Glycosylation of rat NGF receptor ectodomain in the yeast Saccharomyces cerevisiae

AbstractHere we studied the glycosylation of a mammalian protein, the ectodomain of rat nerve growth factor receptor (NGFRe), in Saccharomyces cerevisiae. NGFRe is secreted to the culture medium of S. cerevisiae if it is fused to a polypeptide (hsp150Δ) carrier. The hsp150Δ-carrier has 95 serine and threonine residues, which were extensively O-glycosylated. In spite of 41 potential sites, NGFe lacked O-glycans, whether fused to the carrier or not. Distortion of the conformation of NGFRe by inhibition of disulfide formation did not promote O-glycosylation, whereas N-glycosylation was enhanced. Thus, the serine and threonine residues of the hsp150Δ-NGFRe fusion protein were highly selectively O-glycosylated

Selvitystyö maatilamittaluokan biokaasulaitoksen perustamisesta Helsingin yliopiston Viikin opetus- ja tutkimustilalle

Tämän opinnäytetyön tavoitteena oli selvittää Helsingin yliopiston Viikin opetus- ja tutkimustilalle perustettavan biokaasulaitoksen toimintaedellytyksiä. Työssä kartoitettiin biokaasuprosessiin sopivia, lähiseudulla helposti saatavilla olevia syötteitä, ja selvitettiin kartoituksen tuloksena valittuun syöteseokseen sopivia biokaasulaitosratkaisuja. Syötteiksi valikoituivat Viikin opetus- ja tutkimustilan maatalousyötteiden lisäksi Vanhankaupunginlahden järviruoko, läheisen ratsastuskeskuksen hevosenlanta sekä lähiseudun pienpanimon olutmäski. Useilta biokaasulaitostoimittajilta saatiin tarjouksia biokaasulaitoksista, jotka perustuivat kolmeen eri biokaasun tuotantoprosessiin: märkä- ja kiintomädätykseen, kuivamädätykseen ja kaksivaiheiseen mädätykseen. Kaikki tarjotut teknologiat soveltuivat valitulle syöteseokselle. Biokaasulaitoksen tuottama biokaasu on taloudellisesti kannattavampaa jalostaa metaaniksi kuin tuottaa siitä sähköä ja lämpöä Viikin opetus- ja tutkimustilan tarpeisiin. Koska kysyntä hiilineutraalille metaanille on suurta, jalostettu metaani voitaisiin hyödyntää joko liikkeenpolttoaineena tai kaasunjakeluverkossa. Investointi- ja kannattavuuslaskelmien perusteella biokaasulaitoksesta ei todennäköisesti tulisi kannattavaa, vaikka investointikustannuksiin saataisiinkin Työ- ja elinkeinoministeriön energiatukea

ClpXP Protease Regulates the Signal Peptide Cleavage of Secretory Preproteins in Bacillus subtilis with a Mechanism Distinct from That of the Ecs ABC Transporter

Identification and characterization of a suppressor mutation, sup-15, which partially restored secretion in the protein secretion-deficient Bacillus subtilis ecsA26 mutant, led us to discover a novel function of Clp protease. Inactivation of ClpP improved the processing of the precursor of AmyQ α-amylase exposed on the outer surface of the cytoplasmic membrane. A similar improvement of AmyQ secretion was conferred by inactivation of the ClpX substrate-binding component of the ClpXP complex. In the absence of ClpXP, the transcription of the sipS, sipT, sipV, and lsp signal peptidase genes was elevated two- to fivefold, a likely cause of the improvement of the processing and secretion of AmyQ and complementation of ecs mutations. Specific overproduction of SipT enhanced the secretion. These findings extend the regulatory roles of ClpXP to protein secretion. ClpXP also influenced the processing of the lipoprotein PrsA. A concerted regulation of signal peptidase genes by a ClpXP-dependent activator is suggested. In contrast, Ecs did not affect transcription of the sip genes, pointing to a different mechanism of secretion regulation

Quantitation of the Capacity of the Secretion Apparatus and Requirement for PrsA in Growth and Secretion of α-Amylase in Bacillus subtilis

Regulated expression of AmyQ α-amylase of Bacillus amyloliquefaciens was used to examine the capacity of the protein secretion apparatus of B. subtilis. One B. subtilis cell was found to secrete maximally 10 fg of AmyQ per h. The signal peptidase SipT limits the rate of processing of the signal peptide. Another limit is set by PrsA lipoprotein. The wild-type level of PrsA was found to be 2 × 10(4) molecules per cell. Decreasing the cellular level of PrsA did not decrease the capacity of the protein translocation or signal peptide processing steps but dramatically affected secretion in a posttranslocational step. There was a linear correlation between the number of cellular PrsA molecules and the number of secreted AmyQ molecules over a wide range of prsA and amyQ expression levels. Significantly, even when amyQ was expressed at low levels, overproduction of PrsA enhanced its secretion. The finding is consistent with a reversible interaction between PrsA and AmyQ. The high cellular level of PrsA suggests a chaperone-like function. PrsA was also found to be essential for the viability of B. subtilis. Drastic depletion of PrsA resulted in altered cellular morphology and ultimately in cell death