TITLE: Biotinylation of Membrane Proteins for Binder Selections
AUTHORS: Benedikt T. Kuhn, Iwan Zimmermann, Pascal Egloff, Lea M. Hürlimann, Cedric A.J. Hutter, Christian Miscenic, Roger J.P. Dawson, Markus  A. Seeger, Eric  R. Geertsma

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.</div></div>

[STEPS]
?. [3.1 FX Cloning]
Design FX-cloning compatible primers targeting your gene of interest. This is most conveniently done online at the https://www.fxcloning.org website using a FASTA-formatted sequence including a start and stop codon. Order the primer set optimized toward removal of stable hairpin structures (see Note 1).
As the N- and C-termini of most proteins are comparably long and flexible, we generally do not insert a linker sequence between the target protein and the Avi-tag. Nevertheless, should this be desired, a sequence for a linker is best introduced at this step.
?. [3.1 FX Cloning]
Amplify the gene of interest by PCR. Prepare a  and add the DNA polymerase immediately prior to starting the reaction. Use a touch-down [21] program, e.g., (1)  at ; (2)  at ; (3)  at  (annealing temperature decreased by  per cycle); (4) 15- 30 s/kb at ; repeat (2)–(4) 14 times; (5)  at ; (6)  at ; (7) 15–30 s/kb at ; repeat (5)–(7) 14 times; (8)  at ; (9) unlimited at .
[PCR reaction]
98 °C
98 °C
61 °C
0.5 °C
72 °C
98 °C
53 °C
72 °C
72 °C
10 °C
?. [3.1 FX Cloning]
Analyze the product on a preparative TAE agarose gel. Purify the relevant band using a DNA gel extraction kit and quantify the DNA spectrophotometrically.
?. [3.1 FX Cloning]
Mix  with the extracted insert in a molar ratio of vector:insert of 1:5 (see Note 2).
[pINIT_cat]
Note 2: Alternatively, should subcloning of a sequence-verified open reading frame not be required, proceed with Step 10 and use the purified PCR product to replace the pINIT_cat holding the insert.
?. [3.1 FX Cloning]
Add  and adjust the volume to  with ultrapure water. Add  and incubate for  at  in a PCR machine.
[10× SapI buffer]
9 µl
[ SapI (2 U)]
37 °C
?. [3.1 FX Cloning]
Heat inactivate the SapI for  at . Let the sample cool down and add  and . Incubate for  at .
65 °C
[10 mM ATP]
[T4 DNA ligase (1.25 U)]
65 Room temperature
?. [3.1 FX Cloning]
Heat inactivate the T4 DNA ligase for  at  and transform  to .
65 °C
[ligation mix]
[chemically competent E. coli MC1061 cells (or an alternative CcdB-sensitive strain)]
?. [3.1 FX Cloning]
Plate 10% and 90% aliquots on LB-agar-Cam plates and incubate  at .
37 °C
?. [3.1 FX Cloning]
Use a single colony to inoculate  and cultivate . Isolate the plasmid using a miniprep kit, determine the concentration spectrophotometrically, and verify the insert by DNA sequencing using the pINIT_cat sequencing primers.
[LB-Cam]
?. [3.1 FX Cloning]
Mix  (see Note 3) with pINIT_cat carrying the insert to a molar ratio of vector:pINIT_cat-derivative of 1:5. Add  and adjust the volume to  with ultrapure water. Add  and incubate for  at  in a PCR machine.
[FX cloning Avi-tag expression vector]
[10× SapI buffer]
9 µl
[SapI (2 U)]
37 °C
Note 3: Should an expression and purification strategy for the protein already be established, this combination of tags and fusions proteins should guide the choice for the expression vector. We recommend the production of protein variants with N- and C-terminal Avi-tags as this may allow the presentation of different surfaces of the target protein.
?. [3.1 FX Cloning]
Heat inactivate the SapI for  at . Let the sample cool down and add  and . Incubate for  at .
65 °C
[10 mM ATP]
[T4 DNA ligase (1.25 U)]
65 Room temperature
?. [3.1 FX Cloning]
Heat inactivate the T4 DNA ligase for  at  and transform  to .
65 °C
[ligation mix]
[chemically competent E. coli MC1061 cells]
?. [3.1 FX Cloning]
Plate 10% and 90% aliquots on LB-agar-Amp plates. Incubate the plates  at .
