Biotechnology Journal
Biotechnol. J. 2015, 10, 508–509
DOI 10.1002/biot.201500051
COMMENTARY
www.biotechnology-journal.com
More than one way to skin a cat: In-situ engineering of an antibody through photo-conjugated C2 domain Andrew Kroetsch and Sheldon Park Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, NY, USA See accompanying article by Sara Kanje and Sophia Hober DOI 10.1002/biot.201400808
certain types of antibody engineering to a technology that can be easily performed in bacteria. Although C2 binds antibody tightly, covalently linkage between bound C2 and Fc should further solidify its presumed role as a vehicle for surrogate antibody engineering. Bound C2 can be crosslinked to Fc using a photoactivatable crosslinker, although earlier studies either synthesized C2 chemically or only resulted in moderate crosslinking efficiency [2]. In this Modification of antibody for immobi- 150 kDa of antibody) and can be ex- issue of Biotechnology Journal, Kanje lization and detection can be achieved pressed in bacteria. Therefore, this and Hober concurrently simplified the through chemical crosslinking, but the strategy has a potential to simplify application of the technology and improved its efficiency by process is inherently stobiosynthesizing unnatural chastic and the extent and amino acid-containing C2 location of modification are and by optimizing the posidifficult to control (Fig. 1A). tion of the incorporated Crosslinking should ideally unnatural amino acid [3]. occur in the Fc region to The engineered C2 contains avoid blocking access to the two photoactivatable amino complementarity determinacids, p-benzoylphenylalaing regions (CDR) of the nine (BPA), and crosslinks variable domains [1]. The to Fc with 90% efficiency. bacterial protein C2 is useWhile the current design ful for targeting Fc, since it optimizes crosslinking to binds the domain with high human Fc, the biosynthetic affinity (Fig. 1B). Once C2 is route used for C2 producassociated with the antition makes it straightforbody, it becomes an extenward to repeat the study for sion of the antibody, and other species. any modification to the proAny modification to cotein is tantamount to a modvalently linked C2 should be ification to the antibody. equivalent to directly modiThis is significant from a Figure 1. (A) Chemical labeling leads to non-specific modification (green fying the Fc domain. The practical standpoint be- dots), some of which may interfere with the antibody function. (B) C2 (gray) authors demonstrate one cause one can engineer an binds specifically to the Fc domain and can be crosslinked to Fc through engineered BPAs. (C) C2 may be further engineered to include other useful propersuch example, in which C2 antibody indirectly by engi- ties, such as targeted biotinylation (triangle). The biotinylated C2 and the is genetically fused to a bineering an associated C2, conjugated antibody may be immobilized on a streptavidin coated surface. otin acceptor peptide to alwhich is much smaller in (D) C2 may be engineered instead to contain a fluorescent tag, e.g. GFP low enzymatic biotinylation size (~6 kDa compared to (green). The figures are not to the scale. Chemical modification of an antibody is typically needed for immobilization and functionalization. Targeting the modification to the Fc domain is preferred in this regard in order to preserve antibody-antigen interaction. One rational way to achieve site selectivity is through the C2 domain of protein G that specifically binds to the Fc domain. An antibody can be modified “in situ” by crosslinking C2 that has been engineered to introduce new functionality. The strategy just took a big leap forward with the targeted photocrosslinking technique discussed by Kanje and Hober, who show that C2 purified from bacteria can be crosslinked to Fc through an enzymatically incorporated unnatural amino acid.
508
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnology Journal
Biotechnol. J. 2015, 10, 508–509 www.biotecvisions.com
www.biotechnology-journal.com
are also other Fc-binding proteins and peptides, some as small as one or two helices [5–7]. Although the binding surfaces somewhat overlap, each protein binds Fc in a unique manner and may thus crosslink differently. The property of the labeled antibody may also depend on the conjugated domain. It would be interesting to compare different crosslinking mechanisms and evaluate their performance. By fusing several venues of protein engineering, Kanje and Hober just opened a treasure trove of new investigative leads. The authors declare no financial or commercial conflict of interest.
