Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His₆-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP™ based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni–nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P. angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2 mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2 mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.     

Nitric oxide regulates the 26S proteasome in vascular smooth muscle cells

It is well established that nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation by modulating cell cycle proteins. The 26S proteasome is integral to protein degradation and tightly regulates cell cycle proteins. Therefore, we hypothesized that NO directly inhibits the activity of the 26S proteasome. The three enzymatic activities (chymotrypsin-like, trypsin-like and caspase-like) of the 26S proteasome were examined in VSMC. At baseline, caspase-like activity was approximately 3.5-fold greater than chymotrypsin- and trypsin-like activities. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited all three catalytically active sites in a time- and concentration-dependent manner (P < 0.05). Caspase-like activity was inhibited to a greater degree (77.2% P < 0.05). cGMP and cAMP analogs and inhibitors had no statistically significant effect on basal or NO-mediated inhibition of proteasome activity. Dithiothreitol, a reducing agent, prevented and reversed the NO-mediated inhibition of the 26S proteasome. Nitroso-cysteine analysis following S-nitrosoglutathione exposure revealed that the 20S catalytic core of the 26S proteasome contains 10 cysteines which were S-nitrosylated by NO. Evaluation of 26S proteasome subunit protein expression revealed differential regulation of the α and β subunits in VSMC following exposure to NO. Finally, immunohistochemical analysis of subunit expression revealed distinct intracellular localization of the 26S proteasomal subunits at baseline and confirmed upregulation of distinct subunits following NO exposure. In conclusion, NO reversibly inhibits the catalytic activity of the 26S proteasome through S-nitrosylation and differentially regulates proteasomal subunit expression. This may be one mechanism by which NO exerts its effects on the cell cycle and inhibits cellular proliferation in the vasculature.     

A high-performance liquid chromatography method for the quantification of cysmethynil,an inhibitor of isoprenylcysteine carboxylmethyl transferase,in mouse plasma

Cysmethynil, a newly identified small molecule inhibitor of isoprenylcysteine carboxylmethyl transferase (Icmt) is involved in the post-translational modification of CaaX proteins. Cysmethynil causes cell death in many human cancer cells in vitro, and inhibits tumor growth in the xenograft mouse model in vivo. A HPLC method for the quantification of cysmethynil in mouse plasma was developed and validated. The lower limit of quantification of this method was 0.01 μg/ml. Inter- and intra-day variability ranged from 0.38–8.5% and accuracy was between 86% and 98%. This sensitive method was used to quantify cysmethynil in plasma of mice after intraperitoneal dosing for preliminary pharmacokinetic studies.     

Tandem multimer expression of angiotensin I-converting enzyme inhibitory peptide in Escherichia coli

It is common for small tandem peptide multimer genes to be indirectly inserted into expression vectors and fused with a protein tag. In this study, a multimer of the tandem angiotensin I-converting enzyme inhibitory peptide (ACE-IP) gene was directly transferred to a commercially available vector and the designed gene was expressed as a repeated peptide in Escherichia coli BL21(DE3)pLysS. The process further developed in our study was the construction of six-repeated ACE-IP synthetic genes and their direct insertion. Protein expression in inclusion bodies was confirmed by SDS-PAGE and Western blot. Acid hydrolysis of inclusion bodies produced single-unit peptides through cleavage of the aspartyl-prolyl bonds. This cleaved recombinant peptide (rACE-IP) was purified using immuno-affinity chromatography followed by reversed phase-HPLC. 105–115 mg of the lyophilized recombinant peptide was obtained from 1 L E. coli culture. In vitro biological activity of rACE-IP was indistinguishable from that of the natural peptide produced by hydrolysis in artificial gastric juice or by acidic hydrolysis. The rACE-IP prepared by recombinant DNA technology and solid-phase synthesis methods showed a similar IC₅₀. This strategy could be used for the expression of important peptides, which have N-terminal proline (P) and C-terminal aspartic acid residues (D) for commercial applications, e.g. functional foods and drinks.     

Genome-Wide Association Study Implicates Chromosome 9q21.31 as a Susceptibility Locus for Asthma in Mexican Children

Many candidate genes have been studied for asthma, but replication has varied. Novel candidate genes have been identified for various complex diseases using genome-wide association studies (GWASs). We conducted a GWAS in 492 Mexican children with asthma, predominantly atopic by skin prick test, and their parents using the Illumina HumanHap 550 K BeadChip to identify novel genetic variation for childhood asthma. The 520,767 autosomal single nucleotide polymorphisms (SNPs) passing quality control were tested for association with childhood asthma using log-linear regression with a log-additive risk model. Eleven of the most significantly associated GWAS SNPs were tested for replication in an independent study of 177 Mexican case–parent trios with childhood-onset asthma and atopy using log-linear analysis. The chromosome 9q21.31 SNP rs2378383 (p = 7.10×10⁻⁶ in the GWAS), located upstream of transducin-like enhancer of split 4 (TLE4), gave a p-value of 0.03 and the same direction and magnitude of association in the replication study (combined p = 6.79×10⁻⁷). Ancestry analysis on chromosome 9q supported an inverse association between the rs2378383 minor allele (G) and childhood asthma. This work identifies chromosome 9q21.31 as a novel susceptibility locus for childhood asthma in Mexicans. Further, analysis of genome-wide expression data in 51 human tissues from the Novartis Research Foundation showed that median GWAS significance levels for SNPs in genes expressed in the lung differed most significantly from genes not expressed in the lung when compared to 50 other tissues, supporting the biological plausibility of our overall GWAS findings and the multigenic etiology of childhood asthma.     

Survival of the Fittest: Positive Selection of CD4+ T Cells Expressing a Membrane-Bound Fusion Inhibitor Following HIV-1 Infection

Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.     

Systems approach to studying animal sociality: individual position versus group organization in dynamic social network models

Social networks can be used to represent group structure as a network of interacting components, and also to quantify both the position of each individual and the global properties of a group. In a series of simulation experiments based on dynamic social networks, we test the prediction that social behaviors that help individuals reach prominence within their social group may conflict with their potential to benefit from their social environment. In addition to cases where individuals were able to benefit from improving both their personal relative importance and group organization, using only simple rules of social affiliation we were able to obtain results in which individuals would face a trade-off between these factors. While selection would favor (or work against) social behaviors that concordantly increase (or decrease, respectively) fitness at both individual and group level, when these factors conflict with each other the eventual selective pressure would depend on the relative returns individuals get from their social environment and their position within it. The presented results highlight the importance of a systems approach to studying animal sociality, in which the effects of social behaviors should be viewed not only through the benefits that those provide to individuals, but also in terms of how they affect broader social environment and how in turn this is reflected back on an individual's fitness.