High-level Expression And Functional Characterization Of Active Snake Venom Thrombin Like Enzyme And Chlorophy Ll-binding Protein

In the fields of protein medicine and study of biochemistry, it is very important to obtain a high expression level of a functional protein in a highly active form. As every protein is different, there can be no single strategy for obtaining different recombinant proteins with high activities. Thus, different strategies must be worked out for different proteins of interest in order to obtain high expression levels of functional proteins.In this work, two representative functional proteins, the snake venom thrombin-like enzyme and an algal chlorophyll-binding protein were chosen to investigate the strategies for obtaining high expression and activity for a functional protein, since it was difficult to express these two proteins to a high level and with high activity.In China, snake venom thrombin-like enzyme is a basic medicine for the clinical therapy of occlusive thrombi. However, due to the limited resource of snake venom, production of these enzymes from snake venoms faces a big challenge. Therefore, genetic engineering is one of the best alternatives to solve this problem. Currently, high-scale production of this enzyme is hard to accomplish since the expression and activity of thrombin like enzyme is usually low.Pcb (prochlorophyte chlorophyll (Chl) a/b binding protein) can bind with different chlorophylls. For oxygenic photosynthetic organisms, light can be harvested by Pcb-chlorophyll complexes under different light intensities and nutrition niches. Acaryochloris marina is a cyanobacterium found in deep sea that uses Chl d as its major photopigment. Chl d gives A. marina the critical advantage of using light of longer wavelengths (690-750 nm) that are not absorbed by organisms containing other Chls. Chl a is the predominant pigment in nearly all oxygenic photosynthetic organisms, from cyanobacteria to land plants, whereas its function is mostly replaced by Chl d in Acaryochloris. Introducing the Chl d-bound light-harvesting protein, PcbA, into a Chl a-containing cyanobacterium will not only permit investigation of the mechanism of chlorophyll replacement, but will also provide a system for exploring the functional and evolutionary development of various light-harvesting strategies in photosynthetic organisms.In this study, the expressions of snake venom thrombin-like enzyme and chlorophyll-binding protein PcbA with high activity were investigated. Ⅰ. Expression and function analysis of Gloshedobin in Pichia pastoris.There has been no previous report in the literature concerning the high expression level of an active form of snake venom thrombin-like enzymes in E. coli, even as fusion protein. Pichia pastoris has been widely used in microbial genetic engineering work, as it has the advantage of high expression levels and posttranslational modifications of the expressed proteins. However, previous study investigating the expression level of Gloshedobin in P. pastoris was not satisfactory without fusion-partner strategy. This work focused on the high expression and activity of Gloshedobin expressed as a fusion protein. Hsp70 is a chaperon that has various important functions, including protein folding and protein activation. It has been expressed in P. pastoris to high level (120 mg/L) and exhibits both high activity and stability. Thus Hsp70 was fused to the N-terminus of the Gloshedobin to obtain Hsp70-Glo fusion protein that can be expressed in high level and having high activity in P. pastoris.The expression vector, pPIC9K-Hsp70/tle, with Hsp70 as a fusion partner was constructed and then transformed into P. pastoris. The expression of Hsp70-Glo was confirmed by SDS-PAGE and Western blot. An expression level of 44.5 mg Hsp70-Glo per liter of culture was achieved by methanol induction. It was almost five times higher than recombinant Gloshedobin (10 mg/l) expressed without fusion-partner in P. pastoris. The optimum culture conditions consisted of a complex medium at pH 6.0, the use of 1% methanol during induction, and inducing the culture for 36 hours before harvesting the cells.The Hsp70-Glo was purified from the culture supernatant by two-step column chromatography:ion exchange chromatography on Q Sepharose HP and size exclusion chromatography on Superdex 200. The yield was 67.5% and the specific activity was 33.70 U/mg. The purified enzyme had an apparent molecular mass of 98 kDa according to SDS-PAGE analysis.The amidolytic activity of Hsp70-Glo was determined by cleavage of the synthetic substrate, Na-p-tosyl-Gly-Pro-Arg-p-nitroanilide. The amidolytic activity of Hsp70-Glo was optimal at pH 7.5 and 50℃, and was inhibited by almost 93% and 36%, respectively, by the serine protease inhibitors 1 mM PMSF and 10 mM TLCK, confirming that Gloshedobin belongs to a serine protease family. Benzamidine inhibited 57% of the amidolytic activity of Hsp70-Glo, but EDTA had no effect, indicating that Gloshedobin is not metalloproteinase.Hsp70-Glo preferentially degraded fibrinogen a-chain. The enzyme also degraded fibrinogenβ-chain to a lesser extent, while showing no degradation of the y-chain. A fibrinogen clotting activity of 499.8 U/mg was achieved by the enzyme, which was within the range reported for other thrombin-like enzymes.