Team:Bielefeld-CeBiTec/Parts
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The submission of genes as standardized parts, is a fundamental part of the iGEM engineering biology approach. This page lists all parts our team constructed which have been used for the realization of our project. The aim of our project was to detect hormones of the female cycle using the antibody variants scFvs and nanobodies. Our parts were cloned using E. coli as chassis and using the pTXB1 vector for expression and purification.
In our project we grafted two nanobodies against estradiol and progesterone. The grafting process starts with the grafting of the CDRs 1 to 3 from mAbs (monoclonal antibodies) against our targets estradiol and progesterone onto a nanobody scaffold. This scaffold is 3DWT ( BBa_K3410001 ). Immobilisation on the gold surface will be performed via the immobilization his tag SHGGH [1].
These grafts were cloned for affinity maturation via phagemid display into the phagemid vector pZMB0062. The used vector pZMB0062 is a direct derivative of pAK100 [2]. A special feature of this vector is the trypsin cleavage site (KDIR) between the beta-lactamase gene and gene III. This can be used during a phage display to elute specifically bound phages from the antigen without using a strong pH gradient. This has the advantage that reinfection efficiency is not affected. However, trypsin treatment can make panning more complicated. Especially if antigens with high protease sensitivity, whole cells or tissues are used for selection [3].
In our project, pZMB0062 was used for the phagemid display. Via SfiI restriction digest, the nanobody fragments are cloned into pZMB0062 and are thus fused to the M13 geneIII protein. Subsequently they are presented on the surface of the phage for panning. Through phagemid display the grafted nanobodies undergo affinity maturation.
scFv (single chain fragment variable) are artificially produced antibodies fragments consisting of a variable region of the heavy (VH) and light (VL) chain.The two chains are covalently linked together with a flexible peptide linker. The main advantage is that expression in E. coli is possible and it is possible to improve or change the properties of the scFv's, such as opening or specificity, by protein engineering [4].
All cloning work of the scFv's was performed using the expression vector pTXB1 (figure 7) ( pTXB1-vector) backbone. The vector pTXB1 was chosen due to its suitability for protein expression. By inserting the desired gene sequence upstream of the Mxe intein/Chitin binding domain, the gene of interest can be expressed as a fusion protein. Due to the chitin binding domain, affinity purification of the fusion protein can be performed using chitin bound to a column and the tag can be cleaved off the protein by a thiol-induced cleavage reaction [5], [6].
Figure 8 shows the self-designed scfv fragment for progesterone. In addition to the sequence for the light (VL) and heavy chain (VH), it also contains a linker, which is the connecting element between the two chains. The linker also provides a way to immobilize the expressed scfv on a gold surface. In addition, it contains hydrophilic amino acids such as glycine (G) and serine (S) as well as histidine (H), which mediates the connection between the gold surface and the scfv [1]. Furthermore, the fragment has a Gibson overhang, which was used for cloning into the pTXB1 vector. Additionally the restriction enzymes NdeI and SpeI are marked.
[1] Chain Fragment Variable Antibody for Immunosensors“, Anal. Chem., Bd. 77, Nr. 21, S. 6834–6842, Nov. 2005, doi: 10.1021/ac0507690.
[2] A. Krebber u. a., „Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system“, J. Immunol. Methods, Bd. 201, Nr. 1, S. 35–55, Feb. 1997, doi: 10.1016/S0022-1759(96)00208-6.
[3] „An improved helper phage system for efficient isolation of specific antibody molecules in phage display“. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC101254/ (zugegriffen Okt. 27, 2020).
[4] M. ARSLAN, D. KARADAĞ, und S. KALYONCU, „Protein engineering approaches for antibody fragments: directed evolution and rational design approaches“, Turk. J. Biol., Bd. 43, Nr. 1, S. 1–12, Feb. 2019, doi: 10.3906/biy-1809-28.
[5] E. S. Hosseini, R. Moniri, Y. D. Goli, und H. H. Kashani, „Purification of Antibacterial CHAPK Protein Using a Self-Cleaving Fusion Tag and Its Activity Against Methicillin-Resistant Staphylococcus aureus“, Probiotics Antimicrob. Proteins, Bd. 8, Nr. 4, S. 202–210, Dez. 2016, doi: 10.1007/s12602-016-9236-8.
[6] N. H. Shah und A. J. Stevens, „Identification, Characterization, and Optimization of Split Inteins“, in Expressed Protein Ligation: Methods and Protocols, M. Vila-Perelló, Hrsg. New York, NY: Springer US, 2020, S. 31–54.