References [1] Vashist, S.K., Dixit, C.K., MacCraith, B.D., O’Kennedy, R., Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonancebased immunoassay. Analyst. 2011, 136, 4431–4436. [2] Jung, Y., Lee, J.M., Kim, J.W., Yoon, J. et al., Photoactivable antibody binding protein:
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Site-selective and covalent coupling of antibody. Anal Chem. 2009, 81, 936–942. [3] Kanje, S., Hober, S., In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody binding domain improve site-specific labeling of antibodies. Biotechnol. J. 2015, DIO:10.1002/biot.201400808. [4] Demonte, D., Dundas, C.M., Park, S., Expression and purification of soluble monomeric streptavidin in Escherichia coli. Appl Microbiol Biotechnol. 2014, 98, 6285–6295. [5] Yu, F., Järver, P., Nygren, P-Å., Tailor-making a protein a-derived domain for efficient site-specific photocoupling to Fc of mouse IgG1. PLoS ONE 2013, 8, e56597. [6] Perols, A., Karlström, A.E., Site-specific photoconjugation of antibodies using chemically synthesized IgG-binding domains. Bioconjug Chem 2014, 25, 481–488. [7] Löfblom, J., Feldwisch, J., Tolmachev, V., Carlsson, J. et al., Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS lett 2010, 584, 2670–2680.
Correspondence: Prof. Sheldon Park E-mail: [email protected]
509
COMMENTARY
by the co-expressed biotin ligase. The antibody-C2 complex can be immobilized on a neutravidin-coated surface through the biotin tag while preserving efficient antigen detection (Fig. 1C). Similarly, the C2-labeled antibody is proficient in detecting an antigen captured on an ELISA plate. It will be interesting to see if the technique is amenable to other types of modification, such as fluorescence modification. For example, it may be possible to obtain a nearly homogenous population of fluorescently labeled antibody if C2 is genetically fused to GFP, which would enable more accurate quantification of the antigen based on fluorescence measurement (Fig. 1D). The current study points to several experimental variations that can be easily tested. For example, biotinylated C2 is currently purified by HPLC because in vivo biotinylation is not complete. Instead, affinity purification on recently reported monomeric streptavidin resin [4] may reduce the effort needed to isolate modified C2, streamlining the workflow even more. There
ISSN 1860-6768 · BJIOAM 10 (4) 501–653 (2015) · Vol. 10 · April 2015
Systems & Synthetic Biology · Nanobiotech · Medicine
4/2015 Biofuels Plant Biotechnology Bioenergy
Cover illustration
www.biotechnology-journal.com
Special issue: Renewable Energy Crops, edited by Margit Laimer, Fatemeh Maghuly, Johann Vollmann and Nicolas Carels. Given the growing importance of environmental advantages of biofuel plants reducing greenhouse gas emissions as compared to fossil fuel consumption, second and third generation biofuels today are produced from both annual and perennial non-food biofuel crops, e.g. switchgrass, poplar and Miscanthus, or from new and non-food oil crops, like Jatropha, Camelina and oil palm. To achieve an economic production of biofuels for our future need of sustainable energy, genetic improvement of the plant material must be obtained by a range of different biotechnologies.
Biotechnology Journal – list of articles published in the April 2015 issue. Editorial: Sustainable production of renewable energy from non-food crops Margit Laimer, Fatemeh Maghuly, Johann Vollmann and Nicolas Carels http://dx.doi.org/10.1002/biot.201500100 Forum Perennial plants for biofuel production: Bridging genomics and field research Alexandre Alonso Alves, Bruno G. Laviola, Eduardo F. Formighieri, Nicolas Carels
http://dx.doi.org/10.1002/biot.201400201
Research Article Transgenic switchgrass (Panicum virgatum L.) biomass is increased by overexpression of switchgrass sucrose synthase (PvSUS1) Charleson R. Poovaiah, Mitra Mazarei, Stephen R. Decker, Geoffrey B. Turner, Robert W. Sykes, Mark F. Davis and C. Neal Stewart, Jr.