Ⅱ. Expression and physiological activity of PcbA. Cyanobacterium Synechocystis sp. PCC 6803 only contains Chl a and uses phycobilisome as its major antenna system. It is also an engineered bacterial strain that is used for expressing algal proteins in industry. The foreign gene was inserted into the genomic DNA of Synechocystis sp. PCC 6803 through the use of the expression vector pWS19K, which contains the upstream and downstream sequences of psbAⅢ. Synechocystis sp. PCC 6803 contains two highly expressed psbA genes, psbAⅡand psbAⅢ, both of which code for the D1 protein of photosystemⅡ. Any one of these two active psbA genes can be replaced by another gene without physiological impairment to the organism, thus substituting psbAⅢfor PcbA would have no effect on the activity of photosynthesis. The expressed PcbA may be transported into the thylakoid membrane and associate with photosystemⅡ. This recombinant clone will not only permit the investigation of the mechanism of binding between PcbA and chlorophyll, but will also facilitate the investigation of the function of various light-harvesting strategies in photo synthetic organisms.The PcbA gene with 6xHis-tag sequence at the 3'end was cloned into the vector pWS19K and then transformed into Synechocystis sp. PCC 6803. Through recombination with the host DNA, the PcbA gene was inserted at the psbAⅢsite within the host DNA. The expression of PcbA was confirmed by SDS-PAGE and Western blot using anti-His-tag antibody, which showed an apparent molecular mass of about 38 kDa, consistent with the predicted theoretical molecular mass.The location and association of recombinant PcbA in Synechocystis sp. PCC 6803 were performed. The thylakoid membrane was solubilized by the detergent n-dodecyl-β-D-maltoside and then analysed by SDS-PAGE followed by Western blot using anti his-tag antibody, which showed that the recombinant PcbA was located on the thylakoid membrane. The thylokoid membrane contains two photosystem, PSI and PSII, which can be separated by Blue-Native PAGE. Blue-Native PAGE followed by Western blot of PSI/PSII showed that PcbA was associated with PSII. Second dimension electrophoresis following Blue-Native PAGE also presented the same result.The physiological activity of recombinant PcbA was also investigated. By studying the absorption spectra of both recombinant and wild-type Synechocystis sp. PCC 6803 strains at room-temperature it was found that in the recombinant strain, the relative quantity of phycobilisome decreased by about 40%, while the relative quantity of carotenoid increased by 20% compared to wild type. This suggested that the light-harvesting strategy in Synechocystis sp. PCC 6803 was affected by the presence of recombinant PcbA, and the increase of caroteniod probably led to an increase in the stability of PcbA. The fluorescence emission spectral properties of intact Synechocystis sp. PCC 6803 cells harbouring the recombinant PcbA at room temperature were measured with excitation at 435 nm. An extra shoulder at～705 nm and a dominant peak centered at 720 nm in 77 K was observed in the recombinant strain, suggesting that the newly introduced PcbA antenna complexes had induced a red-shifted peak for bound Chl a. The steady-state excitation spectra clearly showed that almost no excitation peak corresponding to phycobilisome in the recombinant strain when the emission wavelength was chosen at 685 nm (for PSII) and at 720 nm (for PSI). It provided direct evidence that incorporation of PcbA into Synechocystis sp. PCC6803 has changed the cell's light-harvesting strategies, with downregulation of phycobiliproteins and upregulation of the integral membrane-bound antenna system.In conclusion, Hsp70 used as a fusion partner to express Gloshedobin in Pichia Pastoris and synechocysits sp. PCC6803 used as a host to express a foreign chlorophyll-binding resulted in high expression levels of functional proteins. It appears that a foreign protein expressed in a vector that is derived from the host DNA is a good strategy for obtaining a high expression level of a functional protein in a highly active form.