In vitro selection by display methods has been an effective tool for engineering recombinant antibodies. mRNA display based on a cell-free translation system has the advantages of larger library sizes and quicker selection procedures compared with cell-based display methods such as phage display. However, mRNA display has been limited to select single-chain polypeptides such as scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. Here we demonstrated a new way of selecting heterodimeric Fab fragments by using mRNA display combined with emulsion PCR. We designed a pair of complementary 5′ UTR sequences that can link the Fab heavy and light chain genes together by overlap-extension PCR in water-in-oil emulsions. We confirmed that two mRNA-displayed polypeptides for heavy and light chain of a model Fab fragment were associated into the active form and that a specific Fab fragment gene was enriched over 100-fold per round of a model affinity selection followed by the gene-linking emulsion PCR. We further performed directed evolution of Fab fragments with higher binding activity from a randomized Fab fragment library. 1. Introduction In vitro selection by display methods has been an effective tool in the field of protein engineering and especially has been used to engineer recombinant antibodies for various biological applications [1]. Phage display has been widely used in the industry due to its feasibility to select Fab fragments [2]. The Fab fragment of an immunoglobulin is a heterodimer of the N-terminal half of a heavy (H) chain and a complete light (L) chain. Because the Fab is more native-like than the single-chain Fv (scFv), which is the other commonly used recombinant antibody format for in vitro selection, the Fab fragment format makes it able to select more practical antibodies [3]. Other than phage display, cell-free translation-based methods such as ribosome display [4] and mRNA display [5] are being used for in vitro selection of antibodies due to its advantage of permitting speedier selection from larger size libraries than cell-based methods. However, these cell-free translation-based methods are limited to select scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. To overcome this limit, we have recently developed a bicistronic DNA display to select Fab fragments in a cell-free translation system [6]. Bicistronic DNA display relies on in vitro compartmentalization in water-in-oil emulsions [7], and the man-made cell-like compartments make
References
[1]
H. R. Hoogenboom, “Selecting and screening recombinant antibody libraries,” Nature Biotechnology, vol. 23, no. 9, pp. 1105–1116, 2005.
[2]
H. R. Hoogenboom, A. D. Griffiths, K. S. Johnson, D. J. Chiswell, P. Hudson, and G. Winter, “Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains,” Nucleic Acids Research, vol. 19, no. 15, pp. 4133–4137, 1991.
[3]
V. Quintero-Hernández, V. R. Juárez-González, M. Ortíz-León, R. Sánchez, L. D. Possani, and B. Becerril, “The change of the scFv into the Fab format improves the stability and in vivo toxin neutralization capacity of recombinant antibodies,” Molecular Immunology, vol. 44, no. 6, pp. 1307–1315, 2007.
[4]
J. Hanes and A. Plückthun, “In vitro selection and evolution of functional proteins by using ribosome display,” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 10, pp. 4937–4942, 1997.
[5]
I. Fukuda, K. Kojoh, N. Tabata et al., “In vitro evolution of single-chain antibodies using mRNA display,” Nucleic Acids Research, vol. 34, no. 19, article e127, 2006.
[6]
T. Sumida, N. Doi, and H. Yanagawa, “Bicistronic DNA display for in vitro selection of Fab fragments,” Nucleic Acids Research, vol. 37, no. 22, article e147, pp. e147–e147, 2009.
[7]
D. S. Tawfik and A. D. Griffiths, “Man-made cell-like compartments for molecular evolution,” Nature Biotechnology, vol. 16, no. 7, pp. 652–656, 1998.
[8]
M. Nakano, J. Komatsu, S. I. Matsuura, K. Takashima, S. Katsura, and A. Mizuno, “Single-molecule PCR using water-in-oil emulsion,” Journal of Biotechnology, vol. 102, no. 2, pp. 117–124, 2003.
[9]
T. Kojima, Y. Takei, M. Ohtsuka, Y. Kawarasaki, T. Yamane, and H. Nakano, “PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets,” Nucleic Acids Research, vol. 33, no. 17, article no. e150, 2005.
[10]
R. Williams, S. G. Peisajovich, O. J. Miller, S. Magdassi, D. S. Tawfik, and A. D. Griffiths, “Amplification of complex gene libraries by emulsion PCR,” Nature Methods, vol. 3, no. 7, pp. 545–550, 2006.
[11]
F. Diehl, M. Li, Y. He, K. W. Kinzler, B. Vogelstein, and D. Dressman, “BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions,” Nature Methods, vol. 3, no. 7, pp. 551–559, 2006.
[12]
L. Jermutus, A. Honegger, F. Schwesinger, J. Hanes, and A. Plückthun, “Tailoring in vitro evolution for protein affinity or stability,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 1, pp. 75–80, 2001.
[13]
C. Zahnd, S. Spinelli, B. Luginbühl, P. Amstutz, C. Cambillau, and A. Plückthun, “Directed in Vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity,” Journal of Biological Chemistry, vol. 279, no. 18, pp. 18870–18877, 2004.
[14]
B. Luginbühl, Z. Kanyo, R. M. Jones et al., “Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation,” Journal of Molecular Biology, vol. 363, no. 1, pp. 75–97, 2006.
[15]
G. Thom, A. C. Cockroft, A. G. Buchanan et al., “Probing a protein-protein interaction by in vitro evolution,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 20, pp. 7619–7624, 2006.
[16]
H. Persson, H. Wallmark, A. Ljungars, J. Hallborn, and M. Ohlin, “In vitro evolution of an antibody fragment population to find high-affinity hapten binders,” Protein Engineering, Design and Selection, vol. 21, no. 8, pp. 485–493, 2008.
[17]
Y. Shimizu, T. Kanamori, and T. Ueda, “Protein synthesis by pure translation systems,” Methods, vol. 36, no. 3, pp. 299–304, 2005.
[18]
P. A. C. 'T Hoen, S. M. G. Jirka, B. R. Ten Broeke et al., “Phage display screening without repetitious selection rounds,” Analytical Biochemistry, vol. 421, no. 2, pp. 622–631, 2012.
[19]
N. Tabata, Y. Sakuma, Y. Honda et al., “Rapid antibody selection by mRNA display on a microfluidic chip,” Nucleic Acids Research, vol. 37, no. 8, article no. e64, 2009.
[20]
S. R. Nirantar and F. J. Ghadessy, “Compartmentalized linkage of genes encoding interacting protein pairs,” Proteomics, vol. 11, no. 7, pp. 1335–1339, 2011.
[21]
C. Tuerk and L. Gold, “Systemic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase,” Science, vol. 249, no. 4968, pp. 505–510, 1990.
[22]
T. Morii, M. Hagihara, S. I. Sato, and K. Makino, “In vitro selection of ATP-binding receptors using a ribonucleopeptide complex,” Journal of the American Chemical Society, vol. 124, no. 17, pp. 4617–4622, 2002.
[23]
S. I. Sato, M. Fukuda, M. Hagihara, Y. Tanabe, K. Ohkubo, and T. Morii, “Stepwise molding of a highly selective ribonucleopeptide receptor,” Journal of the American Chemical Society, vol. 127, no. 1, pp. 30–31, 2005.
[24]
H. Zhao and W. Zha, “In vitro ‘sexual' evolution through the PCR-based staggered extension process (StEP),” Nature Protocols, vol. 1, no. 4, pp. 1865–1871, 2006.
[25]
J. Olejnik, S. Sonar, E. Krzyma？ska-Olejnik, and K. J. Rothschild, “Photocleavable biotin derivatives: a versatile approach for the isolation of biomolecules,” Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 16, pp. 7590–7594, 1995.