http://dx.doi.org/10.1002/biot.201400499 Research Article In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies
Commentary More than one way to skin a cat: in-situ engineering of an antibody through photo-conjugated C2 domain
Sara Kanje and Sophia Hober
Andrew Kroetsch and Sheldon Park
Research Article Xylan catabolism is improved by blending bioprospecting and metabolic pathway engineering in Saccharomyces cerevisiae
http://dx.doi.org/10.1002/biot.201500051 Review Using Populus as a lignocellulosic feedstock for bioethanol
http://dx.doi.org/10.1002/biot.201400808
Sun-Mi Lee, Taylor Jellison, and Hal S. Alper
http://dx.doi.org/10.1002/biot.201400622
Ilga Porth and Yousry A. El-Kassaby
http://dx.doi.org/10.1002/biot.201400194 Review Camelina as a sustainable oilseed crop: Contributions of plant breeding and genetic engineering Johann Vollmann and Christina Eynck
Biotech Methods Cellulose-based filter aids increase the capacity of depth filters during the downstream processing of plant-derived biopharmaceutical proteins Johannes F. Buyel, Patrick Opdensteinen, Rainer Fischer
http://dx.doi.org/10.1002/biot.201400611
http://dx.doi.org/10.1002/biot.201400200 Research Article Geographic origin is not supported by the genetic variability found in a large living collection of Jatropha curcas with accessions from three continents
Research Article Ca2+ and Mg2+ binding site engineering increases the degradation of polyethylene terephthalate films by polyester hydrolases from Thermobifida fusca
Fatemeh Maghuly, Joanna Jankowicz-Cieslak, Stephan Pabinger, Bradley J. Till and Margit Laimer
Johannes Then, Ren Wei, Thorsten Oeser, Markus Barth, Matheus R. Belisário-Ferrari, Juliane Schmidt and Wolfgang Zimmermann
http://dx.doi.org/10.1002/biot.201400196
http://dx.doi.org/10.1002/biot.201400620
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
Research Article Production of curcuminoids from tyrosine by a metabolically engineered Escherichia coli using caffeic acid as an intermediate Joana L. Rodrigues, Rafael G. Araújo, Kristala L. J. Prather, Leon D. Kluskens, and Ligia R. Rodrigues
http://dx.doi.org/10.1002/biot.201400637 Research Article Buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions: From protein buffer capacity prediction to bioprocess applications
Research Article Indole generates quiescent and metabolically active Escherichia coli cultures Chih-Chin Chen, Rupali Walia, Krishna J. Mukherjee, Subhashree Mahalik and David K. Summers
http://dx.doi.org/10.1002/biot.201400381 Biotech Method Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines Eric Kowarz, Denise Löscher, Rolf Marschalek
http://dx.doi.org/10.1002/biot.201400821
Sven Bahrenburg, Anne R. Karow, Patrick Garidel
http://dx.doi.org/10.1002/biot.201400531 Research Article Over-expression of ICE2 stabilizes cytochrome P450 reductase in Saccharomyces cerevisiae and Pichia pastoris Anita Emmerstorfer, Miriam Wimmer-Teubenbacher, Tamara Wriessnegger, Erich Leitner, Monika Müller, Iwona Kaluzna, Martin Schürmann, Daniel Mink, Günther Zellnig, Helmut Schwab and Harald Pichler
http://dx.doi.org/10.1002/biot.201400780
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
Biotechnol. J. 2015, 10, 508–509
DOI 10.1002/biot.201500051
COMMENTARY
www.biotechnology-journal.com
More than one way to skin a cat: In-situ engineering of an antibody through photo-conjugated C2 domain Andrew Kroetsch and Sheldon Park Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, NY, USA See accompanying article by Sara Kanje and Sophia Hober DOI 10.1002/biot.201400808
certain types of antibody engineering to a technology that can be easily performed in bacteria. Although C2 binds antibody tightly, covalently linkage between bound C2 and Fc should further solidify its presumed role as a vehicle for surrogate antibody engineering. Bound C2 can be crosslinked to Fc using a photoactivatable crosslinker, although earlier studies either synthesized C2 chemically or only resulted in moderate crosslinking efficiency [2]. In this Modification of antibody for immobi- 150 kDa of antibody) and can be ex- issue of Biotechnology Journal, Kanje lization and detection can be achieved pressed in bacteria. Therefore, this and Hober concurrently simplified the through chemical crosslinking, but the strategy has a potential to simplify application of the technology and improved its efficiency by process is inherently stobiosynthesizing unnatural chastic and the extent and amino acid-containing C2 location of modification are and by optimizing the posidifficult to control (Fig. 1A). tion of the incorporated Crosslinking should ideally unnatural amino acid [3]. occur in the Fc region to The engineered C2 contains avoid blocking access to the two photoactivatable amino complementarity determinacids, p-benzoylphenylalaing regions (CDR) of the nine (BPA), and crosslinks variable domains [1]. The to Fc with 90% efficiency. bacterial protein C2 is useWhile the current design ful for targeting Fc, since it optimizes crosslinking to binds the domain with high human Fc, the biosynthetic affinity (Fig. 1B). Once C2 is route used for C2 producassociated with the antition makes it straightforbody, it becomes an extenward to repeat the study for sion of the antibody, and other species. any modification to the proAny modification to cotein is tantamount to a modvalently linked C2 should be ification to the antibody. equivalent to directly modiThis is significant from a Figure 1. (A) Chemical labeling leads to non-specific modification (green fying the Fc domain. The practical standpoint be- dots), some of which may interfere with the antibody function. (B) C2 (gray) authors demonstrate one cause one can engineer an binds specifically to the Fc domain and can be crosslinked to Fc through engineered BPAs. (C) C2 may be further engineered to include other useful propersuch example, in which C2 antibody indirectly by engi- ties, such as targeted biotinylation (triangle). The biotinylated C2 and the is genetically fused to a bineering an associated C2, conjugated antibody may be immobilized on a streptavidin coated surface. otin acceptor peptide to alwhich is much smaller in (D) C2 may be engineered instead to contain a fluorescent tag, e.g. GFP low enzymatic biotinylation size (~6 kDa compared to (green). The figures are not to the scale. Chemical modification of an antibody is typically needed for immobilization and functionalization. Targeting the modification to the Fc domain is preferred in this regard in order to preserve antibody-antigen interaction. One rational way to achieve site selectivity is through the C2 domain of protein G that specifically binds to the Fc domain. An antibody can be modified “in situ” by crosslinking C2 that has been engineered to introduce new functionality. The strategy just took a big leap forward with the targeted photocrosslinking technique discussed by Kanje and Hober, who show that C2 purified from bacteria can be crosslinked to Fc through an enzymatically incorporated unnatural amino acid.
