Resistance to PAI-1 is a factor which confers clinical benefits in thrombolytic therapy. The only US FDA approved PAI-1 resistant drug is Tenecteplase

Urate oxidase is an important enzyme with therapeutic and diagnostic applications. Rasburicase is a recombinant urate oxidase enzyme approved by FDA to use in the treatment of hyperuricemia conditions. Various hosts such as Saccharomyces cerevisiae, Hansenula polymorpha and Escherichia coli have been used to express the enzyme. Today, Pichia pastoris is considered as an important host for heterologous protein expression since it has beneficial characteristics such as strong promoters, simple scale up, post translational modifications, high cell density cultivation and simple genetic manipulation. In this study, Aspergillus flavus urate oxidase gene was cloned in pPICZαA expression vector and expressed in P. pastoris. The recombinant urate oxidase was expressed in secretory form and was confirmed through RT-PCR, SDS-PAGE analysis and western blotting. The enzyme activity was determined using a colorimetric assay. A production yield of 0.43 U/ml of culture supernatant was obtained.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-395) contains supplementary material, which is available to authorized users.

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Aims: A novel chimeric-truncated form of tissue-type plasminogen activator (t-PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed-batch processes. Methods and Results: The expression cassette for the novel t-PA was prepared in pET-28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase ® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t-PA Assay Kit. The fed-batch fermentation mode, coupled with a Ni-NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46Á66 IU mg À1 ) compared to traditional batch mode (35Á8 IU mg À1 ). Conclusions:Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed-batch cultivation methods showed the potential to replace miss-folded formats of protein with proper folded, soluble form with improved potency. Significance and Impact of the Study: Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post-translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.

The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments.

In Saccharomyces cerevisiae, Nce102 encodes a 173 amino acid transmembrane protein, which acts as a key player in eisosome assembly and plasma membrane organization. Here, we describe the characterization of Nce102 homologue in the human pathogen, Aspergillus fumigatus. Our results demonstrated that AfuNce102 is continuously expressed during fungal growth. In addition, microscopic examination of an AfuNce102-GFP-expressing transformant confirmed the localization of the fusion protein to the endoplasmic reticulum with higher density fluorescence at the tip of the mycelium. During conidiogenesis, the protein was localized to the conidiophores and the conidia. Abnormal conidiation of AfuNce102 deletion mutant suggests a potential role for AfuNce102 in sporulation process.

Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis–cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.

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