Recombinant Antibodies for Specific Detection of Clostridial [Fe-Fe] Hydrogenases

Overview

Journal Sci Rep

Specialty Science

Date 2016 Oct 28

PMID 27786270

Citations 1

Authors

Rahul Mangayil

Matti Karp

Urpo Lamminmaki

Ville Santala

Affiliations

Soon will be listed here.

Abstract

Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases.

Citing Articles

Sip2: A Serum-Induced Protein That Is Essential to Serum Survival, Acid Resistance, Intracellular Replication, and Host Infection.

Li M, Sun L Front Microbiol. 2018; 9:1084.

PMID: 29887847 PMC: 5980991. DOI: 10.3389/fmicb.2018.01084.

References

Saini D, Kala M, Jain V, Sinha S . Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor. BMC Biotechnol. 2005; 5:33. PMC: 1351172. DOI: 10.1186/1472-6750-5-33. View

Akhtar M, Jones P . Deletion of iscR stimulates recombinant clostridial Fe-Fe hydrogenase activity and H2-accumulation in Escherichia coli BL21(DE3). Appl Microbiol Biotechnol. 2008; 78(5):853-62. DOI: 10.1007/s00253-008-1377-6. View

Bruggeman Y, Boogert A, van Hoek A, Jones P, Winter G, Schots A . Phage antibodies against an unstable hapten: oxygen sensitive reduced flavin. FEBS Lett. 1996; 388(2-3):242-4. DOI: 10.1016/0014-5793(96)00544-3. View

Dwyer M, Lu W, Dwyer J, Kossiakoff A . Biosynthetic phage display: a novel protein engineering tool combining chemical and genetic diversity. Chem Biol. 2000; 7(4):263-74. DOI: 10.1016/s1074-5521(00)00102-2. View

Huovinen T, Syrjanpaa M, Sanmark H, Brockmann E, Azhayev A, Wang Q . Two ScFv antibody libraries derived from identical VL-VH framework with different binding site designs display distinct binding profiles. Protein Eng Des Sel. 2013; 26(10):683-93. DOI: 10.1093/protein/gzt037. View

Haque A, Tonks N . The use of phage display to generate conformation-sensor recombinant antibodies. Nat Protoc. 2012; 7(12):2127-43. PMC: 3712638. DOI: 10.1038/nprot.2012.132. View

Gorwa M, Croux C, Soucaille P . Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. J Bacteriol. 1996; 178(9):2668-75. PMC: 177994. DOI: 10.1128/jb.178.9.2668-2675.1996. View

Swanson K, Ratzloff M, Mulder D, Artz J, Ghose S, Hoffman A . [FeFe]-hydrogenase oxygen inactivation is initiated at the H cluster 2Fe subcluster. J Am Chem Soc. 2015; 137(5):1809-16. DOI: 10.1021/ja510169s. View

Stephenson M, Stickland L . Hydrogenase: a bacterial enzyme activating molecular hydrogen: The properties of the enzyme. Biochem J. 1931; 25(1):205-14. PMC: 1260629. DOI: 10.1042/bj0250205. View

10.

Brockmann E, Lamminmaki U, Saviranta P . Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage. Biochim Biophys Acta. 2005; 1724(1-2):146-54. DOI: 10.1016/j.bbagen.2005.04.012. View

11.

Fack F, Song D, Queitsch I, Petersen G, Bautz E . Epitope mapping by phage display: random versus gene-fragment libraries. J Immunol Methods. 1997; 206(1-2):43-52. DOI: 10.1016/s0022-1759(97)00083-5. View

12.

Chung W, Sack M, Carter R, Spiegel H, Fischer R, Hirst T . Phage-display derived single-chain fragment variable (scFv) antibodies recognizing conformational epitopes of Escherichia coli heat-labile enterotoxin B-subunit. J Immunol Methods. 2008; 339(2):115-23. DOI: 10.1016/j.jim.2008.08.005. View

13.

Buhrke T, Lenz O, Krauss N, Friedrich B . Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 is based on limited access of oxygen to the active site. J Biol Chem. 2005; 280(25):23791-6. DOI: 10.1074/jbc.M503260200. View

14.

Stripp S, Goldet G, Brandmayr C, Sanganas O, Vincent K, Haumann M . How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms. Proc Natl Acad Sci U S A. 2009; 106(41):17331-6. PMC: 2765078. DOI: 10.1073/pnas.0905343106. View

15.

Santala V, Lamminmaki U . Production of a biotinylated single-chain antibody fragment in the cytoplasm of Escherichia coli. J Immunol Methods. 2004; 284(1-2):165-75. DOI: 10.1016/j.jim.2003.10.008. View

16.

Hertveldt K, Belien T, Volckaert G . General M13 phage display: M13 phage display in identification and characterization of protein-protein interactions. Methods Mol Biol. 2008; 502:321-39. DOI: 10.1007/978-1-60327-565-1_19. View

17.

Cohen J, Kim K, Posewitz M, Ghirardi M, Schulten K, Seibert M . Molecular dynamics and experimental investigation of H(2) and O(2) diffusion in [Fe]-hydrogenase. Biochem Soc Trans. 2005; 33(Pt 1):80-2. PMC: 2587414. DOI: 10.1042/BST0330080. View

18.

Girbal L, Abendroth G, Winkler M, Benton P, Meynial-Salles I, Croux C . Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities. Appl Environ Microbiol. 2005; 71(5):2777-81. PMC: 1087525. DOI: 10.1128/AEM.71.5.2777-2781.2005. View

19.

Schirrmann T, Meyer T, Schutte M, Frenzel A, Hust M . Phage display for the generation of antibodies for proteome research, diagnostics and therapy. Molecules. 2011; 16(1):412-26. PMC: 6259421. DOI: 10.3390/molecules16010412. View

20.

Smith G . Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985; 228(4705):1315-7. DOI: 10.1126/science.4001944. View