508
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnology Journal
Biotechnol. J. 2015, 10, 508–509 www.biotecvisions.com
www.biotechnology-journal.com
are also other Fc-binding proteins and peptides, some as small as one or two helices [5–7]. Although the binding surfaces somewhat overlap, each protein binds Fc in a unique manner and may thus crosslink differently. The property of the labeled antibody may also depend on the conjugated domain. It would be interesting to compare different crosslinking mechanisms and evaluate their performance. By fusing several venues of protein engineering, Kanje and Hober just opened a treasure trove of new investigative leads. The authors declare no financial or commercial conflict of interest.
References [1] Vashist, S.K., Dixit, C.K., MacCraith, B.D., O’Kennedy, R., Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonancebased immunoassay. Analyst. 2011, 136, 4431–4436. [2] Jung, Y., Lee, J.M., Kim, J.W., Yoon, J. et al., Photoactivable antibody binding protein:
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Site-selective and covalent coupling of antibody. Anal Chem. 2009, 81, 936–942. [3] Kanje, S., Hober, S., In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody binding domain improve site-specific labeling of antibodies. Biotechnol. J. 2015, DIO:10.1002/biot.201400808. [4] Demonte, D., Dundas, C.M., Park, S., Expression and purification of soluble monomeric streptavidin in Escherichia coli. Appl Microbiol Biotechnol. 2014, 98, 6285–6295. [5] Yu, F., Järver, P., Nygren, P-Å., Tailor-making a protein a-derived domain for efficient site-specific photocoupling to Fc of mouse IgG1. PLoS ONE 2013, 8, e56597. [6] Perols, A., Karlström, A.E., Site-specific photoconjugation of antibodies using chemically synthesized IgG-binding domains. Bioconjug Chem 2014, 25, 481–488. [7] Löfblom, J., Feldwisch, J., Tolmachev, V., Carlsson, J. et al., Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS lett 2010, 584, 2670–2680.
Correspondence: Prof. Sheldon Park E-mail: [email protected]
509
COMMENTARY
by the co-expressed biotin ligase. The antibody-C2 complex can be immobilized on a neutravidin-coated surface through the biotin tag while preserving efficient antigen detection (Fig. 1C). Similarly, the C2-labeled antibody is proficient in detecting an antigen captured on an ELISA plate. It will be interesting to see if the technique is amenable to other types of modification, such as fluorescence modification. For example, it may be possible to obtain a nearly homogenous population of fluorescently labeled antibody if C2 is genetically fused to GFP, which would enable more accurate quantification of the antigen based on fluorescence measurement (Fig. 1D). The current study points to several experimental variations that can be easily tested. For example, biotinylated C2 is currently purified by HPLC because in vivo biotinylation is not complete. Instead, affinity purification on recently reported monomeric streptavidin resin [4] may reduce the effort needed to isolate modified C2, streamlining the workflow even more. There
ISSN 1860-6768 · BJIOAM 10 (4) 501–653 (2015) · Vol. 10 · April 2015
Systems & Synthetic Biology · Nanobiotech · Medicine
4/2015 Biofuels Plant Biotechnology Bioenergy
Cover illustration
www.biotechnology-journal.com
Special issue: Renewable Energy Crops, edited by Margit Laimer, Fatemeh Maghuly, Johann Vollmann and Nicolas Carels. Given the growing importance of environmental advantages of biofuel plants reducing greenhouse gas emissions as compared to fossil fuel consumption, second and third generation biofuels today are produced from both annual and perennial non-food biofuel crops, e.g. switchgrass, poplar and Miscanthus, or from new and non-food oil crops, like Jatropha, Camelina and oil palm. To achieve an economic production of biofuels for our future need of sustainable energy, genetic improvement of the plant material must be obtained by a range of different biotechnologies.
