Exogenous protein expression in Xenopus Laevis Oocyte, Basic procedure

The oocytes of the South African clawed frog Xenopus laevis have been widely used as a reliable system for the expression and characterization of different types of proteins, including ion channels and membrane receptors. The large size and resilience of these oocytes make them easy to handle and to microinject with different molecules such as natural mRNAs, cRNAs, and antibodies. A variety of methods can then be used to monitor the expression of the proteins encoded by the microinjected mRNA/cRNA, and to perform a functional characterization of the heterologous polypeptides. In this chapter, after describing the equipment required to maintain X. laevis in the laboratory and to set up a microinjection system, we provide detailed procedures for oocyte isolation, micropipet and cRNA preparation, and oocyte microinjection. A method for the labeling of oocyte-synthesized proteins and for the immunological detection of the heterologous polypeptides is also described

Pichia pastoris as a host for secretion of toxic saporin chimeras

Most of the targeting moieties, such as antibody fragments or growth factor domains, used to construct targeted toxins for anticancer therapy derive from secretory proteins. These normally fold in the oxidative environment of the endoplasmic reticulum, and hence their folding in bacterial cells can be quite inefficient. For instance, only low amounts of properly folded antimetastatic chimera constituted by the amino-terminal fragment of human urokinase (ATF) fused to the plant ribosome-inactivating protein saporin could be recovered. ATF-saporin was instead secreted efficiently when expressed in eukaryotic cells protected from autointoxication with neutralizing anti-saporin antibodies. Pichia pastoris is a microbial eukaryotic host where these domains can fold into a transport-competent conformation and reach the extracellular medium. We show here that despite some host toxicity codon-usage optimization greatly increased the expression levels of active saporin but not those of an active-site mutant SAP-KQ in GS115 (his4) strain. The lack of any toxicity associated with expression of the latter confirmed that toxicity is due to saporin catalytic activity. Nevertheless, GS115 (his4) cells in flask culture secreted 3.5 mg/L of a histidine-tagged ATF-saporin chimera showing an IC(50) of 6 x 10(-11) M against U937 cells, thus demonstrating the suitability of this expression platform for secretion of toxic saporin-based chimeras

Engineering a switchable toxin: the potential use of PDZ domains in the expression, targeting and activation of modified saporin variants

A critical problem in studying ribosome-inactivating proteins (RIPs) lies in the very limited possibility to produce them in heterologous systems. In fact, their inherent toxicity for the producing organism nearly always prevents their recombinant expression. In this study, we designed, expressed and characterized an engineered form of the RIP saporin (SapVSAV), bearing a C-terminal extra sequence that is recognized and bound by the second PDZ domain from murine PTP-BL protein (PDZ2). The coexpression of SapVSAV and PDZ2 in Escherichia coli BL21 cells greatly enhances the production of the toxin in a soluble form. The increase of production was surprisingly not due to protection from bacterial intoxication, but may arise from a stabilization effect of PDZ2 on the toxin molecule during biosynthesis. We found that once purified, SapVSAV is stable but is not toxic to free ribosomes, while it is fully active against human cancer cells. This strategy of co-expression of a toxin moiety and a soluble PDZ domain may represent a new system to increase the production of recombinant toxic proteins and could allow the selection of new extra sequences to target PDZ domains inside specific mammalian cellular domains

Head trauma in primary cranial dystonias: a multicentre case–control study

BACKGROUND: The relationship between prior trauma and primary adult-onset dystonia is not well understood. Previous uncontrolled observations and exploratory case-control studies have yielded contradictory results. OBJECTIVE: To analyse the association between cranial dystonia and prior head trauma. METHODS: An ad hoc multicentre case-control study was performed using a semistructured interview to collect detailed information on the history of head trauma before disease onset in five Italian tertiary referral centres for movement disorders. The presence of a history of head trauma and of post-traumatic sequelae (loss of consciousness, bone fractures, scalp/facial wounds) before disease onset was recorded from 177 patients with primary adult-onset cranial dystonia and from 217 controls with primary hemifacial spasm matched by age strata and sex. Differences between groups were assessed by Mann-Whitney U test and Fisher's exact test, and the relationship between prior head trauma and case/control status was analysed by multivariate logistic regression models. RESULTS: No association was found between vault/maxillofacial trauma and cranial dystonia. Most reported traumas occurred several years before disease onset. None of the main post-traumatic sequelae altered the chance of developing cranial dystonia compared with patients with primary hemifacial spasm, nor did head trauma modify the age at onset of cranial dystonia. CONCLUSIONS: These results do not support prior head trauma as a possible environmental factor modifying the risk of developing late-onset cranial dystonia. The lack of association may have pathogenetic and medical-forensic implications