporting the interpretation of STM images. First, the growth of Pd on an Ag(111) surface proceeds in a multilayer mode, leading to the formation of a columnar structure. Then, upon Ag deposition on this structure, Ag and Pd partially mix and form a two-dimensional AgPd alloy on top of the columns. Finally, an atomically flat Ag(111) surface is restored, and two-dimensional growth continues. An interpretation of this peculiar growth mode including interfacial alloying is proposed based on thermodynamic and kinetic arguments

Selective growth of CdTe on patterned CdTe/Si(211)

The authors have studied selective growth of cadmium telluride on Si(211) by molecular beam epitaxy (MBE). Patterned substrates were produced by optical lithography of MBE-grown CdTe/As/Si(211). Photoemission microscopy was used as the main tool to study selective growth. This is very powerful because Si or SiO2 can be very easily distinguished from areas covered with even small amounts of CdTe due to contrast from work function differences. It was found that CdTe grows on CdTe without sticking on bare Si areas if the temperature is sufficiently high. Based on the analysis of the temperature dependence of the growth rate of CdTe, we suggest that different physisorption energies on Si and CdTe are the main cause of this selective growth. (C) 2008 American Vacuum Society