37 °C
?. [3.1 FX Cloning]
Use a single colony to inoculate  and cultivate  at .
[LB-Amp]
37 °C
?. [3.1 FX Cloning]
Archive the culture as a glycerol stock at  (see Note 4). This stock can serve for inoculation of expression cultures based on the araBAD promoter (see Notes 5 & 6).
-80 °C
Note 4: For other expression systems that require fresh transformations for expression cultures, e.g., those based on the T7 promoter, the stock serves as a plasmid source. No additional verification by DNA sequencing is required following the subcloning of a sequence-verified open reading frame from pINIT_cat to an FX cloning expression vector.
Note 5: The FX-cloning expression vectors for fused Avi-tag allow recombinant expression in E. coli under the control of the PBAD promotor with decaHis-tag. Instead of subcloning a sequence-verified ORF from pINITIAL to an FX cloning Avi-tag expression vector, PCR products can also be cloned immediately into an FX cloning Avi-tag expression vector. This requires sequence verification of each expression vector. If multiple expression vectors are constructed, subcloning from pINITIAL prevents excessive sequencing. If one aims for only a single Avi-tagged construct, we recommend starting with pBXNH3CA (Addgene #47069), which adds a cleavable N-terminal decaHis-tag and a C-terminal Avi-tag to the protein. In our hands, this vector resulted in good expression levels for a number of ABC transporters as well as maltose-binding protein (MBP) and GFP.
Note 6: Should expression in alternative pro- or eukaryotic systems be preferred, the PBAD-based expression vectors may serve as facile intermediates for fusing the Avi-tag sequences.
?. [3.2 BirA-Based In Vitro Biotinylation]
Recombinantly express the target protein using previously established procedures [22] (see Note 7). Purify the Avi-tagged target protein (see Note 8)and determine the protein concentration spectrophotometrically.
Note 7: If the Avi-tagged target protein is expressed in E. coli and if the Avi-tag sequence is located in the cytoplasm, the Avi-tag will be biotinylated in vivo by virtue of the natively expressed BirA. The degree of biotinylation varies from case to case (subject to availability of biotin, level of target protein overexpression and accessibility of Avi-tag), but is often incomplete. The degree of in vivo biotinylation may be increased by co-expression of BirA and supplementation of the medium with biotin [14]. However, due to the relevance of complete biotinylation of the Avi-tag, our protocol ignores in vivo biotinylation of the target within E. coli and assures full biotinylation by performing an additional in vitro step. If required, the degree of native biotinylation can be assessed as outlined in Section 3.5 .
Note 8: The BirA-based biotinylation protocol describes the procedure for His-tagged target protein but can in principle be adapted to protocols involving other affinity-tags. The use of strep-tags [23] or fusions with streptavidin-binding-protein (SBP) [24] should be avoided as biotinylation of Avi-tags by endogenous BirA, which may reach a very high degree depending on the experimental conditions and target protein, will prevent elution from the respective columns.
?. [3.2 BirA-Based In Vitro Biotinylation]
Add 3C protease to a molar ratio of 1:10 to cleave off the decaHis-tag while dialyzing the sample for  at  to remove excess imidazole (see Note 9).
4 °C
Note 9: Although BirA is inhibited by NaCl (over 100 mM) and glycerol (over 1%) [14], we generally use buffers containing 150 mM NaCl and 10% glycerol if the target membrane protein requires this for maintaining a well-folded state. We compensate for the reduced BirA activity by biotinylating for prolonged periods (overnight).
?. [3.2 BirA-Based In Vitro Biotinylation]
Adjust the target protein concentration to  –  (either by dilution or using a concentrator unit). Add biotin to a molar ratio of target protein:biotin of 1:1.5, 5 mM ATP, 10 mM MgOAc and BirA to a molar ratio of target protein:BirA of 20:1 (see Note 10). Incubate the sample  at  (see Note 11).
4 °C
Note 10: Addition of extra amount of detergent might be necessary to keep the detergent concentration well above the CMC.
Note 11: The BirA-based biotinylation reaction can also be performed for   at   if the target protein is stable under these conditions. For most membrane proteins we recommend keeping the sample at  .