Parts
Overview
| Part | Description | Design |
|---|---|---|
| BBa_K3410001 | nanobody scaffold suitable for grafting | Neele Kusch and Stephan Weber |
| BBa_K3410002 | selfmade grafted nanobody against estradiol | Neele Kusch and Stephan Weber |
| BBa_K3410004 | selfmade grafted nanobody against progesteron | Neele Kusch and Stephan Weber |
| BBa_K3410005 | selfmade grafted scfvagainst progesteron | Laura Keller and Philipp Kühnel | BBa_K3410000 | Flavin-binding fluorescent protein (FbFP B2) | Melina Müller and Tim Kettler | BBa_K3410007 | Flavin-binding fluorescent protein (FbFP B2)wildtype | Melina Müller and Tim Kettler |
Nanobodies
Figure 1: Map of the used nanobody scaffold 3DWT. Marked in purple are the CDRs 1 to 3 and in grey the framework regions. This map was generated with SnapGene.
Figure 2: Map of the grafted estradiol nanobody. The CDRs 1 to 3 (purple) were taken from the estradiol mAb 10G6D6 and grafted onto the scaffold 3DWT (grey). For cloning purposes, SfiI restriction sites were added at 5´ and 3´-ends. Furthermore, to immobilize the nanobody after affinity maturation onto the gold surface of our chip, a his-tag was added at the 3´-end. This map was created with SnapGene.
Figure 3: Map of the grafted progesterone nanobody. The CDRs 1 to 3 (purple) were taken from the progesterone mAb 15G12C12G11 and grafted onto the scaffold 3DWT (grey). For cloning purposes, SfiI restriction sites were added at 5´ and 3´-ends. Furthermore, to immobilize the nanobody after affinity maturation onto the gold surface of our chip, a his tag was added at the 3´-end. This map was created with SnapGene.
Figure 4: Plasmid map of the pZMB0062 vector without an insert. Emphasized are the restriction sites of the restriction enzyme SfiI and the KDIR trypsin cleavage site. The plasmid map was generated via SnapGene.
Figure 5: Map of pZMB0062 with the estradiol nanobody fragment (highlighted in brown) as insert. Cloning was done via SfiI restriction digest. This map was created using SnapGene.
Figure 6: Map of pZMB0062 with the progesterone nanobody fragment (highlighted in brown) as insert. Cloning was done via SfiI restriction digest. This map was created using SnapGene.
ScFv
All cloning work of the scFv's was performed using the expression vector pTXB1 (figure 7) ( pTXB1-vector) backbone. The vector pTXB1 was chosen due to its suitability for protein expression. By inserting the desired gene sequence upstream of the Mxe intein/Chitin binding domain, the gene of interest can be expressed as a fusion protein. Due to the chitin binding domain, affinity purification of the fusion protein can be performed using chitin bound to a column and the tag can be cleaved off the protein by a thiol-induced cleavage reaction [5], [6].
Figure 7: Plasmid map of the pTXB1 vector with inserted progesterone scfv fragment. The plasmid contains the selection marker AmpR and Mxe intein/chitin binding domain, which is used for purification of the expressed scfv. The restriction sites of NdeI and SpeI are highlighted. The plasmid map was generated via SnapGene.
Figure 8 shows the self-designed scfv fragment for progesterone. In addition to the sequence for the light (VL) and heavy chain (VH), it also contains a linker, which is the connecting element between the two chains. The linker also provides a way to immobilize the expressed scfv on a gold surface. In addition, it contains hydrophilic amino acids such as glycine (G) and serine (S) as well as histidine (H), which mediates the connection between the gold surface and the scfv [1]. Furthermore, the fragment has a Gibson overhang, which was used for cloning into the pTXB1 vector. Additionally the restriction enzymes NdeI and SpeI are marked.
Figure 8 Schematic illustration of the used Scfv fragment. The 1312 bp gene fragment is shown with the respective Gibson overlaps and the heavy and light chains connected by a linker. Additionally marked are the restriction enzymes NdeI and SpeI.
References
[2] A. Krebber u. a., „Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system“, J. Immunol. Methods, Bd. 201, Nr. 1, S. 35–55, Feb. 1997, doi: 10.1016/S0022-1759(96)00208-6.
[3] „An improved helper phage system for efficient isolation of specific antibody molecules in phage display“. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC101254/ (zugegriffen Okt. 27, 2020).
[4] M. ARSLAN, D. KARADAĞ, und S. KALYONCU, „Protein engineering approaches for antibody fragments: directed evolution and rational design approaches“, Turk. J. Biol., Bd. 43, Nr. 1, S. 1–12, Feb. 2019, doi: 10.3906/biy-1809-28.
[5] E. S. Hosseini, R. Moniri, Y. D. Goli, und H. H. Kashani, „Purification of Antibacterial CHAPK Protein Using a Self-Cleaving Fusion Tag and Its Activity Against Methicillin-Resistant Staphylococcus aureus“, Probiotics Antimicrob. Proteins, Bd. 8, Nr. 4, S. 202–210, Dez. 2016, doi: 10.1007/s12602-016-9236-8.
[6] N. H. Shah und A. J. Stevens, „Identification, Characterization, and Optimization of Split Inteins“, in Expressed Protein Ligation: Methods and Protocols, M. Vila-Perelló, Hrsg. New York, NY: Springer US, 2020, S. 31–54.