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Original Research Article

DTT 2023; 2(2): 71-79

Published online September 30, 2023 https://doi.org/10.58502/DTT.23.0013

Copyright © The Pharmaceutical Society of Korea.

Mass Production and Characterization of Recombinant Human Grim-19 in Escherichia coli

Gaurab Subedi, Sung Jean Park

College of Pharmacy and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon, Korea

Correspondence to:Sung Jean Park, psjnmr@gachon.ac.kr

Received: April 4, 2023; Revised: May 10, 2023; Accepted: May 16, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

The gene associated with retinoid-interferon-induced mortality 19 (Grim-19, also known as NADH dehydrogenase 1 α subunit 13) induces apoptotic cell death in response to interferon-β (IFN-β) and retinoic acid (RA) treatment. The Grim-19/Stat3 complex suppresses the essential genes that enhance cell growth. In addition, Grim-19 inhibits oncoproteins and viral gene products during tumorigenesis, which suggests that the development of biologics of Grim-19 could be considered. Although various cellular functions of Grim-19 were identified, limited information on the structural characteristics of Grim-19 is available. To study the structural characteristics of Grim-19 and to discover the possible biologics of Grim-19, the preparation of a large amount of Grim-19 is necessary. Grim-19 is the subunit of the mitochondrial membrane protein, ubiquinone, which is also found in the nucleus and cytoplasm. Thus, the stable recombinant protein of Grim-19 is not easily obtainable because of the aggregation of the protein. Here we successfully established an efficient expression system in E. coli by designing various E. coli vectors. Most of the E. coli vectors showed acceptable expression levels of Grim-19 in E. coli while they mainly produced an insoluble form of Grim-19 which could not be refolded sufficiently in vitro. Only the NusA-tagged Grim-19 fusion protein can be obtained as the soluble form in E. coli. The CD analysis showed Grim-19 mainly contains the α-helical structure with a melting temperature, 47℃.

KeywordsGrim-19, NusA, NADH dehydrogenase, biologics

Grim-19, a novel cell death regulatory gene, plays important roles in cell apoptosis, embryogenesis, and mitochondrial respiratory chain. Grim-19 was originally identified as the cell death-associated factor induced by exposure to interferon-β (IFN-β) and retinoic acid (RA) (Moreira et al. 2011). IFN-β and RA signaling facilitates that Grim-19 forms a complex with High temperature required A2 (HtrA2). The secreted Grim-19/HtrA2 complex binds to X-linked inhibitors of Apoptosis protein (XIAP), suppressing XIAP activity (Ma et al. 2007) and enhancing apoptosis. Grim-19 also interacts with STAT3 to regulate the activity of STAT3, resulting in enhanced apoptosis (Lufei et al. 2003; Zhang et al. 2003). The Grim-19/Stat3 complex suppresses the expression of essential genes that enhance cell growth (Kong et al. 2019). Thus, the induced overexpression of Grim-19 in cells or the application of Grim-19 biologics into cells can enhance cell sensitivity to IFN-RA-induced death.

In addition, Grim-19 is responsible for various biological events by interacting with other proteins. Grim-19 is also essential for early embryonic development because deletion of Grim-19 homolog in mice causes lethality at embryonic day 9.5 (Huang et al. 2004). As a functional component of mitochondrial complex I (the NADH dehydrogenase 1 α subunit 13, NDUFA13), Grim-19 is necessary for mitochondrial respiratory chain complex I assembly and ATP synthesis activity (Fearnley et al. 2001; Lu and Cao 2008). While Grim-19 is primarily localized to the mitochondria, there is evidence to suggest that a significant fraction of Grim-19 is also present in the nucleus and cytoplasm (Nallar and Kalvakolanu 2017). Studies have shown that Grim-19 can shuttle between the nucleus and cytoplasm in response to cellular stress or signaling events. For instance, the complexation of Grim-19 with the TAD Domain of STAT3 occurs in Nucleus (Lufei et al. 2003). Grim-19 binds p16 and arrests G₁/S transition in the cell cycle (Sun et al. 2010). Nucleotide oligomerization domain 2 (NOD2) also interacts with Grim-19, which controls pathogen invasion of intestinal epithelial cells (Barnich et al. 2005).

Grim-19 has been shown to play a role in inhibiting the growth of cancer cells (Nallar and Kalvakolanu 2017). Low expression of Grim-19 was found in human carcinoma in the kidney, lung, and liver (Alchanati et al. 2006; Wen et al. 2013) and mutation of Grim-19 localized in thyroid tumor. The function of Grim-19 as a tumor suppressor and one of the biologics candidates for cancer therapeutics was well shown in research (Angell et al. 2000); Antisense expression of Grim-19 strongly reduced IFN/RA-induced cancer cell death by reducing the intracellular levels of Grim-19 protein. Like this, the activity of growth inhibition and apoptosis of cancer cells by Grim-19 was reported (Zhou et al. 2009; Okamoto et al. 2010; Hao et al. 2012). Grim-19 has also been shown to inhibit viral gene products during viral infections (Yeo et al. 2008). This is because Grim-19 is a component of the mitochondrial respiratory chain, and has been shown to interact with viral proteins that target mitochondria during infection. By inhibiting these viral proteins, Grim-19 may help to limit viral replication and spread. Studies have shown that the expression of Grim-19 is reduced in patients with hepatitis C virus (HCV), suggesting that it may be an important factor in the body’s ability to fight the virus (Kim et al. 2017). Developing Grim-19-based biologics could therefore provide a promising avenue for the treatment of viral infections as well as anti-tumor agents.

