Production of Nanobody 35

Abstract

Nanobody 35 (Nb35) binds the heterotrimeric G protein complex and stabilizes it against GTPγS-induced dissociation. This nanobody is widely employed in GPCR structural studies. Here, we give details of its recombinant production.

Materials and reagents

1. E. coli BL21 (DE3) cells (TIANGEN, China Catalog number: CB105-02).

2. Plasmids encoding Nb35 protein.

3. TB culture medium with 100 mg/mL ampicillin (Sangon Biotech, China, catalog number: A100339-0025), 2 mM MgCl₂ (Sinopharm Chemical Reagent Co., Ltd., China, catalog number: 10012818), 0.1% (w/v) glucose (Sigma, USA, catalog number: G8270-5KG).

4. LB culture medium with 100 mg/mL ampicillin.

5. PMSF (Diamond, China, catalog number: A100754-0025).

6. 0.22 μm filters (Millipore, USA, catalog number: SLGVR33RB).

7. 10 kDa molecular weight cutoff (MWCO) concentrator (Merck Millipore, USA, catalog number: UFC501024).

8. Na₂HPO₄ (Sinopharm Chemical Reagent Co., Ltd, China, catalog number: 20040618).

9. NaH₂PO₄ (Sinopharm Chemical Reagent Co., Ltd, China, catalog number: 20040818).

Equipment

1. Shaker

2. Conical flask

3. Culture dish

4. Magneticstirrer

5. Centrifuge

6. Superclean bench

7. Autoclave

8. UV-visible spectrophotometer

9. Beaker

Procedure

Expression of Nb35

1. The Nb35 plasmid was transformed into BL21(DE3) competent cells as the host strain and plated onto LB agar plates, followed by incubation at 37°C for 16 hours.
2. A single colony was picked and inoculated into 10 mL of sterile LB liquid medium. The following filter-sterilized (using 0.22 μm filters) components were added: 1 mL of 20% glucose, 10 μL of 1M MgCl₂, and 10 μL of ampicillin. The culture was incubated at 37°C with shaking at 180 rpm for 16 hours (overnight).
3. The 10 mL overnight culture from step 2 was added to a pre-sterilized 2L TB medium. The following filter-sterilized (using 0.22 μm filters) components were added: 10 mL 20% glucose, 4 mL of 1M MgCl₂, and 2 mL of ampicillin. The culture was incubated at 37°C while shaking at 180 rpm. When the OD₆₀₀ reached 0.7-1.2, protein expression was induced by adding 2 mL of filter-sterilized 1M IPTG. The temperature was then reduced to 28°C and incubation continued at 180 rpm for 16 hours.
4. Following induction, the Nb35 culture was centrifuged at 4°C and 4,000 rpm for 20 minutes. The supernatant was discarded. The pellet was resuspended in PBS and centrifuged again under the same conditions (4°C, 4,000 rpm, 20 minutes). The supernatant was discarded, and the cell pellet was stored at -80°C.

Purification of Nb35

Table 1 Formula of Nb35 purification buffers (1L)

1. Thaw Nb35 cell culture collected previously at room temperature. Transfer to a 1 L beaker. Add 100 mL of TES lysis buffer and protease inhibitor PMSF (100× stock). Stir overnight at 4°C on a magnetic stirrer.
2. Add 120 mL of TES/4 solution. Continue stirring at 4°C for 2 hours.
3. Centrifuge at 4°C, 12,000 rpm for 30 minutes. During centrifugation: wash 2 mL of Ni affinity resin with 40 mL of ddH₂O, followed by 40 mL of filter-sterilized (0.22 μm) Nb35 SEC buffer.
4. After centrifugation, incubate the supernatant with the prepared Ni resin in 50 mL centrifuge tubes for 2 hours at 4°C. Then centrifuge at 550× g for 10 minutes at 4°C.
5. Discard the supernatant. Transfer the Ni resin to a gravity-flow column.
6. At 4°C, wash the Ni resin with 40 mL of Phosphate 1 buffer, followed by 40 mL of Phosphate 2 buffer.
7. Finally, elute the protein using 20 mL of Nb35 Elute buffer, and collect the eluate in a 250 mL conical flask placed on ice.
8. Combine the eluted protein fractions and concentrate to approximately 5 mL using a 10 kDa molecular weight cutoff (MWCO) concentrator.
9. Equilibrate a HiLoad 16/600 Superdex 200 pg column with at least 150 mL of Nb35 SEC buffer. Load the concentrated Nb35 sample from step 8 onto the column for size-exclusion chromatography (SEC). Collect the peak fractions containing highly pure Nb35 protein. Flash-freeze the purified protein in liquid nitrogen with 30% (v/v) glycerol and store at -80°C for future use.

Data analysis

Purified protein can be analyzed by SDS-PAGE. As shown in the representative data below, the protein was separated by size-exclusion chromatography (SEC) with an elution peak at 102.27 mL, and subsequent SDS-PAGE analysis confirmed its purity.

Figure 1. Results of Nb35 purification.