Topology of the C-terminal fragment of human presenilin 1.

Mutations of human presenilin 1 (PS1) have been genetically linked to early-onset familial Alzheimer's disease. PS1 contains 10 hydrophobic regions (HRs) sufficiently long to be alpha-helical membrane spanning segments. Most previous topology studies agree that the N-terminus of PS1 is cytosolic and HRs 1-6 span the membrane but HR 7 does not. However, whether HRs 8 and 9 are membrane spanning segments remains controversial. Here we study the topology and biogenesis of this region of PS1 using a reporter gene fusion approach, where portions of the PS1 sequence containing possible membrane spanning segments were fused up- or downstream of a reporter sequence whose translocation into the endoplasmic reticulum could be monitored via its glycosylation. We provide strong evidence, supported by cysteine accessibility studies in full-length PS1, that HRs 8 and 9 are indeed membrane spanning and that the integration of HR 8 into the membrane is dependent on the presence of HR 9. We also explain how our results reconcile previous apparently divergent conclusions regarding the topology of HRs 8 and 9.

SUMO fusion technology for difficult-to-express proteins.

The demands of structural and functional genomics for large quantities of soluble, properly folded proteins in heterologous hosts have been aided by advancements in the field of protein production and purification. Escherichia coli, the preferred host for recombinant protein expression, presents many challenges which must be surmounted in order to over-express heterologous proteins. These challenges include the proteolytic degradation of target proteins, protein misfolding, poor solubility, and the necessity for good purification methodologies. Gene fusion technologies have been able to improve heterologous expression by overcoming many of these challenges. The ability of gene fusions to improve expression, solubility, purification, and decrease proteolytic degradation will be discussed in this review. The main disadvantage, cleaving the protein fusion, will also be addressed. Focus will be given to the newly described SUMO fusion system and the improvements that this technology has advanced over traditional gene fusion systems.

Use of Ssp dnaB derived mini-intein as a fusion partner for production of recombinant human brain natriuretic peptide in Escherichia coli.

To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be released by proteolytic or chemical reagents. In this report, a modified Ssp dnaB mini-intein linked with a chitin binding domain tag was used as a fusion partner for production of human brain natriuretic peptide (hBNP), a hormone for the treatment of congestive heart failure. The fusion protein was expressed as an inclusion body in Escherichia coli. After refolding, the fusion protein was purified with a chitin affinity column, and dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH in the chitin column to 7.0 at 25 degrees C for 16 h, which led to the release and separation of hBNP from its fusion partner. The hBNP sample was further purified with reverse phase HPLC and its biological activity was assayed in vitro. It was found that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC(50) of (1.24+/-0.32)x10(-6)mg/ml, which was similar to that of the synthetic BNP standard. The expression strategy described here promises to produce small peptides without use of proteolytic or chemical reagents.

Expression of anti-Z-DNA single chain antibody variable fragment on the filamentous phage surface

We describe the expression of an anti-Z-DNA single chain variable region antibody fragment (scFv) on a filamentous phage surface. Four vectors for phage display were constructed. Two of them are able to display multiple copies of the antibody fragment, and the others can be used to make monovalent libraries. The vectors use different promoter/leader sequences to direct the expression of the fused proteins. All were able to promote the assembly of fusion virion particles. In this paper we also show the affinity selection (biopanning) of those phage-antibodies based on the capacity of their products to recognize the antigen. We used biotinylated Z-DNA and the selection was performed in a solution phase fashion. The data presented here indicate that these vectors can be further used to construct anti-nucleic acid antibody fragment libraries that can be used to study the basis of nucleic acid-protein interaction and its role in autoimmunity mechanisms.

Influência do pH na atividade hemolítica de uma amostra aviária de vírus influnza A / Influence of pH on hemolytic activity of an avian sample of influenza a virus

A atividade de fusäo, depende de pH, de uma amostra aviária de Vírus Influenza A com eritrócitos de galinhas, foi investigada usando oteste de hemólise (HL). Este teste é baseado na quantificaçäo de hemoglobina (Hb) liberada como consequência da fusäo vírus/eritrócito. A atividade hemolítica desta preparaçäo viral pode ser demonstrada na faixa de pH ácido (5.0-5.8), o que está relacionado à ocorrência de uma mudança conformacional nas moléculasda hemaglutinina (HA) sob estas condiçöes. A atividade fusogênica era rapidamente inativada após incubaçäo do vírus, em pH ácido, a 37 graus C, na ausência de eritrócitos, sugerindo que a mudança conformacional da HA do vírus pode ser responsável pela ativaçäo e/ou inativaçäo da capacidade fusogênica. O estudo da reaçäo de HL mostra que elevando-se a concentraçäo de eritrócitos, enquanto a concentraçäo de vírus permanece constante, pode-se observar um aumento na liberaçäo de hemoglobina (Hb), o qual sugere que, provavelmente, os vírus tiveram a chance de encontrar um maior número de células susceptíveis. Uma vez que a Hb em soluçäo muda de tonalidade em funçäo do pH em que se encontra, um fator de correçäo foi utilizado após o ensaio colorimétrico.