?. [3.2 BirA-Based In Vitro Biotinylation]
Remove His-tagged BirA, HRV 3C protease, and potential remaining contaminants from the sample by reverse IMAC and collect the flow-through holding the biotinylated target protein.
?. [3.2 BirA-Based In Vitro Biotinylation]
Perform size exclusion chromatography (SEC) to remove soluble aggregates and excess of biotin from the sample (see Note 12). Determine the degree of biotinylation as outlined in Section 3.5.
Note 12: Removal of free biotin is often crucial for downstream processes. In case no size exclusion chromatography is performed, use dialysis or a desalting column to remove the excess biotin from the sample.
?. [3.2 BirA-Based In Vitro Biotinylation]
Proceed with the selection of binders such as nanobodies and sybodies (see Note 13) or store the target protein (see Notes 14 & 15).
Note 13: The outcome of the binder selection depends to a very large extent on the quality of the target protein used. Productive binder selections are expected if: (1) the SEC profile of the biotinylated target protein is monodisperse and very similar to that obtained for non-biotinylated target protein; (2) the fraction of non-biotinylated target protein is less than 10%; and (3) in case of chemical biotinylation: over-biotinylation is excluded, ideally as assessed by mass spectrometry.
Note 14: If possible, the biotinylated target protein is supplemented with 10% glycerol, aliquoted, snap-frozen in liquid nitrogen, and stored at  . To assess if freezing is tolerated by the target protein, compare a frozen/thawed and untreated sample by SEC. If no discernible aggregation or protein loss is observed, freezing can be considered as tolerated.
Note 15: We routinely freeze biotinylated membrane proteins for storage purposes. Having thus far analyzed more than a dozen membrane proteins in this manner, we never experienced aggregation problems due to freezing. Frozen biotinylated proteins remain stable at   for several years.
?. [3.3 BirA-Based In Vivo Biotinylation]
Generate mammalian expression vectors for the gene of interest in pC031 or pC039 to obtain a fusion protein with an N- or C-terminal Avi-tag (see Notes 16 & 17).
Note 16: The preferred location of the Avi tag on the target protein depends on the quality and quantity of the fusion protein that can be obtained. Both parameters are most easily assessed by using fluorescence-detection size-exclusion chromatography (FSEC) analysis [25].
Note 17: Biotinylation of Avi-tags during cultivation can be achieved in several additional expression systems [14,26,27]. These procedures also require the co-expression of BirA and growth medium supplemented with biotin.
?. [3.3 BirA-Based In Vivo Biotinylation]
Split an Expi293 subculture (see Note 18), typically grown to 3-5 × 106 cells/mL, into a 3 L Fernbach shaking flask and adjust to a final volume of  with a density of 0.7 × 106 cells/mL.
[Expi293 medium]
Note 18: Numerous expression screenings provided the tendency that Expi293 is more successful for expressing membrane proteins. As an alternative we recommend Freestyle 293-F cells. Implementation of the latter will require small adaptations of the described workflow for which we refer to the instructions from the supplier.
?. [3.3 BirA-Based In Vivo Biotinylation]
Incubate the culture for  at , under humidified atmosphere and 5% CO2 in a shaking incubator.
37 °C
?. [3.3 BirA-Based In Vivo Biotinylation]
On the day of the transient transfection, adjust the culture to  with a density of 3.4 × 106 cells/mL by adding Expi293 medium and/or removing cells.
830 mL
?. [3.3 BirA-Based In Vivo Biotinylation]
Add  (final concentration of ) (see Note 19).
[biotin solution]
Note 19: Supplementing the medium with biotin is optional. Over the course of many years and targets we observed virtually complete biotinylation even in the absence of supplemented biotin.
?. [3.3 BirA-Based In Vivo Biotinylation]
Pipet  into a 100 mL sterile Schott bottle. Add , shake gently, and incubate for  at .
[Opti-MEM]
[ExpiFectamine transfection reagent]
37 Room temperature
?. [3.3 BirA-Based In Vivo Biotinylation]
Pipet  into a second 100 mL Schott bottle and add the two plasmid batches in a final amount of  to , target protein expression plasmid:BirA expression plasmid, respectively. Shake gently and incubate for  at .
