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A fusion of Psb32 from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePsb32) with superfolder GFP was created for enhanced solubility and improved detection and purification. The fusion protein readily formed large hexagonal crystals belonging to space group P6122. A full data set extending to 2.3 Å resolution was collected a...
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Citations
... However, the green fluorescent protein (GFP) and its mutant form (GFPmut3B), enhanced yellow fluorescent protein (eYFP), and Cerulean work well as reporter proteins in cyanobacteria (Huang et al. 2010;Liu et al. 2013). To increase the solubility of these foreign proteins, a superfolder (sf) variant has been developed for all the fluorescent proteins (Liauw et al. 2015). These proteins show greater half-lives and therefore, are not suitable to study the real-time effect. ...
Cyanobacteria are attractive hosts that can be engineered for the photosynthetic production of fuels, fine chemicals, and proteins from CO2. Moreover, the responsiveness of these photoautotrophs towards different environmental signals, such as light, CO2, diurnal cycle, and metals make them potential hosts for the development of biosensors. However, engineering these hosts proves to be a challenging and lengthy process. Synthetic biology can make the process of biological engineering more predictable through the use of standardized biological parts that are well characterized and tools to assemble them. While significant progress has been made with model heterotrophic organisms, many of the parts and tools are not portable in cyanobacteria. Therefore, efforts are underway to develop and characterize parts derived from cyanobacteria. In this review, we discuss the reported parts and tools with the objective to develop cyanobacteria as cell factories or biosensors. We also discuss the issues related to characterization, tunability, portability, and the need to develop enabling technologies to engineer this “green” chassis.
... The fluorescent protein variants still produce fluorescence even with incubation of SDS-PAGE loading buffer [22], and they are visually identified on the SDS-PAGE gel, due to the evolved GFP variants with robust folding [24]. Other GFP variants as the N-and C-terminal fusion partners also show fluorescence on the SDS-PAGE gel [25,26]. Here, two GFP fusion proteins were constructed, in which two TEVp cut sites are placed to increase cleavage efficiency, based on published paper [27]. ...
It is documented that the tobacco etch virus protease (TEVp) variant TEVp(3M) is less efficient in cleaving the fusion protein bound to Ni-NTA resin at relatively low temperature. Here, we determined that, using the GFP fusion substrate bound to Ni-NTA or Strep-tactin agarose, activity of the TEVp(5M) variant was higher than that of the other TEVp construct, and about 15% higher than that of the TEVp(3M). The GST fusion proteins immobilized on Strep-tactin agarose or Glutathione Sepharose were efficiently cleaved by purified TEVp(5M) at the specified condition using GFP reporter for visual track and detection. After on-column cleavage of three fusion constructs using the cognate TEVp(5M) constructs, two target proteins with relatively high purity were separated from Ni-NTA or Amylose agarose. With elution of the buffer containing 1 M NaCl, maize sulfiredoxin was released from Ni-NTA resin via on-column cleavage. Our results confirmed that TEVp(5M) efficiently cleaved the fusion proteins bound to the four affinity matrices. By combination with appropriate affinity handles, the cognate TEVp(5M) mediating tag removal enabled purification and cleavage of the fusion proteins, removal of the protease, and separation of the target proteins from the affinity resin to be accomplished in one step.
... The fact that Synechocystis contains up to 200 copies of its genome [16] and that foreign DNA is taken up spontaneously prompted us to use stable genome integration via homologues recombination for enzyme expression. The corresponding plasmids are compatible to a set of E. coli expression vectors that have been created in a previous study [17], thus allowing easy exchange of genes for expression in different host organisms. To facilitate the quantification of the enzymes in crude extracts, we created fusion proteins with super-folder GFP [18], an enhanced derivative of the well-known green fluorescent protein. ...
... TTTGTAGAGCTCTTTTCC or GCCTGCAGTTAGCT-GCCACCGGACTCAT (reverse primer) and cloned into the pNHIS-GFP-TEV plasmid [19] via the SfoI and KpnI restriction enzyme sites. The resulting plasmids pNHG-Est and pNHG-Amd were used for transformation of E. coli overexpression C43 cell (Lucigen) and protein expression was performed according to literature [17]. For the determination of dry cell weight (DCW), 15 mL of the culture supernatant was added in triplicate to pre-weighed reaction tubes and centrifuged for 4 min at 13000 rpm and 4°C. ...
Global resource depletion poses a dramatic threat to our society and creates a strong demand for alternative resources that do not compete with the production of food. Meeting this challenge requires a thorough rethinking of all steps of the value chain regarding their sustainability resource demand and the possibility to substitute current, petrol-based supply-chains with renewable resources. This regards also the production of catalysts for chemical synthesis. Phototrophic microorganisms have attracted considerable attention as a biomanufacturing platform for the sustainable production of chemicals and biofuels. They allow the direct utilization of carbon dioxide and do not compete with food production. Photosynthetic enzyme production of catalysts would be a sustainable supply of these important components of the biotechnological and chemical industries. This paper focuses on the usefulness of recombinant cyanobacteria for the photosynthetic expression of enantioselective catalysts. As a proof of concept, we used the cyanobacterium Synechocystis sp. PCC 6803 for the heterologous expression of two highly enantioselective enzymes.
We investigated the expression yield and the usefulness of cyanobacterial cell extracts for conducting stereoselective reactions. The cyanobacterial enzyme expression achieved protein yields of 3% of total soluble protein (%TSP) while the expression in E. coli yielded 6-8% TSP. Cell-free extracts from a recombinant strain expressing the recombinant esterase ST0071 from the thermophilic organism Sulfolobus tokodai ST0071 and arylmalonate decarboxylase from Bordetella bronchiseptica showed excellent enantioselectivity (>99%ee) and yield (>91%) in the desymmetrisation of prochiral malonates.
We were able to present the proof-of-concept of photoautotrophic enzyme expression as a viable alternative to heterotrophic expression hosts. Our results show that the introduction of foreign genes is straightforward. Cell components from Synechocystis did not interfere with the stereoselective transformations, underlining the usability of photoautotrophic organisms for the production of enzymes. Given the considerable commercial value of recombinant biocatalysts, cyanobacterial enzyme expression has thus the potential to complement existing approach to use phototrophic organisms for the production of chemicals and biofuels.