Grim-19 is composed of 144 amino acids and the 3D structure of full length is not determined, yet. A recent cryo-EM study showed great architecture of the human mitochondrial respiratory megacomplex that contains a short part of Grim-19 (Guo et al. 2017); 22 residues (from K7 to R28) of Grim-19 stuck on one side of the complex forming a long loop (Fig. 1A). Based on the predicted structure by AlphaFold, Grim-19 seem to form a long stretch of α-helix with a N-terminal loop (Fig. 1B) that is responsible to the binding to the respiratory complex (Jumper et al. 2021).

Figure 1.The structure of the mitochondrial respiratory system. (A) The megacomplex consists of 18 chains of respiratory proteins including a part of Grim-19 (red). (B) The predicted structure of Grim-19 obtained by AlphaFold. The binding site of Grim-19 is supposed to be the N-terminal loop (red) of the predicted structure.

Although the biological function of Grim-19 has been mainly investigated, protein-level studies including mass production in vitro, the development of biologics, and structural characterization are highly limited. This may be closely related with that the stable recombinant protein of Grim-19 is not easily obtainable because of easy aggregation of the protein. Here we successfully produced the soluble and stable Grim-19 in E. coli by designing various E. coli vectors and characterizing the features of the secondary structure of Grim-19.

Materials

Restriction endonucleases and T4 DNA Ligase were purchased from New England Biolabs (Beverly, MA, USA). EmeraldAmp GT PCR Master Mix was purchased from TaKaRa Bio, Inc. (Shiga, Japan). pET vectors and pVFT4S were purchased from Novagen (Madison, WI, USA). pGEX-4T-1 vector was obtained from GE Healthcare Life Sciences (Pittsburgh, USA). pCold1 and the chaperone expression vectors, pG-KJE8 and pG-TF2 were purchased from Takara Korea Biomedical Inc. (Seoul, Korea). Cloned Pfu DNA Polymerase was obtained from Stratagen (La Jolla, CA, USA). Oligonucleotides used for PCR amplification reaction and DNA sequencing were obtained from Cosmogenetech (Seoul, Korea). The Kits for plasmid extraction and DNA purification were purchased from Cosmogenetech (Seoul, Korea) and GeneAll (Seoul, Korea). For the purification of the protein, Ni SepharoseTM High Performance, anion and cation exchange column (5 mL, HiTrap Q, SP HP) from GE Healthcare Life Sciences were purchased. All Materials of reagent were biotechnological grade.

Construction of plasmid

The coding regions of Grim-19 were inserted into pET-15b, pCold1, pGEX-4T-1, pET-30a (GB1), pET-32a, pET-44a (+), and pVFT4S vectors. The plasmids were generated by inserting the PCR fragment containing the Grim-19 sequence into the appropriately digested vector. The seven constructs are summarized in Fig. 2. The constructed plasmid was confirmed by DNA sequencing.

Figure 2.The seven constructs of Grim-19 used in this study. GB1, domain B1 of Immunoglobulin G-binding protein G; Trx, thioredoxin; NusA, N-utilization substance A; MBP, E. coli maltose binding protein; GST, glutathione-S-transferase.

General expression of Grim-19 in various vectors

Grim-19 constructs were transformed into appropriate E. coli host strains such as BL21, BL21 (DE3), and BL21 (DE3) codonPlus-RIPL (Seo et al. 2021). The transformed BL21 (DE3) cells were cultured in 100 mL of LB broth media with ampicillin (50 µg/mL), respectively. After incubation at 37 or 15℃ until OD600 0.6, proteins were induced by adding Isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. After 4 hours of incubation at 37℃ or two days of incubation at 15℃, cells were harvested by centrifugation with 8,000 rpm at 4℃ for 15 min (Beckman JA).

Expression of Grim-19 with chaperone

Grim-19 in the pCold1 or pGEX-4T-1 vectors was co-expressed with E. coli chaperones, dnaK, grpE, groEl, and groES from the vector pG-KJE8 or groES, groEL, and tig from pG-Tf2. Liquid LB media was prepared to contain both 20 µg/mL of chloramphenicol and 50 µg/mL of ampicillin. Expression of chaperons was achieved by adding 0.5 mg/mL L-arabinose and 5 ng/mL tetracycline; the plasmid pG-KJE8 was treated with both L-arabinose and tetracycline for chaperones induction. The chaperones coded in the pG-Tf2 vector were induced by tetracycline. Grim-19 constructs (pCold1 and pGEX-4T-1) were transformed into chaperone-competent cell BL21 that harbored pG-KJE8 or pG-Tf2. Cells were incubated at 37℃ until OD600 to 0.4 and then transferred to 15℃. When OD600 reached to 0.6, protein expression was induced by adding 0.5 mM IPTG, 0.5 mg/mL L-arabinose, and 5 ng/mL tetracycline. Cells were cultured with shaking at 15℃ for 24 to 48 hours.