[Opti-MEM]
1 mg
0.1 mg
37 Room temperature
?. [3.3 BirA-Based In Vivo Biotinylation]
Mix the contents of both bottles, filter sterilize, and incubate for  –  at  to form the transfection complex.
37 Room temperature
?. [3.3 BirA-Based In Vivo Biotinylation]
Add  to the Fernbach shaking flask with  for a final volume of . Incubate further at  and 5% CO2 with mild shaking.
[formed transfection complex]
[cell culture]
950 mL
37 °C
?. [3.3 BirA-Based In Vivo Biotinylation]
Add sterile  and  from the ExpiFectamine 293 transfection kit at  –  post-transfection and continue incubation.
[Enhancer 1]
[Enhancer 2]
?. [3.3 BirA-Based In Vivo Biotinylation]
Incubate for a total time of approximately  -  post-transfection depending on the most optimal condition for protein expression. Harvest the cells by centrifugation at , flash freeze the pellet in liquid nitrogen, and store at .
Centrifuge: 3000 34, 15 min
-80 °C
?. [3.3 BirA-Based In Vivo Biotinylation]
Purify the biotinylated Avi-tagged target protein (see Note 8) and determine the protein concentration spectrophotometrically. Determine the degree of biotinylation as outlined in Section 3.5. Proceed with the selection of binders such as nanobodies and sybodies (see Note 13) or store the target protein (see Notes 14 & 15).
Note 8: The BirA-based biotinylation protocol describes the procedure for His-tagged target protein but can in principle be adapted to protocols involving other affinity-tags. The use of strep-tags [23] or fusions with streptavidin-binding-protein (SBP) [24] should be avoided as biotinylation of Avi-tags by endogenous BirA, which may reach a very high degree depending on the experimental conditions and target protein, will prevent elution from the respective columns.
Note 13: The outcome of the binder selection depends to a very large extent on the quality of the target protein used. Productive binder selections are expected if: (1) the SEC profile of the biotinylated target protein is monodisperse and very similar to that obtained for non-biotinylated target protein; (2) the fraction of non-biotinylated target protein is less than 10%; and (3) in case of chemical biotinylation: over-biotinylation is excluded, ideally as assessed by mass spectrometry.
Note 14: If possible, the biotinylated target protein is supplemented with 10% glycerol, aliquoted, snap-frozen in liquid nitrogen, and stored at  . To assess if freezing is tolerated by the target protein, compare a frozen/thawed and untreated sample by SEC. If no discernible aggregation or protein loss is observed, freezing can be considered as tolerated.
Note 15: We routinely freeze biotinylated membrane proteins for storage purposes. Having thus far analyzed more than a dozen membrane proteins in this manner, we never experienced aggregation problems due to freezing. Frozen biotinylated proteins remain stable at   for several years.
?. [3.4 Chemical Biotinylation]
Recombinantly express the target protein using previously established procedures [22]. Purify the target protein and employ preparative SEC using PBS, supplemented with the required detergent, as buffer (see Note 20). Determine the protein concentration spectrophotometrically.
Note 20: It is very important that compounds containing primary amines are absent from the purified protein sample for chemical biotinylation. A frequent source of primary amines stems from Tris-buffers. IMAC-purified protein is not pure enough regarding biogenic amines to be used for NHS coupling.
?. [3.4 Chemical Biotinylation]
Concentrate the target protein to  - .
?. [3.4 Chemical Biotinylation]
Dissolve EZ-Link Sulfo-NHS-LC-Biotin in highly pure DMSO to a concentration of  (see Note 21).
Note 21: The NHS moiety of EZ-Link Sulfo-NHS-LC-Biotin reacts with water and is thereby inactivated. We therefore highly recommend preparing the Sulfo-NHS-LC-Biotin solution freshly. Keep solid EZ-Link Sulfo-NHS-LC-Biotin under argon at   for prolonged storage.
?. [3.4 Chemical Biotinylation]
Add EZ-Link Sulfo-NHS-LC-Biotin to the target protein in fivefold molar excess and incubate at  for  under gentle agitation (see Note 22).
25 °C
Note 22: In case the target protein is unstable at  , the biotinylation reaction can be carried out at  . In this case, increase the biotin-target protein ratio to 10:1 and incubate for   instead of  .
