Using real-time PCR (LightCycler) and immunohistochemistry, we have analyzed expression of key components of the vitamin D system in basal cell carcinomas (BCCs) and normal human skin (NS).
The highly acclaimed prospect of renewable lignocellulosic biocommodities as obvious replacement of their fossilbased counterparts is burgeoning within the last few years. However, the use of the abundant lignocellulosic biomass provided by nature to produce value-added products, especially bioethanol, still faces significant challenges. One of the crucial challenging factors is in association with the expression levels, stability, and cost-effectiveness of the cellulose-degrading enzymes (cellulases). Interestingly, several recommendable endeavors in the bid to curb these challenges are in pursuance. However, the existing body of literature has not well provided the updated roadmap of the advancement and key players spearheading the current success. Moreover, the description of enzyme systems and emerging paradigms with high prospects, for example, the cell-surface display system has been ill-captured in the literature. This review focuses on the lignocellulosic biocommodity pathway, with emphasis on cellulase and hemicellulase systems. The paradigm shift towards cell-surface display system and its emerging recommendable developments have also been discussed. The attempts in supplementing cellulase with other enzymes, accessory proteins, and chemical additives have also been discussed. Moreover, some of the prominent and influential discoveries in the cellulase fraternity have been discussed.
The glioma ampli®ed sequence 41 (GAS41) was previously isolated by microdissection mediated cDNA capture from the glioblastoma multiforme cell line TX3868 and shown to be frequently ampli®ed in human gliomas. We determined the complete cDNA sequence of the GAS41 gene, demonstrated that the GAS41 protein is evolutionarily conserved, speci®cally at the Nterminus, and identi®ed the yeast transcription factor tf2f domain within the GAS41 sequence. A human multiple-tissue Northern blot revealed ubiquitous expression of GAS41 with the highest expression in human brain. After generating polyclonal antibodies we found GAS41 protein expression in the nucleus of the TX3868 cell line by Western blot analysis and immuno¯uores-cence microscopy. The nuclear localization was con®rmed for several human tumors including gliomas of dierent grades of malignancy. In neuroblastoma however, GAS41 was found in the nucleoli but not in the nucleoplasm. Yeast two-hybrid screening of the TX3868 cell line identi®ed the nuclear mitotic apparatus protein (NuMA), the KIAA1009 protein, and prefoldin subunit 1 (PFDN1) as potential interacting partners of GAS41. We generated a polyclonal antibody against the KIAA1009 protein and we demonstrated that the KIAA1009 protein is a nuclear protein, which appears to be co-localized with the GAS41 protein and NuMA.
BackgroundHuman cytochrome P450 (CYP) enzymes mediate the first step in the breakdown of most drugs and are strongly involved in drug–drug interactions, drug clearance and activation of prodrugs. Their biocatalytic behavior is a key parameter during drug development which requires preparative synthesis of CYP related drug metabolites. However, recombinant expression of CYP enzymes is a challenging bottleneck for drug metabolite biosynthesis. Therefore, we developed a novel approach by displaying human cytochrome P450 1A2 (CYP1A2) and cytochrome P450 reductase (CPR) on the surface of Escherichia coli.ResultsTo present human CYP1A2 and CPR on the surface, we employed autodisplay. Both enzymes were displayed on the surface which was demonstrated by protease and antibody accessibility tests. CPR activity was first confirmed with the protein substrate cytochrome c. Cells co-expressing CYP1A2 and CPR were capable of catalyzing the conversion of the known CYP1A2 substrates 7-ethoxyresorufin, phenacetin and the artificial substrate luciferin-MultiCYP, which would not have been possible without interaction of both enzymes. Biocatalytic activity was strongly influenced by the composition of the growth medium. Addition of 5-aminolevulinic acid was necessary to obtain a fully active whole cell biocatalyst and was superior to the addition of heme.ConclusionWe demonstrated that CYP1A2 and CPR can be co-expressed catalytically active on the cell surface of E. coli. It is a promising step towards pharmaceutical applications such as the synthesis of drug metabolites.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0427-5) contains supplementary material, which is available to authorized users.
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