Purification and refolding of Grim-19

After sonication of bacterial cell pellets, the inclusion bodies were collected and re-suspended in 60 mL of 6 M guanidine hydrochloride. After incubation in a cold room overnight and then the supernatant was loaded onto Ni SepharoseTM High-Performance resin using the open column. Fractions were eluted in 50 mM Tris-HCl (pH 8.5), 500 mM NaCl, and 10% glycerol with an imidazole gradient from 150 mM to 300 mM. Pooled Grim-19 was refolded with by stepwise dialysis; the dialysis buffer was sequentially switched with the refolding buffers (50 mM Tris-HCl and 500 mM NaCl, pH 8) containing different concentrations of guanidine hydrochloride (from 6 to 0 M) for at 24 hours in a cold room.

Purification of NusA tagged Grim-19

The bacterial cell pellet was suspended in 100 mL of the lysis buffer (50 mM Tris-HCl pH 8.5, 500 mM NaCl, 10% glycerol, and 40 mM imidazole). The bacterial cell lysis was performed by sonication (pulse on 2 sec, off 7 sec, 5 min, 45% amplitude, and 4℃). After centrifugation of the lysate (45 min 15,000 rpm, 4℃), the supernatant was loaded onto Ni SepharoseTM High-Performance resin using the open column. Fractions were eluted in the elution buffer containing 50 mM Tris-HCl 8.5, 500 mM NaCl, and 10% glycerol with an imidazole gradient from 100 mM and 500 mM. Further purification was accomplished by ion exchange chromatography with a HiTrap Q HP 5 mL and HiTrap 5 mL SP HP column (GE Healthcare) in the buffer containing 50 mM Tris-HCl, pH 8.5, 25 mM NaCl, and 10% glycerol. The NusA tagged Grim-19 did not bind and flew through the column. The binding pass fraction was collected and dialyzed with PreScission protease to cleave the NusA tag. The cleaved Grim-19 was further purified using S-100 size exclusion chromatography. The final Grim-19 was concentrated using an Amicon Ultra centrifugal filter 10,000 MWCO (Millipore) and stored at −80℃ until use.

Multi-angle light scattering coupled with size exclusion chromatography

Multi-angle light scattering (MALS) was used to determine the oligomeric states of NusA-tagged Grim-19 and Grim-19. The size-exclusion column, BioSep SEC-s3000 column (Phenomenex) was run on the 1260 Infinity HPLC system (Agilent Technologies). The laser scattering data were obtained in the MALS machine, miniDAWN-TREOS with emission at 657.4 nm (Wyatt Technology) and analyzed by ASTRA 6.0.1.10 software (Wyatt Technology). 100 µM of NusA tagged Grim-19 and Grim-19 were injected under the running buffer containing 50 mM Tris-HCl, pH 8, and 50 mM NaCl. All experiments were performed at room temperature.

Circular dichroism (CD)

The CD spectra were obtained in the Applied Photophysics Chirascan spectrometer (Surrey, UK). The thickness of the cuvette used was 0.1 mm. The measured wavelength range was from 280 to 190 nm. The measurement was repeated three times and averaged. The background buffer signals were eliminated. Tm Scan for melting temperature determination was measured by gradually changing the temperature from 20℃ to 80℃. The concentration of Grim-19 was 3 µM and the buffer used was 25 mM Sodium phosphate (pH 7.4) without any salt.

E. coli expression of various Grim-19 constructs and refolding of inclusion bodies

Seven constructs as shown in Fig. 2 were designed to express the soluble Grim-19. The expression level of all constructs in E. coli was appreciable (Fig. 3). Among the constructs, MBP fused Grim-19 and GB1 fused Grim-19 were most highly expressed. However, all these constructs showed insoluble aggregates after sonication, suggesting inclusion bodies may be formed during expression in E. coli at 37℃. The codon-optimized E. coli such as BL21 (DE3)-RIPL was not helpful to increase the soluble fraction of Grim-19, either.

Figure 3.Expression results of recombinant Grim-19 at 37℃. The expressed proteins are depicted with rectangles. Lane M; molecular weight marker, Lane 1; before induction, Land 2; expression of His-tag Grim-19 (pET-15b), Lane 3; expression of His-tag Grim-19 (pCold1), Lane 4; expression of GB1 tagged Grim-19, Lane 5; expression of Trx tagged Grim-19, Lane 6; expression of NusA tagged Grim-19, Lane 7; expression of MBP tagged Grim-19, Lane 8; expression of GST tagged Grim-19.

To obtain the soluble protein, the inclusion bodies of His-tagged Grim-19 were refolded by serial dilution of Guanidine-HCl; resolved Grim-19 by 6M Guanidine-HCl was sequentially transferred until the revolved solution became 1M Guanidine-HCl solution. Then the remaining Guanidine-HCl was completely removed by dialysis with the buffer solutions containing the solubilizing materials such as Tween 80, Glycine, Proline, and Triton X-100. However, the refolding process was not successful, resulting in the re-aggregation of the protein (Fig. 4).

