Tang R et al. (2021), mem-iLID, a fast and economic protein purificat...

XB-ART-58207

Biosci Rep 2021 Jul 30;417:. doi: 10.1042/BSR20210800.

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mem-iLID, a fast and economic protein purification method.

Tang R , Yang S , Nagel G , Gao S .

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Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

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Species referenced: Xenopus laevis
Genes referenced: ctrl

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	Figure 1. Light-assisted protein purification based on iLID/SspB after Xenopus oocyte expression(A) SspB (the original SspB, which is also called SspB_nano) and iLID were fused with YFP and expressed as soluble fraction, or fused with Lyn11 and expressed as membrane fraction, separately. The general purification process was shown in the schematic diagram. The detailed protocol was described in the ‘Materials and methods’ part. (B) The amount of YFP-tagged proteins bound to the membrane fractions after 30 min in the dark or blue light illumination. The red arrow indicated the corresponding step of the tested samples. For each reaction, membrane fraction from 25 oocytes was mixed with soluble fraction containing a final 20 nM YFP-tagged proteins in 300 μl buffer A. n=3, error bars = SEM, all individual data points are shown. (C) Illumination time-dependent binding efficiency of Lyn11-LOV-A and YFP-SspB. Illumination was performed with blue light. Each reaction contained a soluble fraction of 40 nM YFP-SspB and membrane fraction from 25 oocytes in 300 μl buffer A. n=3, error bars = SEM, all individual data points are shown. (D) Time-dependent release efficiency of YFP-SspB from the membrane expressing Lyn11-LOV-A in the dark. The binding was induced by 20 min blue light of (C). The blue arrow indicated the corresponding step of the tested samples. n=3, error bars = SEM, all individual data points are shown.
	Figure 2. Light-assisted protein purification after E. coli expression(A) Expressions of YFP-SspB (original SspB, SspB_nano) and H1021-LOV-A were induced by 0.5 mM IPTG and confirmed in SDS/PAGE. A total of 50 ml E. coli culture was washed and homogenized in 1 ml buffer A for the crude extraction of membrane fraction and soluble fraction. The extracted membrane fractions were resuspended in 1 ml buffer A. Five microliters of each sample was loaded to the SDS/PAGE. Total: the whole cell lysate after ultrasonic homogenization. MF, membrane fraction. SF, soluble fraction. The YFP-SspB (45 kDa) band in soluble fraction and H1021-LOV-A (24 kDa) band in membrane fraction were indicated by black arrows. (B) The amounts of different YFP-SspB versions bound the membrane fractions containing H1021-LOV-A were compared after 20 min binding in the dark or blue light illumination. In each reaction, the membrane fraction from 2.5 ml E. coli culture was mixed with different concentrations of YFP-SspB in 300 μl buffer A. Michaelis–Menten curves were fitted. n=3, all individual data points are shown. (C) The amounts of purified proteins with different YFP-SspB versions were compared. The membrane fraction from 10 ml E. coli was mixed with soluble fraction containing 4 μM YFP-SspB for the purification. The purified proteins were collected in 200 μl buffer A. Illumination time is 20 min and releasing time in the dark is 30 min. n=3, error bars = SEM, all individual data points are shown. (D) Ten microliters purified YFP-SspB from (C) were tested in the SDS/PAGE. The YFP-SspB band was indicated by the black arrow. D, binding in the dark. L, binding in the light.
	Figure 3. Generation and comparison of new monomeric SspB mutants(A) The extracted and E. coli whole cell lysate expressing YFP-SspB_milli and YFP-SspB_milli-8M in a native PAGE gel. The whole cell lysates were prepared from 50 ml E. coli culture resuspended and homogenized in 1 ml buffer A. Five microliters from each sample was loaded to the native PAGE. The gel was stained by EZBlue. Total: the whole cell lysate after ultrasonic homogenization. BSA was loaded as a marker, which is 66 kDa in monomeric state and 132 kDa in dimeric state (: monomer, : dimer). (B) The binding efficacy of YFP-SspB_milli (dimeric) and YFP-SspB_milli-8M (monomeric) to H1021-LOV-A after 20 min blue illumination. In each reaction, the membrane fraction from 2.5 ml E. coli was mixed with soluble fractions with different concentrations