In the present study, fusion genes composed of Thermotoga maritima MSB8 nitrilase and Bacillus subtilis 168 outer coat protein CotG were constructed with various peptide linkers and displayed on B. subtilis DB 403 spores. The successful display of CotG-nit fusion proteins on the spore surface of B. subtilis was verified by Western blot analysis and activity measurement. It was demonstrated that the fusion with linker GGGGSEAAAKGGGGS presented the highest thermal and pH stability, which is 2.67- and 1.9-fold of the fusion without linker. In addition, fusion with flexible linker (GGGGS)3 demonstrated better thermal and pH stability than fusions with linkers GGGGS and (GGGGS)2. Fusion with rigid linker (EAAAK) demonstrated better thermal stability than fusions with linkers (EAAAK)2 and (EAAAK)3. Fusions with linker (EAAAK)2 demonstrated better pH stability than fusions with linkers (EAAAK) and (EAAAK)3. In the presence of 1 m<smlcap>M</smlcap> dithiothreitol, 1% (v/v) sodium dodecyl sulfate, and 20% (v/v) ethanol, the optimal linkers of the fusions were MGSSSN, GGGGSEAAAKGGGGS, and (GGGGS)3, respectively. In summary, our results showed that optimizing the peptide linkers with different type, length, and amino acid composition of the fusion proteins would be an efficient way to maintain the stability of fusion proteins and thus improve the nitrilase display efficiency, which could provide an effective method for rational design peptide linkers of displayed nitrilase on B. subtilis.

Amet N, Lee HF, Shen WC: Insertion of the designed helical linker led to increased expression of tf-based fusion proteins. Pharm Res 2009;26:523-528.
Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T: Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng 2001;14:529-532.
Argos P: An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol 1990;211:943-958.
Bai Y, Shen WC: Improving the oral efficacy of recombinant granulocyte colony-stimulating factor and transferrin fusion protein by spacer optimization. Pharm Res 2006;23:2116-2121.
Bhaskara RM, de Brevern AG, Srinivasan N: Understanding the role of domain-domain linkers in the spatial orientation of domains in multi-domain proteins. J Biomol Struct Dyn 2013;31:1467-1480.
Chen H, Tian R, Ni Z, Zhang Q, Zhang T, Chen Z, Chen K, Yang S: Surface display of the thermophilic lipase Tm1350 on the spore of Bacillus subtilis by the CotB anchor protein. Extremophiles 2015a;19:799-808.
Chen HY, Chen Z, Ni Z, Tian R, Zhang TX, Jia JR, Chen KP, Yang SL: Display of Thermotoga maritima msb8 nitrilase on the spore surface of Bacillus subtilis using out coat protein CotG as the fusion partner. J Mol Catal B Enzym 2016;123:73-80.
Chen HY, Zhang TX, Jia JR, Vastermark A, Tian R, Ni Z, Chen Z, Chen KP, Yang SL: Expression and display of a novel thermostable esterase from Clostridium thermocellum on the surface of Bacillus subtilis using the CotB anchor protein. J Ind Microbiol Biot 2015b;42:1439-1448.
Chen XY, Zaro JL, Shen WC: Fusion protein linkers: property, design and functionality. Adv Drug Deliver Rev 2013;65:1357-1369.
Chen Z, Chen H, Ni Z, Tian R, Zhang T, Jia J, Yang S: Expression and characterization of a novel nitrilase from hyperthermophilic Bacterium Thermotoga maritima MSB8. J Microbiol Biotechnol 2015c;25:1660-1669.
Cutting SM, Horn PBV: Genetic analysis; in Harwood CR, Cutting SM (eds): Molecular Biological Methods for Bacillus. Chichester, John Wiley &amp; Sons, 1990, pp 33-35.
George RA, Heringa J: An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng 2002;15:871-879.
Ghosh S, Zhang PF, Li YQ, Setlow P: Superdormant spores of bacillus species have elevated wet-heat resistance and temperature requirements for heat activation. J Bacteriol 2009;191:5584-5591.
Hoedemaeker F, Signorelli T, Johns K, Kuntz D, Rose D: A single chain Fv fragment of P-glycoprotein-specific monoclonal antibody C219 design, expression, and crystal structure at 2.4 A resolution. J Biol Chem 1997;272:29784-29789.
Huang Z, Li G, Zhang C, Xing XH: A study on the effects of linker flexibility on acid phosphatase PHoC-GFP fusion protein using a novel linker library. Enzyme Microb Tech 2016;83:1-6.
Isticato R, Cangiano G, Tran HT, Ciabattini A, Medaglini D, Oggioni MR, De Felice M, Pozzi G, Ricca E: Surface display of recombinant proteins on Bacillus subtilis spores. J Bacteriol 2001;183:6294-6301.
Karginov AV, Hahn KM: Allosteric activation of kinases: design and application of RapR kinases; in Bonifacino JS, et al (eds): Current Protocols in Cell Biology. Hoboken, Wiley &amp; Sons, 2011.
Liu Y, Li S, Xu H, Wu L, Xu Z, Liu J, Feng X: Efficient production of D-tagatose using a food-grade surface display system. J Agric Food Chem 2014;62:6756-6762.
Lu P, Feng MG: Bifunctional enhancement of a beta-glucanase-xylanase fusion enzyme by optimization of peptide linkers. Appl Microbiol Biotechnol 2008;79:579-587.
Lu P, Feng MG, Li WF, Hu CX: Construction and characterization of a bifunctional fusion enzyme of Bacillus-sourced beta-glucanase and xylanase expressed in Escherichia coli. FEMS Microbiol Lett 2006;261:224-230.
Martins LO, Soares CM, Pereira MM, Teixeira M, Costa T, Jones GH, Henriques AO: Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J Biol Chem 2002;277:18849-18859.
Nicholson WL, Setlow P: Sporulation, germination and outgrowth; in Harwood CR, Cutting SM (eds): Molecular Biological Methods for Bacillus. Chichester, John Wiley &amp; Sons, 1990, pp 391-450.
Petriz J, Gottesman MM, Aran JM: An MDR-EGFP gene fusion allows for direct cellular localization, function and stability assessment of P-glycoprotein. Curr Drug Deliv 2004;1:43-56.
Qu YY, Wang JW, Zhang ZJ, Shi SN, Li DX, Shen WL, Shen E, Zhou JT: Catalytic transformation of hodas using an efficient meta-cleavage product hydrolase-spore surface display system. J Mol Catal B Enzym 2014;102:204-210.
Rosenblum MG, Cheung LH, Liu Y, Marks JW 3rd: Design, expression, purification, and characterization, in vitro and in vivo, of an antimelanoma single-chain Fv antibody fused to the toxin gelonin. Cancer Res 2003;63:3995-4002.
Volkel T, Korn T, Bach M, Müller R, Kontermann RE: Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies. Protein Eng 2001;14:815-823.
Yu K, Liu CC, Kim BG, Lee DY: Synthetic fusion protein design and applications. Biotechnol Adv 2015;33:155-164.
Zhu D, Mukherjee C, Yang Y, Rios BE, Gallagher DT, Smith NN, Biehl ER, Hua L: A new nitrilase from Bradyrhizobium japonicum USDA 110. Gene cloning, biochemical characterization and substrate specificity. J Biotechnol 2008;133:327-333.
You do not currently have access to this content.