Structural and thermodynamic analysis of the GFP:GFP‐nanobody complex

Kubala, Marta H.; Kovtun, Oleksiy; Alexandrov, Kirill; Collins, Brett M.

doi:10.1002/pro.519

Cited by 360 publications

(401 citation statements)

References 38 publications

Supporting

Mentioning

389

Contrasting

Order By: Relevance

“…The size of GFP specific VHH is only ~2 × 4 nm and the binding site of the GFP is located on the longer side of the nanobody [8]. Despite the very short Debye length of approximately 1 nm in 100 mM ionic strength, a small signal due to GFP binding was still observed even in high ionic strength buffers ( Figure 1E,F).…”

Section: Discussionmentioning

confidence: 96%

“…Therefore, shorter receptors that enable analyte binding closer to the surface, such as antibody fragments or aptamers, have been proposed. Camelid heavy-chain VHH fragment (also called nanobody) receptors are among the shortest available biological receptors (~13 kDa, <3 nm) [7] and are much smaller and structurally simpler than common whole antibodies (~150 kDa, ~15 nm) or Fab fragments (~50 kDa, ~7-8 nm) [8]. In addition, the nanobodies are easily produced and stable in a range of different conditions [7].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Label-Free Immunodetection in High Ionic Strength Solutions Using Carbon Nanotube Transistors with Nanobody Receptors

Filipiak

Rother

Andoy

et al. 2017

Proceedings of Eurosensors 2017, Paris, France, 3–6 September 2017

View full text Add to dashboard Cite

Nanomaterial-based field-effect transistors (FETs) have been proposed for real-time, label-free detection of various biological species. However, screening of the analyte charge by electrolyte ions (Debye screening) has so far limited their use in physiological samples. Here, this challenge is overcome by combining FETs based on single-walled semiconducting carbon nanotube networks (SWCNTs) with a novel surface functionalization comprising: (1) short nanobody receptors, and (2) a polyethylene glycol layer (PEG). Nanobodies are stable, easy-to-produce, short biological receptors (~2-4 nm) that enable analyte binding closer to the sensor surface. The addition of PEG enhances the signal in high ionic strength environment. Using green fluorescent protein (GFP) as a model antigen, high selectivity and sub-picomolar detection limit with a dynamic range exceeding 4 orders of magnitude is demonstrated in physiological solutions. The presented immunoassay is fast, label-free, does not require any sample pre-treatment or washing steps.

show abstract

Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Label-Free Immunodetection in High Ionic Strength Solutions Using Carbon Nanotube Transistors with Nanobody Receptors

Filipiak

Rother

Andoy

et al. 2017

Proceedings of Eurosensors 2017, Paris, France, 3–6 September 2017

View full text Add to dashboard Cite

show abstract

“…4 coated with an antibody against GFP [12] -GFP-nanobody, which binds Citrine with a similar affinity as GFP [12]. Captured proteins were eluted as previously described [13] and subjected to trypsin digestion.…”

Section: A C C E P T E D Accepted Manuscriptmentioning

confidence: 99%

Mammalian farnesyltransferase α subunit regulates vacuolar protein sorting-associated protein 4A (Vps4A) – dependent intracellular trafficking through recycling endosomes

Kubala

Norwood

Gómez

et al. 2015

Biochemical and Biophysical Research Communications

Self Cite

View full text Add to dashboard Cite

The protein farnesyltransferase (FTase) mediates posttranslational modification of proteins with isoprenoid lipids. FTase is a heterodimer and although the β subunit harbors the active site, it requires the α subunit for its activity. Here we explore the other functions of the FTase α subunit in addition to its established role in protein prenylation. We found that in the absence of the β subunit, the α subunit of FTase forms a stable autonomous dimeric structure in solution. We identify interactors of FTase α using mass spectrometry, followed by rapid in vitro analysis using the Leishmania tarentolae cell -free system.

show abstract

“…We directly assessed the efficiency of YHRC into pSUMO--YHRC by cloning six different genes encoding: Nanobody GFP [36,37], Rpo41 T920--L1217 --HA, DHFR mutD --FLAG, [38], 3HA--stGnd1 [39], Sis1 [40] and Ydj1 [41]. First, we defined DNA sequences in pSUMO--YHRC suitable for homologous recombination ( Fig.…”

Section: Yhrc Of Single Fragments In Psumo--yhrcmentioning

confidence: 99%

“…Nanobody GFP is a single domain antibody fragment (VHH) derived from a llama antibody that binds to GFP with nanomolar affinity and has been described under several different names including GFP--binding protein 1 (GBP1), Enhancer and GFP--nanobody [29,36,37,42]. Solubility tags similar to His6--SUMO are known to facilitate high--level expression of nanobodies in the cytoplasm of E. coli [43].…”

Section: Expression and Purification Of Functional Nanobody Gfpmentioning

confidence: 99%

A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination

Holmberg

Gowda

Andréasson

2014

Protein Expression and Purification

View full text Add to dashboard Cite

A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination.Protein Expression and Purification, http://dx.doi.org/10.1016/j. pep.2014.03.002 Access to the published version may require subscription. N.B. When citing this work, cite the original published paper. Permanent link to this version AbstractProduction of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single--gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET--based bacterial expression vector pSUMO--YHRC. The vector supports cloning for untagged expression as well as fusions to His6--SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single--step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs. Protein expression/Protein purification/cloning/recombination cloning SUMO/nanobody/ Introduction Straightforward cloning and production of biologically active proteins is central for studies in biochemistry and biophysics. During recent years, several improvements in recombinant protein expression and purification techniques have been developed. Yet many laboratories still depend on unreliable cloning techniques based on ligation of endonuclease restriction products. Especially, when cloning multiple DNA fragments into one expression vector traditional methodology requires time--consuming reiteration of the process and the cloning step frequently becomes limiting in the protein production process. Current cloning methodology relies on PCR amplification of the relevant gene using two primers designed to facilitate the subsequent cloning step. Traditionally, unique restriction sites in the flanking primer regions are digested and the PCR product is ligated into a vector containing compatible cohesive ends. Similarly, in TA--cloning a single adenosine residue added at each end of the PCR product by Taq DNA polymerase facilitates ligation into a restricted vector carrying complementary thymidine residues [1]. An alternative to these methods is ligation independent cloning (LIC). In LIC flanking sequence extensions are added to the PCR product and are by enzymatic modification converted to single stranded overhangs that are annealed to a vector carrying complementary ends [2]. Cloning of multipl...

show abstract

Structural and thermodynamic analysis of the GFP:GFP‐nanobody complex

Cited by 360 publications

References 38 publications

Label-Free Immunodetection in High Ionic Strength Solutions Using Carbon Nanotube Transistors with Nanobody Receptors

Label-Free Immunodetection in High Ionic Strength Solutions Using Carbon Nanotube Transistors with Nanobody Receptors

Mammalian farnesyltransferase α subunit regulates vacuolar protein sorting-associated protein 4A (Vps4A) – dependent intracellular trafficking through recycling endosomes

A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination

Contact Info

Product

Resources

About