Figure 4.Refolding results of His-tagged Grim-19 at 4℃. The pellets (Lane 1, 3, 5, and 7) and supernatants (Lane 2, 4, 6, and 8) are shown after the final step. All Grim-19 was found in the pellets. The final dialysis buffer contains various solubilizing agents such as proline (Lane 1 and 2), Glycine (Lane 3 and 4), Triton X-100 (Lane 5 and 6), and Tween 80 (Lane 7 and 8).

Optimization of expression conditions

The lowered IPTG concentration was not effective for the improvement of protein solubility. We also monitored the temperature effect on solubility enhancement; expression at reduced temperatures, 25℃ and 15℃ was less effective, resulting in the production of insoluble aggregates. However, the biggest construct, NusA fused Grim-19 was successfully obtained from soluble fraction through cultivation at 15℃.

To improve the expression of soluble Grim-19, the co-expression of several E. coli chaperones with Grim-19 were performed as well. Grim-19 inserted into the pCold1 or pGEX-4T-1 vectors were co-transformed with either pG-KJE8 containing dnaK, grpE, groEl, and groES or pG-Tf2 containing groES, groEL, and tig. As a result, the chaperones increased the soluble fraction of Grim-19 while the amount of soluble Grim-19 was insufficient to proceed with further protein purification. Fig. 5 shows the soluble fraction of His-tagged Grim-19 and GST-Grim-19, which revealed that co-expression with chaperones was moderately effective only in His-tagged Grim-19. The solubility of the proteins in various conditions is summarized in Table 1.

Figure 5.Co-expression with E. coli chaperone. His-tagged (Lane 1 and 2) or GST fused (Lane 3 and 4) Grim-19 was co-expressed with GroEL, GroES, and tig encoded in pG-TF2. In the supernatant fraction after lysis, a moderate level of His-tagged Grim-19 was found (Lane 2) while that of GST-fused Grim-19 was very low (Lane 4). The pellets after lysis are shown in Lane 1 (His-tagged Grim-19) and Lane 3 (GST-fused Grim-19).

Table 1 Solubility of Grim-19 in various expression conditions

Protein tags (vectors)Solubility (expression temperature)
37℃15℃
Single expressionHis-tag (pET-15b)BadBad
His-tag (pCold1)BadBad
GB1 (pET-30a)BadBad
Trx (pET-32a)BadBad
NusA (pET-44a)BadGood
MBP (pVFT4S)BadBad
GST (pGEX-4T-1)BadBad
Co-expression with E. coli chaperonesHis-tag (pCold1) + groEL, groES, tig (pG-TF2)NT*Less than one-third of total Grim-19**
His-tag (pCold1) + dnaK, J, grpE, groEL, groES (pG-KJE8)Less than one-third of total Grim-19
GST (pGEX-4T-1) + groEL, groES, tig (pG-TF2)Bad
GST (pGEX-4T-1) + dnaK, J, grpE, groEL, groES (pG-KJE8)Bad

*NT: not tried.

**The amount of protein in pellets or supernatant was estimated by the density of protein bands as shown in Fig. 5.


As described above, only NusA fused Grim-19 could be sufficiently obtained in the soluble fraction. Thus we used this construct to purify and characterize Grim-19 hereafter. The purification was performed with a combination of several chromatographic methods including Ni affinity chromatography, ion exchange chromatography, and size exclusion chromatography. The final yield of Grim-19 was about 5 mg for 1 L culture and with a purity of more than 95% (Fig. 6A).

Figure 6.The purification of NusA fused Grim-19 and its structural characteristic. (A) SDS PAGE results for each purification step. Lane 1; induction in E. coli, Lane 2; pellets after lysis, Lane 3; supernatant after lysis, Lane 4; purified NusA fused Grim-19, Lane 5; enzyme cleavage, Lane 6; purified Grim-19. (B) CD spectroscopy of Grim-19. NusA fused Grim-19 is represented by black dots and cleaved Grim-19 by red dots. (C) Melting of Grim-19. NusA fused Grim-19 is represented by black dots and cleaved Grim-19 by red dots.

Structural characteristic of Grim-19

NusA fused Grim-19 seems to be monomeric in solution, as judged by multi-angle light scattering (MALS) data while NusA cleaved Grim-19 showed time-dependent multimerization and became unstable in the buffer without 10% glycerol. Based on the hydrodynamic molecular weight, cleaved Grim-19 may form a trimeric state (Table 2).

Table 2 Oligomeric states of Grim-19 constructs by MALS analysis

ConstructsMw (kDa, theoretical)Mw (kDa, observed)Polydispersity (Mw/Mn)Fraction of mass (% of Total)
NusA fused Grim-197670.4 ± 5.1 (monomer)1.00297
Cleaved Grim-191615.2 ± 3.2 (monomer)1.01167
50.1 ± 7.4 (Trimer)1.00318

To characterize the structural features of Grim-19 fused NusA, the CD spectrum was measured. The structures of NusA fused Grim-19 and cleaved Grim-19 were compared at the same concentration of the proteins. Fig. 6B shows two negative minima around 208 nm and 222 nm, suggesting that all proteins adopt the helical structure, similarly (Park et al. 2021). The secondary structural composition calculated by the software, CDNN revealed more than 90% of the secondary components are α-helices, which is in good agreement with the predicted structure as shown in Fig. 1. The NusA also adopts α-helical structure (Gopal et al. 2001).

