An unusual halotolerant α-type carbonic anhydrase from the alga Dunaliella salina functionally expressed in Escherichia coli
Section snippets
Strains, plasmids, biochemicals, and chemicals
The E. coli hosts BL21(DE3)pLysS [(F− ompT hsdSB (rB−mB−) gal dcm (DE3) pLysS (CmR)] and Origami B(DE3)pLysS [(F−ompThsdSB(rB−mB−) lacY1 gor522::Tn10 (TcR) trxB::kan (DE3) pLysS (CmR)], and the expression vectors pET28c and pET21c were obtained from Novagen (Madison, WI). Taq DNA polymerase (Taqzol) was purchased from Talron (Israel). Restriction enzymes were obtained from Biolabs (England). Electrophoretically purified human erythrocyte carbonic anhydrase (hCA-I) was obtained from Sigma.
Expression and purification of Dca
Our early attempts to express Dca in E. coli BL21(DE3)pLysS cells yielded mostly an insoluble recombinant protein. To increase the yield of soluble, active enzyme, we screened the effects of different parameters such as growth medium (M9 minimal medium, LB, or 2× LB), IPTG concentrations (0.1–2 mM), growth temperature (25–37 °C), cell disruption methods (sonication, freezing, and thawing in the presence or absence of detergents such as Triton X-100 or N-lauryl sarcosine and lysozyme) (data not
Acknowledgements
This study was supported by the Minerva Foundation (Germany), the Nikken-Sohonsha Corp. Hashima City, Japan, and Nature Beta Technologies, Eilat, Israel. J.L.S. is the Pickman Professor of Structural Biology.
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Cited by (36)
Stabilization of Bovine carbonic anhydrase II through rational site-specific immobilization
2018, Biochemical Engineering JournalHalotolerant carbonic anhydrase with unusual N-terminal extension from marine Hydrogenovibrio marinus as novel biocatalyst for carbon sequestration under high-salt environments
2018, Journal of CO2 UtilizationCitation Excerpt :In this regard, tolerance to these mineral salts (that is, halotolerance), along with thermostability, should be one of the most important properties for the successful application of CA to CO2 capture. Nevertheless, there have been only a few studies on halotolerant CAs, almost exclusively on an α-type CA (dsCA) from Dunaliella salina [13,19–21]. Thus, expanding the pool of halotolerant CAs might be a key starting point for the design and development of practical biocatalysts for CO2 capture under high-salt conditions.
Identification of the carbonic anhydrases from the unicellular green alga Dunaliella salina strain CCAP 19/18
2016, Algal ResearchCitation Excerpt :This implies that dCAI and dCAII can enhance photosynthetic CO2 assimilation by facilitating an increase of inorganic carbon during CO2-limiting hypersaline conditions. Due to these results, Dunaliella CAs have been considered as key enzymes for salt tolerating mechanisms [9,24,25,52]. In this study, we investigated genomic and EST sequences of D. salina strain CCAP 19/18 (obtained from US DOE/USDA Joint Genome Institute Project ID#16719) and identified eight novel D. salina CAs (DsCAs).
Dunaliella salina Hsp90 is halotolerant
2015, International Journal of Biological MacromoleculesCitation Excerpt :Consequently, the surface Dunaliella proteins but not the intracellular proteins are expected to be highly tolerant to salts since the intracellular proteins are not exposed to the hypersaline environments. The best studied examples of the halotolerance of surface proteins are the two carbonic anhydrases isolated from D. salina (dCA I and dCA II) [18–23]. These two dCAs locate at the plasma membrane of D. salina and exhibit exceptional salt tolerance.
Increased expression level and catalytic activity of internally-duplicated carbonic anhydrase from Dunaliella species by reconstitution of two separate domains
2012, Process BiochemistryCitation Excerpt :The halo-tolerant characteristic of CA has some advantages for industrial application. However, the amount of acquired CA fractions from cell extract or the amount of recombinant CA in bacterial expression system is not sufficient in our study as well as previous one [7]. When full-length heterogeneous proteins are expressed in E. coli, low yields are major problem due to the aggregation and insolubility of protein which come from some factors including large size, susceptibility to proteases, intrinsic segmental flexibility or requirements for post-translational modifications [9,10].
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Present address: Institute of Plant Genomics and Biotechnology, Southern Crop Improvement Facility, Texas A&M University, College Station, TX 77843, USA.