RESUMEN
Exploitation of potential new targets for drug and vaccine development has an absolute requirement for multimilligram quantities of soluble protein. While recombinant expression of full-length proteins is frequently problematic, high-yield soluble expression of functional subconstructs is an effective alternative, so long as appropriate termini can be identified. Bioinformatics localizes domains, but doesn't predict boundaries with sufficient accuracy, so that subconstructs are typically found by trial and error. Combinatorial Domain Hunting (CDH) is a technology for discovering soluble, highly expressed constructs of target proteins. CDH combines unbiased, finely sampled gene-fragment libraries, with a screening protocol that provides "holistic" readout of solubility and yield for thousands of protein fragments. CDH is free of the "passenger solubilization" and out-of-frame translational start artifacts of fusion-protein systems, and hits are ready for scale-up expression. As a proof of principle, we applied CDH to p85alpha, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications.
Asunto(s)
Técnicas Químicas Combinatorias , Estructura Terciaria de Proteína , Proteínas/química , Biblioteca de Genes , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Proteínas/aislamiento & purificación , SolubilidadRESUMEN
Two enhancer regions upstream of the apolipoprotein(a) (apo(a)) gene were amplified and sequenced from subjects who were known to express abnormally high or low amounts of lipoprotein(a) (Lp(a)). No base changes were found in the DHII region situated 28 kb from the apo(a) gene. Three common base substitutions were found in the DHIII region about 20 kb from the gene. One, an A to G change at position -1230, increased the activity of reporter-gene constructs approximately 2.5-fold. The other two, a C to A change at -1617 and a G to T change at -1712, decreased reporter activity by 30 and 40%, respectively. The sites at -1230 and -1617 were in linkage disequilibrium with each other and also with a polymorphic site near the DHII enhancer and sites in the apo(a) promoter and gene. The rarer G variant at -1230 was associated with smaller alleles. After correcting for the effect of allele size, values of Lp(a) concentration for alleles associated with the G variant at -1230 were 70% higher than those associated with the more common A variant. In contrast, the corrected values for alleles associated with the rare T variant at -1712 were 40% lower than those associated with the common G variant. Thus, overall the changes observed in this enhancer could influence apo(a) gene transcription up to 4-fold and could provide a significant contribution to the variation in Lp(a) concentrations in plasma.
Asunto(s)
Apolipoproteínas A/genética , Elementos de Facilitación Genéticos/genética , Desequilibrio de Ligamiento , Polimorfismo Genético , Variación Genética , Humanos , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Población BlancaRESUMEN
A high plasma concentration of lipoprotein(a) [Lp(a)] confers an increased risk for the development of coronary heart disease. Hormones, such as oestrogen, are some of the few compounds known to reduce plasma Lp(a) levels. A putative enhancer region, located at the DHII DNase I hypersensitive site approx. 28 kb upstream of the apolipoprotein(a) [apo(a)] gene, contains a number of sequences similar to the binding half-sites for nuclear hormone receptors, such as the oestrogen receptor and the peroxisome proliferator-activated receptor (PPAR). The 180 bp core DHII enhancer increased the activity of the apo(a) promoter by over 7-fold in reporter-gene assays in HepG2 cells in vitro. Almost 60% of this increase was lost in the presence of co-transfected oestrogen receptor and oestrogen. In contrast, co-transfection with PPARalpha increased the effect of the DHII enhancer on apo(a) transcriptional activity by approx. 70% and could overcome the inhibitory effect of the oestrogen receptor on apo(a) transcription. Gel mobility-shift assays showed that oestrogen receptor protein bound to one half of a sequence corresponding to a predicted oestrogen receptor response element. PPARalpha also bound to this site and competed with oestrogen receptors for binding. In addition, PPARalpha bound to a separate site that comprised part of a direct repeat of nuclear hormone receptor half-sites. The results suggest that nuclear hormones affect plasma Lp(a) concentrations by binding to the sequences within the DHII enhancer, thereby altering the amount by which the enhancer increases the transcription of the apo(a) gene.