of YFP-SspB (different versions) in 300 μl buffer A. Michaelis–Menten curves were fitted. n=3, all individual data points are shown. (C) Sequences of the mutation part (α-helix1) for the newly designed SspB_nano mutations. The changed amino acids were labeled in red. (D) Comparing the binding abilities of different YFP-SspB mutants to H1021-LOV-A after 20 min in the dark or blue light illumination. In each reaction, the membrane fraction from 2.5 ml E. coli was mixed with different concentrations of YFP-SspB mutants in 300 μl buffer A. Michaelis–Menten curves were fitted. n=3, all individual data points are shown. (E) Comparing the purification efficacy of YFP-SspB_milli and YFP-SspB_nano-3M. The membrane fraction extracted from 5 ml E. coli was mixed with soluble fractions containing different initial concentrations of YFP-SspB in 300 μl buffer A for the purification. Illumination time is 20 min and releasing time in the dark is 30 min. The final purified proteins were collected in 200 μl buffer A. n=3, error bars = SEM. All individual data points are shown. (F) The purified YFP-SspB_milli and YFP-SspB_nano-3M from (E) were checked in a native PAGE gel. A total of 200 ng of each sample was loaded (: monomer, **: dimer), BSA was loaded as a marker.
	Figure 4. Characterizations of the H1021-LOV-A/SspB_nano-3M(A) Illumination time-dependent binding efficiency between H1021-LOV-A and YFP-SspB_nano-3M. Each reaction contained 4 μM YFP-SspB and membrane fraction from 5 ml E. coli in 300 μl buffer A. n=3, error bars = SEM, all individual data points are shown. (B) Time (in the dark)-dependent release efficiency of the YFP-SspB_nano-3M bound to H1021-LOV-A. The binding was stimulated by 20 min blue light. The released (purified) proteins were collected in 200 μl buffer A. n=3, error bars = SEM, all individual data points are shown. (C) Purification of YFP-SspB_nano-3M under different conditions. Each reaction contained 4 μM YFP-SspB and membrane fraction from 5 ml E. coli in 300 μl buffer A. Purified proteins were collected in 200 μl buffer A. A total of 20 μl samples were loaded in SDS/PAGE.
	Figure 5. bPAC purification and activity analysis(A) Light-gated cAMP production abilities of bPAC-YFP-SspB_nano and bPAC-YFP-SspB_nano-3M. Reactions were illuminated by blue light (473 nm, 0.3 mW/mm2). Details of the in vitro reaction were described in the ‘Materials and methods’ part. n=3, error bars = SEM. All individual data points are shown. (B) The purified bPAC-YFP-SspB_nano-3M in SDS/PAGE. A total of 100 oocytes injected with bPAC-YFP-SspB_nano-3M were homogenized in 300 μl buffer, and then mixed with the membrane extract from 5 ml E. coli for the purification. The purified protein was collected in 200 μl buffer. Total: the total soluble extract from oocytes expressing bPAC-YFP-SspB_nano-3M, loading amount: 5 μl. D: released proteins after binding in the dark, loading amount: 20 μl. L: released proteins after binding in the light, loading amount: 20 μl. (C) Confirmation of the purified bPAC-YFP-SspB_nano-3M by Western blot. ctrl: oocyte without cRNA injection. Otherwise, the loading condition was similar to (C). Details of the Western blot were described in the ‘Materials and methods’ part. (D) cAMP production by purified bPAC-YFP-SspB_nano-3M. Samples were illuminated by blue light (473 nm, 0.3 mW/mm2). Details of the in vitro reaction were described in the ‘Materials and methods’ part. n=3, error bars = SEM. All individual data points are shown.
	Figure 6. DNA polymerase purification and activity estimation(A) Confirmation of IPTG-induced SspB_nano-3M tagged DNA polymerase (∼120 kDa) expression and purification in SDS/PAGE. A total of 50 ml E. coli was homogenized by 1 ml buffer B, and the pellet part was resuspended by 1 ml buffer B before the Gel loading. Total: the whole cell lysate, loading amount: 5 μl. Pellet: sediment fraction, loading amount: 5 μl. SF: soluble fraction, loading amount: 5 μl. D: released proteins after binding in the dark, loading amount: 20 μl. L: released proteins after binding in the light, loading amount: 20 μl. (B) Agarose gel electrophoresis of PCR products with the purified and commercial DNA polymerase. Lanes 1–3: PCR products using 2, 1 and 0.5 μl of the purified DNA polymerase. Lane 4: PCR products using 0.2 μl (0.4 U) commercial Phusion DNA polymerase.

References [+] :

Achmüller, N(pro) fusion technology to produce proteins with authentic N termini in E. coli. 2007, Pubmed