The melting temperatures of the two proteins were slightly different from each other. Melting of NusA fused Grim-19 and cleaved Grim-19 similarly began around 37℃ while the 50% denaturation temperatures were 47℃ for cleaved Grim-19 and 51℃ for NusA fused Grim-19 (Fig. 6C).

To improve the solubility of the expressed Grim-19 in E. coli, various constructs and expression conditions were investigated in this study. Intact Grim-19 and Grim-19 fused with various proteins showed good expression in E. coli while most of the constructs could not yield soluble Grim-19 except the largest fusion tag, NusA, and co-expression with E. coli chaperones. This result suggested that Grim-19 is easily aggregated during protein synthesis, which may be related to the long helical structure as predicted by Alphofold. The hydrophobic residues such as Ile, Leu, and Val are exposed to a solvent environment on the surface of helices (Fig. 7). Intermolecular hydrophobic interactions between these residues appear to be critical for the aggregation of Grim-19. Resultantly, this aggregation of Grim-19 may be inhibited by fusion with the huge protein, NusA, and binding to chaperone; the soluble NusA tag may prevent the association of Grim-19 by steric hindrance and the chaperones may mask the solvent-exposed hydrophobic residues by making complex with Grim-19 in E. coli. The fusion with NusA was only effective at low-temperature cultivation, which reveals that the expression rate of NusA fused Grim-19 is also important for the proper folding of the protein. The stability of NusA fused Grim-19 is much higher over time than the NusA-free form. Incubation of cleaved Grim-19 at 4℃ overnight leads to the multimerization of the protein.

Figure 7.The location of hydrophobic residues in Grim-19. Ile (blue), Leu (green), and Val (orange) are depicted in the structure of Grim-19. The structure shown is predicted by AlphaFold.

CD analysis showed the α-helical propensity of Grim-19, which corresponds to the structure predicted by AlphaFold. This structural feature may be maintained in NusA fused Grim-19 since both the CD spectra of cleaved Grim-19 and NusA fused Grim-19 were very similar. However, NusA fused Grim-19 appears to be thermodynamically more stable, which may be contributed to the intrinsically high stability of NusA.

Grim-19 may be a potential therapeutic target for the treatment of cancer, viral infections, and neurological disorders. In addition, it is possible to use Grim-19 as a biologic. Developing a biologic based on Grim-19 would require extensive research to determine its efficacy, safety, and potential side effects. To develop a protein-based biologic, mass protein production is typically required. This is because biologics are typically administered at relatively high doses, and therefore require large quantities of the active protein. The process of producing large quantities of protein for use in biologics is often complex and time-consuming. This study established a mass production system and highly pure protein can be obtained as a soluble form. The established system may help study structure as well as for discovering Grim-19-related therapeutic agents at the protein level.

This work was supported by grants from the National Research Foundation of Korea (NRF-2021R1F1A1061607 and 2020R1A6A1A0304370812). This work was also supported by the Gachon University research fund of 2022 (GCU-202209030001).

The authors declare that they have no conflict of interest.

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Article

Original Research Article

DTT 2023; 2(2): 71-79

Published online September 30, 2023 https://doi.org/10.58502/DTT.23.0013

Copyright © The Pharmaceutical Society of Korea.

Mass Production and Characterization of Recombinant Human Grim-19 in Escherichia coli

Gaurab Subedi, Sung Jean Park

College of Pharmacy and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon, Korea

Correspondence to:Sung Jean Park, psjnmr@gachon.ac.kr

Received: April 4, 2023; Revised: May 10, 2023; Accepted: May 16, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The gene associated with retinoid-interferon-induced mortality 19 (Grim-19, also known as NADH dehydrogenase 1 α subunit 13) induces apoptotic cell death in response to interferon-β (IFN-β) and retinoic acid (RA) treatment. The Grim-19/Stat3 complex suppresses the essential genes that enhance cell growth. In addition, Grim-19 inhibits oncoproteins and viral gene products during tumorigenesis, which suggests that the development of biologics of Grim-19 could be considered. Although various cellular functions of Grim-19 were identified, limited information on the structural characteristics of Grim-19 is available. To study the structural characteristics of Grim-19 and to discover the possible biologics of Grim-19, the preparation of a large amount of Grim-19 is necessary. Grim-19 is the subunit of the mitochondrial membrane protein, ubiquinone, which is also found in the nucleus and cytoplasm. Thus, the stable recombinant protein of Grim-19 is not easily obtainable because of the aggregation of the protein. Here we successfully established an efficient expression system in E. coli by designing various E. coli vectors. Most of the E. coli vectors showed acceptable expression levels of Grim-19 in E. coli while they mainly produced an insoluble form of Grim-19 which could not be refolded sufficiently in vitro. Only the NusA-tagged Grim-19 fusion protein can be obtained as the soluble form in E. coli. The CD analysis showed Grim-19 mainly contains the α-helical structure with a melting temperature, 47℃.