?. [3.4 Chemical Biotinylation]
Perform SEC to remove excess of biotin from the sample (see Note 12).
Note 12: Removal of free biotin is often crucial for downstream processes. In case no size exclusion chromatography is performed, use dialysis or a desalting column to remove the excess biotin from the sample.
?. [3.4 Chemical Biotinylation]
Determine the biotinylation pattern of the biotinylated target protein by mass-spectrometry (Fig. 1, see Note 23)). In case mass spectrometry analysis is not available or cannot be carried out due to the target’s high molecular weight, determine the degree of biotinylation as outlined in Section 3.5.
Note 23: A typical pattern contains different species containing either none, one, or several biotin moieties per target protein (Fig.1) Ideally, the non-biotinylated species should not exceed 10% of the total species (in the example of GFP labeling shown in Fig.1, non-biotinylated target accounts for around 5%). In case of over- or under-biotinylation, the biotin-target protein ratio needs to be adjusted accordingly, while keeping the target protein concentration and incubation time constant.
?. [3.4 Chemical Biotinylation]
Proceed with the selection of binders such as nanobodies and sybodies (see Note 13) or store the target protein (see Notes 14 & 15).
Note 13: The outcome of the binder selection depends to a very large extent on the quality of the target protein used. Productive binder selections are expected if: (1) the SEC profile of the biotinylated target protein is monodisperse and very similar to that obtained for non-biotinylated target protein; (2) the fraction of non-biotinylated target protein is less than 10%; and (3) in case of chemical biotinylation: over-biotinylation is excluded, ideally as assessed by mass spectrometry.
Note 14: If possible, the biotinylated target protein is supplemented with 10% glycerol, aliquoted, snap-frozen in liquid nitrogen, and stored at  . To assess if freezing is tolerated by the target protein, compare a frozen/thawed and untreated sample by SEC. If no discernible aggregation or protein loss is observed, freezing can be considered as tolerated.
Note 15: We routinely freeze biotinylated membrane proteins for storage purposes. Having thus far analyzed more than a dozen membrane proteins in this manner, we never experienced aggregation problems due to freezing. Frozen biotinylated proteins remain stable at   for several years.
?. [3.5 Assessing Degree of Biotinylation]
Mix two aliquots of  with 5× SDS-PAGE sample buffer.
[biotinylated target protein]
?. [3.5 Assessing Degree of Biotinylation]
Add streptavidin to one of the aliquots in a 1:1 molar ratio of target protein:streptavidin (see Note 24).
Note 24: The biotinylation of target protein can be quantified by mobility shift in SDS-PAGE upon addition of streptavidin to the sample. Streptavidin remains folded and bound to the biotinylated target protein under conventional SDS-PAGE conditions [28]. Membrane protein samples are usually not boiled before SDS-PAGE. However, when boiling the sample is required add streptavidin afterward.
?. [3.5 Assessing Degree of Biotinylation]
Analyze the control (no addition) and test (streptavidin addition) samples in adjacent lanes on SDS-PAGE.
?. [3.5 Assessing Degree of Biotinylation]
Stain the gel with Coomassie Brilliant Blue R-250 and quantify the band intensities with the ImageJ software and calculate the degree of biotinylation (see Notes 25 & 26).
Note 25: Due to the tetrameric architecture of streptavidin with four biotin-binding sites, multiple protein bands may be observed, e.g., (1) free streptavidin (53 kDa), (2) streptavidin associated with a single target protein, and (3) streptavidin associated with multiple (up to four) target proteins. In our hands, it is more straightforward to use the intensity loss of the target protein band upon streptavidin addition relative to the control sample for quantification. For more precise quantification we recommend mass spectrometry to analyze the degree of biotinylation. For qualitative analysis of target protein biotinylation, western blotting using a streptavidin-HRP conjugate can be employed.
Note 26: Incomplete biotinylation might be advantageous regarding oligomeric proteins. Similar to the presence of multiple biotin labels on monomeric proteins, the occurrence of multiple biotin groups per oligomeric protein complex may restrict its flexibility upon immobilization and thereby decrease the variation and amount of protein surface accessible to the binders. We recommend a pull-down of biotinylated target protein with immobilized streptavidin and compare the pull-down efficiency with the mobility shift in SDS-PAGE to quantify the biotinylation per oligomeric unit.