Biotechnology Journal – list of articles published in the April 2015 issue. Editorial: Sustainable production of renewable energy from non-food crops Margit Laimer, Fatemeh Maghuly, Johann Vollmann and Nicolas Carels http://dx.doi.org/10.1002/biot.201500100 Forum Perennial plants for biofuel production: Bridging genomics and field research Alexandre Alonso Alves, Bruno G. Laviola, Eduardo F. Formighieri, Nicolas Carels
http://dx.doi.org/10.1002/biot.201400201
Research Article Transgenic switchgrass (Panicum virgatum L.) biomass is increased by overexpression of switchgrass sucrose synthase (PvSUS1) Charleson R. Poovaiah, Mitra Mazarei, Stephen R. Decker, Geoffrey B. Turner, Robert W. Sykes, Mark F. Davis and C. Neal Stewart, Jr.
http://dx.doi.org/10.1002/biot.201400499 Research Article In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies
Commentary More than one way to skin a cat: in-situ engineering of an antibody through photo-conjugated C2 domain
Sara Kanje and Sophia Hober
Andrew Kroetsch and Sheldon Park
Research Article Xylan catabolism is improved by blending bioprospecting and metabolic pathway engineering in Saccharomyces cerevisiae
http://dx.doi.org/10.1002/biot.201500051 Review Using Populus as a lignocellulosic feedstock for bioethanol
http://dx.doi.org/10.1002/biot.201400808
Sun-Mi Lee, Taylor Jellison, and Hal S. Alper
http://dx.doi.org/10.1002/biot.201400622
Ilga Porth and Yousry A. El-Kassaby
http://dx.doi.org/10.1002/biot.201400194 Review Camelina as a sustainable oilseed crop: Contributions of plant breeding and genetic engineering Johann Vollmann and Christina Eynck
Biotech Methods Cellulose-based filter aids increase the capacity of depth filters during the downstream processing of plant-derived biopharmaceutical proteins Johannes F. Buyel, Patrick Opdensteinen, Rainer Fischer
http://dx.doi.org/10.1002/biot.201400611
http://dx.doi.org/10.1002/biot.201400200 Research Article Geographic origin is not supported by the genetic variability found in a large living collection of Jatropha curcas with accessions from three continents
Research Article Ca2+ and Mg2+ binding site engineering increases the degradation of polyethylene terephthalate films by polyester hydrolases from Thermobifida fusca
Fatemeh Maghuly, Joanna Jankowicz-Cieslak, Stephan Pabinger, Bradley J. Till and Margit Laimer
Johannes Then, Ren Wei, Thorsten Oeser, Markus Barth, Matheus R. Belisário-Ferrari, Juliane Schmidt and Wolfgang Zimmermann
http://dx.doi.org/10.1002/biot.201400196
http://dx.doi.org/10.1002/biot.201400620
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
Research Article Production of curcuminoids from tyrosine by a metabolically engineered Escherichia coli using caffeic acid as an intermediate Joana L. Rodrigues, Rafael G. Araújo, Kristala L. J. Prather, Leon D. Kluskens, and Ligia R. Rodrigues
http://dx.doi.org/10.1002/biot.201400637 Research Article Buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions: From protein buffer capacity prediction to bioprocess applications
Research Article Indole generates quiescent and metabolically active Escherichia coli cultures Chih-Chin Chen, Rupali Walia, Krishna J. Mukherjee, Subhashree Mahalik and David K. Summers
http://dx.doi.org/10.1002/biot.201400381 Biotech Method Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines Eric Kowarz, Denise Löscher, Rolf Marschalek
http://dx.doi.org/10.1002/biot.201400821
Sven Bahrenburg, Anne R. Karow, Patrick Garidel
http://dx.doi.org/10.1002/biot.201400531 Research Article Over-expression of ICE2 stabilizes cytochrome P450 reductase in Saccharomyces cerevisiae and Pichia pastoris Anita Emmerstorfer, Miriam Wimmer-Teubenbacher, Tamara Wriessnegger, Erich Leitner, Monika Müller, Iwona Kaluzna, Martin Schürmann, Daniel Mink, Günther Zellnig, Helmut Schwab and Harald Pichler
http://dx.doi.org/10.1002/biot.201400780
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
