A fast and efficient procedure to produce scFvs specific for large macromolecular complexes

Wu, Si; Ke, Ailong; Doudna, Jennifer A.

doi:10.1016/j.jim.2006.10.005

Cited by 20 publications

(13 citation statements)

References 21 publications

(25 reference statements)

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“…Soluble scFv fragments protein expression levels for these clones were in the range of 0.044 to 1.8 mg/liter of culture. These yields are in general agreement with published yields in the range of 0.1 to 5.0 mg per liter of E. coli culture for clones isolated from the Tomlinson libraries (Kennel et al, 2004; Wu et al, 2007; Lobova et al, 2008). SDS-PAGE analysis for these four clones show bands corresponding approximately to 26–28 kD molecular weight when compared to the 25 kD band of the protein standard ladder run in parallel with the samples (Fig.…”

Section: Resultssupporting

confidence: 89%

“…These amber stop codons, while still allowing phage/scFv production in TG1 cells, prevent the expression of full-length scFvs in the nonsuppressor HB2151 E. coli strain. We are currently using molecular biology techniques (Wu et al, 2007) to repair these clones containing randomly generated amber codons. However, while work is ongoing to repair the amber codons in the sequences of the J library clones, we proceeded to produce soluble scFv fragments using selected clones isolated from the I library with no amber stop codons.…”

Section: Resultsmentioning

confidence: 99%

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Isolation of scFv fragments specific for monokine induced by interferon-gamma (MIG) using phage display

Eteshola

2010

Journal of Immunological Methods

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Iterative affinity selection procedures were used to isolate a number of single chain Fv (scFv) antibody fragment clones from naïve Tomlinson I + J phage display libraries that specifically recognize and bind a chemokine, monokine induced by interferon-gamma (MIG/CXCL9). MIG is an important transplant rejection/biology chemokine protein. ELISA-based affinity characterization results indicate that selectants preferentially bind to MIG in the presence of key biopanning component materials and closely related chemokine proteins. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific MIG binding capability with potential applications in transplant rejection monitoring, and other biomedical applications where detection of MIG level is important.

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Isolation of scFv fragments specific for monokine induced by interferon-gamma (MIG) using phage display

Eteshola

2010

Journal of Immunological Methods

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“…However, during the 20-min incubation of this assay, Ffh-FtsY complexes are not stably formed in the absence of the 4.5S RNA (Shepotinovskaya et al 2003). scFv IB11 is specific to Ffh alone or in the SRP complex, but does not recognize FtsY (Wu et al 2006). Both antibodies' specificities are unaffected by the GNRA tetraloop since they show similar levels of detection for complexes containing either the wild-type or UUCG 4.5S RNA (data not shown).…”

Section: Binding Affinity Of 45s Rna Tetraloop Mutants To Ffhmentioning

confidence: 93%

“…To test this possibility, SRP-FtsY ternary complex formation for wild-type and UUCG 4.5S RNA was monitored by enzyme-linked immunosorbent assay (ELISA) using single-chain antibodies (scFv) IA02 and IB11 (Wu et al 2006). The antibody scFv IA02 specifically recognizes the SRP-FtsY complex, and not SRP, Ffh, or FtsY alone (Wu et al 2006). This recognition is independent of the RNA since IA02 can detect Ffh-FtsY complexes (data not shown).…”

Section: Binding Affinity Of 45s Rna Tetraloop Mutants To Ffhmentioning

confidence: 99%

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

Siu

Spanggord²,

Doudna³

2006

RNA

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The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRPreceptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP-receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP-receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP-receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo.

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“…The flexible tertiary structure adopting elongated shapes and inter-domain movements within RNA makes crystal packing even more difficult (1). Assistive methods for crystallization are constantly pursued to alleviate these complications (2,3). Chaperone-assisted RNA crystallography (CARC) is one such assistive method which employs a monoclonal antibody fragment to bind RNAs of interest and aid in crystallization.…”

mentioning

confidence: 99%

Improvement of the crystallizability and expression of an RNA crystallization chaperone

Ravindran

Héroux

2011

Journal of Biochemistry

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Crystallizing RNA has been an imperative and challenging task in the world of RNA research. Assistive methods such as chaperone-assisted RNA crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by forming crystal contacts and providing initial phasing information. Despite the early successes, the crystallization of large RNAFab complex remains a challenge in practice. The possible reason for this difficulty is that the Fab scaffold has not been optimized for crystallization in complex with RNA. Here, we have used the surface entropy reduction (SER) technique for the optimization of "C209 P4P6/Fab2 model system. Protruding lysine and glutamate residues were mutated to a set of alanines or serines to construct Fab2SMA or Fab2SMS. Expression with the shake flask approach was optimized to allow large scale production for crystallization. Crystal screening shows that significantly higher crystal-forming ratio was observed for the mutant complexes. As the chosen SER residues are far away from the CDR regions of the Fab, the same set of mutations can now be directly applied to other Fabs binding to a variety of ribozymes and riboswitches to improve the crystallizability of FabRNA complex.Keywords: chaperone assisted RNA crystallography/ Fab/protein engineering/shake flask expression/ surface entropy reduction.With every new discovery of a functional RNA, there is a sprint to obtain its crystal structure and elucidate the structurefunction relationship. The dynamic RNA research is mired by the difficulty in obtaining RNA crystals due to its structural property. The negatively charged phosphate backbone of RNA disfavours good crystal forming lattices. Compared to proteins, RNAs have fewer functional groups and have weaker tertiary interactions. The flexible tertiary structure adopting elongated shapes and inter-domain movements within RNA makes crystal packing even more difficult (1). Assistive methods for crystallization are constantly pursued to alleviate these complications (2, 3). Chaperone-assisted RNA crystallography (CARC) is one such assistive method which employs a monoclonal antibody fragment to bind RNAs of interest and aid in crystallization. Synthetic antigenbinding fragments (Fabs) selected from a phage display library can bind to large functional RNAs recognizing tertiary structures. These Fabs have large surface area, participate extensively in crystal contacts and provide good initial phasing information (4, 5).YSGX synthetic library is a reduced genetic codon library that generates randomized CDRs using codons enriched in tyrosines, serines and glycines (6). This was used to obtain the first RNAFab complex crystal structure (1.95 Å ) of ÁC209 P4P6 RNA binding to Fab2 (1, 4). ÁC209 P4P6 RNA is a single site mutant form of the P4P6 RNA of the Tetrahymena group I intron which catalyses its own excision from precursor RNA. The single site mutation increases the tertiary stability by reducing the conformational f...

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A fast and efficient procedure to produce scFvs specific for large macromolecular complexes

Cited by 20 publications

References 21 publications

Isolation of scFv fragments specific for monokine induced by interferon-gamma (MIG) using phage display

Isolation of scFv fragments specific for monokine induced by interferon-gamma (MIG) using phage display

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

Improvement of the crystallizability and expression of an RNA crystallization chaperone

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