Keywords: Grim-19, NusA, NADH dehydrogenase, biologics

Introduction

Grim-19, a novel cell death regulatory gene, plays important roles in cell apoptosis, embryogenesis, and mitochondrial respiratory chain. Grim-19 was originally identified as the cell death-associated factor induced by exposure to interferon-β (IFN-β) and retinoic acid (RA) (Moreira et al. 2011). IFN-β and RA signaling facilitates that Grim-19 forms a complex with High temperature required A2 (HtrA2). The secreted Grim-19/HtrA2 complex binds to X-linked inhibitors of Apoptosis protein (XIAP), suppressing XIAP activity (Ma et al. 2007) and enhancing apoptosis. Grim-19 also interacts with STAT3 to regulate the activity of STAT3, resulting in enhanced apoptosis (Lufei et al. 2003; Zhang et al. 2003). The Grim-19/Stat3 complex suppresses the expression of essential genes that enhance cell growth (Kong et al. 2019). Thus, the induced overexpression of Grim-19 in cells or the application of Grim-19 biologics into cells can enhance cell sensitivity to IFN-RA-induced death.

In addition, Grim-19 is responsible for various biological events by interacting with other proteins. Grim-19 is also essential for early embryonic development because deletion of Grim-19 homolog in mice causes lethality at embryonic day 9.5 (Huang et al. 2004). As a functional component of mitochondrial complex I (the NADH dehydrogenase 1 α subunit 13, NDUFA13), Grim-19 is necessary for mitochondrial respiratory chain complex I assembly and ATP synthesis activity (Fearnley et al. 2001; Lu and Cao 2008). While Grim-19 is primarily localized to the mitochondria, there is evidence to suggest that a significant fraction of Grim-19 is also present in the nucleus and cytoplasm (Nallar and Kalvakolanu 2017). Studies have shown that Grim-19 can shuttle between the nucleus and cytoplasm in response to cellular stress or signaling events. For instance, the complexation of Grim-19 with the TAD Domain of STAT3 occurs in Nucleus (Lufei et al. 2003). Grim-19 binds p16 and arrests G₁/S transition in the cell cycle (Sun et al. 2010). Nucleotide oligomerization domain 2 (NOD2) also interacts with Grim-19, which controls pathogen invasion of intestinal epithelial cells (Barnich et al. 2005).

Grim-19 has been shown to play a role in inhibiting the growth of cancer cells (Nallar and Kalvakolanu 2017). Low expression of Grim-19 was found in human carcinoma in the kidney, lung, and liver (Alchanati et al. 2006; Wen et al. 2013) and mutation of Grim-19 localized in thyroid tumor. The function of Grim-19 as a tumor suppressor and one of the biologics candidates for cancer therapeutics was well shown in research (Angell et al. 2000); Antisense expression of Grim-19 strongly reduced IFN/RA-induced cancer cell death by reducing the intracellular levels of Grim-19 protein. Like this, the activity of growth inhibition and apoptosis of cancer cells by Grim-19 was reported (Zhou et al. 2009; Okamoto et al. 2010; Hao et al. 2012). Grim-19 has also been shown to inhibit viral gene products during viral infections (Yeo et al. 2008). This is because Grim-19 is a component of the mitochondrial respiratory chain, and has been shown to interact with viral proteins that target mitochondria during infection. By inhibiting these viral proteins, Grim-19 may help to limit viral replication and spread. Studies have shown that the expression of Grim-19 is reduced in patients with hepatitis C virus (HCV), suggesting that it may be an important factor in the body’s ability to fight the virus (Kim et al. 2017). Developing Grim-19-based biologics could therefore provide a promising avenue for the treatment of viral infections as well as anti-tumor agents.

Grim-19 is composed of 144 amino acids and the 3D structure of full length is not determined, yet. A recent cryo-EM study showed great architecture of the human mitochondrial respiratory megacomplex that contains a short part of Grim-19 (Guo et al. 2017); 22 residues (from K7 to R28) of Grim-19 stuck on one side of the complex forming a long loop (Fig. 1A). Based on the predicted structure by AlphaFold, Grim-19 seem to form a long stretch of α-helix with a N-terminal loop (Fig. 1B) that is responsible to the binding to the respiratory complex (Jumper et al. 2021).

Figure 1. The structure of the mitochondrial respiratory system. (A) The megacomplex consists of 18 chains of respiratory proteins including a part of Grim-19 (red). (B) The predicted structure of Grim-19 obtained by AlphaFold. The binding site of Grim-19 is supposed to be the N-terminal loop (red) of the predicted structure.

Although the biological function of Grim-19 has been mainly investigated, protein-level studies including mass production in vitro, the development of biologics, and structural characterization are highly limited. This may be closely related with that the stable recombinant protein of Grim-19 is not easily obtainable because of easy aggregation of the protein. Here we successfully produced the soluble and stable Grim-19 in E. coli by designing various E. coli vectors and characterizing the features of the secondary structure of Grim-19.

