Shaofang Liu

Molecular cloning and stress-dependent expression of a gene encoding Δ12-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Δ12 unsaturated fatty acids was identified which prompted us to speculate about the involvement of Δ12 fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Δ12-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15°C and 3.9-fold at 4°C compared to the alga at 25°C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Δ12 fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC α and β subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.

Purification of 6×His-tagged phycobiliprotein using zinc-decorated silica-coated magnetic nanoparticles

Zinc-decorated silica-coated magnetic nanoparticles (ZnSiMNPs) were prepared by adsorbing zinc onto colloidal silica. These nanoparticles were used for the rapid purification of 6×His-tagged recombinant phycobiliprotein. The surface changes in the magnetic nanoparticles after zinc adsorption were characterized by transmission electron microscopy. The adsorption of the 6×His-tagged phycobiliprotein onto ZnSiMNPs in 10 mM PBS at 25°C was found to follow the Langmuir isotherm. ZnSiMNPs could be used to extract 6×His-tagged phycobiliprotein from lysate to single-band purity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No spectral variation was observed in the purified phycobiliprotein. Thus, ZnSiMNPs served as a useful tool for the magnetic separation and delivery of the 6×His-tagged phycobiliprotein.

Biosynthesis and Immobilization of Biofunctional Allophycocyanin

The holo-allophycocyanin-α subunit, which has various reported pharmacological uses, was biosynthesized with both Strep-II-tag and His-tag at the N-terminal in Escherichia coli. The streptavidin-binding ability resulting from the Strep II-tag was confirmed by Western blot. Additionally, the metal-chelating ability deriving from the His-tag not only facilitated its purification by immobilized metal-ion affinity chromatography but also promoted its immobilization on Zn (II)-decorated silica-coated magnetic nanoparticles. The holo-allophycocyanin-α subunit with streptavidin-binding ability was thereby immobilized on magnetic nanoparticles. Magnetic nanoparticles are promising as drug delivery vehicles for targeting and locating at tumors. Thus, based on genetic engineering and nanotechnology, we provide a potential strategy to facilitate the biomodification and targeted delivery of pharmacological proteins.