Materials and Methods

Materials

Restriction endonucleases and T4 DNA Ligase were purchased from New England Biolabs (Beverly, MA, USA). EmeraldAmp GT PCR Master Mix was purchased from TaKaRa Bio, Inc. (Shiga, Japan). pET vectors and pVFT4S were purchased from Novagen (Madison, WI, USA). pGEX-4T-1 vector was obtained from GE Healthcare Life Sciences (Pittsburgh, USA). pCold1 and the chaperone expression vectors, pG-KJE8 and pG-TF2 were purchased from Takara Korea Biomedical Inc. (Seoul, Korea). Cloned Pfu DNA Polymerase was obtained from Stratagen (La Jolla, CA, USA). Oligonucleotides used for PCR amplification reaction and DNA sequencing were obtained from Cosmogenetech (Seoul, Korea). The Kits for plasmid extraction and DNA purification were purchased from Cosmogenetech (Seoul, Korea) and GeneAll (Seoul, Korea). For the purification of the protein, Ni SepharoseTM High Performance, anion and cation exchange column (5 mL, HiTrap Q, SP HP) from GE Healthcare Life Sciences were purchased. All Materials of reagent were biotechnological grade.

Construction of plasmid

The coding regions of Grim-19 were inserted into pET-15b, pCold1, pGEX-4T-1, pET-30a (GB1), pET-32a, pET-44a (+), and pVFT4S vectors. The plasmids were generated by inserting the PCR fragment containing the Grim-19 sequence into the appropriately digested vector. The seven constructs are summarized in Fig. 2. The constructed plasmid was confirmed by DNA sequencing.

Figure 2. The seven constructs of Grim-19 used in this study. GB1, domain B1 of Immunoglobulin G-binding protein G; Trx, thioredoxin; NusA, N-utilization substance A; MBP, E. coli maltose binding protein; GST, glutathione-S-transferase.

General expression of Grim-19 in various vectors

Grim-19 constructs were transformed into appropriate E. coli host strains such as BL21, BL21 (DE3), and BL21 (DE3) codonPlus-RIPL (Seo et al. 2021). The transformed BL21 (DE3) cells were cultured in 100 mL of LB broth media with ampicillin (50 µg/mL), respectively. After incubation at 37 or 15℃ until OD600 0.6, proteins were induced by adding Isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. After 4 hours of incubation at 37℃ or two days of incubation at 15℃, cells were harvested by centrifugation with 8,000 rpm at 4℃ for 15 min (Beckman JA).

Expression of Grim-19 with chaperone

Grim-19 in the pCold1 or pGEX-4T-1 vectors was co-expressed with E. coli chaperones, dnaK, grpE, groEl, and groES from the vector pG-KJE8 or groES, groEL, and tig from pG-Tf2. Liquid LB media was prepared to contain both 20 µg/mL of chloramphenicol and 50 µg/mL of ampicillin. Expression of chaperons was achieved by adding 0.5 mg/mL L-arabinose and 5 ng/mL tetracycline; the plasmid pG-KJE8 was treated with both L-arabinose and tetracycline for chaperones induction. The chaperones coded in the pG-Tf2 vector were induced by tetracycline. Grim-19 constructs (pCold1 and pGEX-4T-1) were transformed into chaperone-competent cell BL21 that harbored pG-KJE8 or pG-Tf2. Cells were incubated at 37℃ until OD600 to 0.4 and then transferred to 15℃. When OD600 reached to 0.6, protein expression was induced by adding 0.5 mM IPTG, 0.5 mg/mL L-arabinose, and 5 ng/mL tetracycline. Cells were cultured with shaking at 15℃ for 24 to 48 hours.

Purification and refolding of Grim-19

After sonication of bacterial cell pellets, the inclusion bodies were collected and re-suspended in 60 mL of 6 M guanidine hydrochloride. After incubation in a cold room overnight and then the supernatant was loaded onto Ni SepharoseTM High-Performance resin using the open column. Fractions were eluted in 50 mM Tris-HCl (pH 8.5), 500 mM NaCl, and 10% glycerol with an imidazole gradient from 150 mM to 300 mM. Pooled Grim-19 was refolded with by stepwise dialysis; the dialysis buffer was sequentially switched with the refolding buffers (50 mM Tris-HCl and 500 mM NaCl, pH 8) containing different concentrations of guanidine hydrochloride (from 6 to 0 M) for at 24 hours in a cold room.

Purification of NusA tagged Grim-19

The bacterial cell pellet was suspended in 100 mL of the lysis buffer (50 mM Tris-HCl pH 8.5, 500 mM NaCl, 10% glycerol, and 40 mM imidazole). The bacterial cell lysis was performed by sonication (pulse on 2 sec, off 7 sec, 5 min, 45% amplitude, and 4℃). After centrifugation of the lysate (45 min 15,000 rpm, 4℃), the supernatant was loaded onto Ni SepharoseTM High-Performance resin using the open column. Fractions were eluted in the elution buffer containing 50 mM Tris-HCl 8.5, 500 mM NaCl, and 10% glycerol with an imidazole gradient from 100 mM and 500 mM. Further purification was accomplished by ion exchange chromatography with a HiTrap Q HP 5 mL and HiTrap 5 mL SP HP column (GE Healthcare) in the buffer containing 50 mM Tris-HCl, pH 8.5, 25 mM NaCl, and 10% glycerol. The NusA tagged Grim-19 did not bind and flew through the column. The binding pass fraction was collected and dialyzed with PreScission protease to cleave the NusA tag. The cleaved Grim-19 was further purified using S-100 size exclusion chromatography. The final Grim-19 was concentrated using an Amicon Ultra centrifugal filter 10,000 MWCO (Millipore) and stored at −80℃ until use.

Multi-angle light scattering coupled with size exclusion chromatography

Multi-angle light scattering (MALS) was used to determine the oligomeric states of NusA-tagged Grim-19 and Grim-19. The size-exclusion column, BioSep SEC-s3000 column (Phenomenex) was run on the 1260 Infinity HPLC system (Agilent Technologies). The laser scattering data were obtained in the MALS machine, miniDAWN-TREOS with emission at 657.4 nm (Wyatt Technology) and analyzed by ASTRA 6.0.1.10 software (Wyatt Technology). 100 µM of NusA tagged Grim-19 and Grim-19 were injected under the running buffer containing 50 mM Tris-HCl, pH 8, and 50 mM NaCl. All experiments were performed at room temperature.

Circular dichroism (CD)

The CD spectra were obtained in the Applied Photophysics Chirascan spectrometer (Surrey, UK). The thickness of the cuvette used was 0.1 mm. The measured wavelength range was from 280 to 190 nm. The measurement was repeated three times and averaged. The background buffer signals were eliminated. Tm Scan for melting temperature determination was measured by gradually changing the temperature from 20℃ to 80℃. The concentration of Grim-19 was 3 µM and the buffer used was 25 mM Sodium phosphate (pH 7.4) without any salt.

Results

E. coli expression of various Grim-19 constructs and refolding of inclusion bodies

Seven constructs as shown in Fig. 2 were designed to express the soluble Grim-19. The expression level of all constructs in E. coli was appreciable (Fig. 3). Among the constructs, MBP fused Grim-19 and GB1 fused Grim-19 were most highly expressed. However, all these constructs showed insoluble aggregates after sonication, suggesting inclusion bodies may be formed during expression in E. coli at 37℃. The codon-optimized E. coli such as BL21 (DE3)-RIPL was not helpful to increase the soluble fraction of Grim-19, either.

Figure 3. Expression results of recombinant Grim-19 at 37℃. The expressed proteins are depicted with rectangles. Lane M; molecular weight marker, Lane 1; before induction, Land 2; expression of His-tag Grim-19 (pET-15b), Lane 3; expression of His-tag Grim-19 (pCold1), Lane 4; expression of GB1 tagged Grim-19, Lane 5; expression of Trx tagged Grim-19, Lane 6; expression of NusA tagged Grim-19, Lane 7; expression of MBP tagged Grim-19, Lane 8; expression of GST tagged Grim-19.

To obtain the soluble protein, the inclusion bodies of His-tagged Grim-19 were refolded by serial dilution of Guanidine-HCl; resolved Grim-19 by 6M Guanidine-HCl was sequentially transferred until the revolved solution became 1M Guanidine-HCl solution. Then the remaining Guanidine-HCl was completely removed by dialysis with the buffer solutions containing the solubilizing materials such as Tween 80, Glycine, Proline, and Triton X-100. However, the refolding process was not successful, resulting in the re-aggregation of the protein (Fig. 4).

Figure 4. Refolding results of His-tagged Grim-19 at 4℃. The pellets (Lane 1, 3, 5, and 7) and supernatants (Lane 2, 4, 6, and 8) are shown after the final step. All Grim-19 was found in the pellets. The final dialysis buffer contains various solubilizing agents such as proline (Lane 1 and 2), Glycine (Lane 3 and 4), Triton X-100 (Lane 5 and 6), and Tween 80 (Lane 7 and 8).

Optimization of expression conditions

The lowered IPTG concentration was not effective for the improvement of protein solubility. We also monitored the temperature effect on solubility enhancement; expression at reduced temperatures, 25℃ and 15℃ was less effective, resulting in the production of insoluble aggregates. However, the biggest construct, NusA fused Grim-19 was successfully obtained from soluble fraction through cultivation at 15℃.

To improve the expression of soluble Grim-19, the co-expression of several E. coli chaperones with Grim-19 were performed as well. Grim-19 inserted into the pCold1 or pGEX-4T-1 vectors were co-transformed with either pG-KJE8 containing dnaK, grpE, groEl, and groES or pG-Tf2 containing groES, groEL, and tig. As a result, the chaperones increased the soluble fraction of Grim-19 while the amount of soluble Grim-19 was insufficient to proceed with further protein purification. Fig. 5 shows the soluble fraction of His-tagged Grim-19 and GST-Grim-19, which revealed that co-expression with chaperones was moderately effective only in His-tagged Grim-19. The solubility of the proteins in various conditions is summarized in Table 1.

Figure 5. Co-expression with E. coli chaperone. His-tagged (Lane 1 and 2) or GST fused (Lane 3 and 4) Grim-19 was co-expressed with GroEL, GroES, and tig encoded in pG-TF2. In the supernatant fraction after lysis, a moderate level of His-tagged Grim-19 was found (Lane 2) while that of GST-fused Grim-19 was very low (Lane 4). The pellets after lysis are shown in Lane 1 (His-tagged Grim-19) and Lane 3 (GST-fused Grim-19).

Table 1 . Solubility of Grim-19 in various expression conditions.

Protein tags (vectors)Solubility (expression temperature)
37℃15℃
Single expressionHis-tag (pET-15b)BadBad
His-tag (pCold1)BadBad
GB1 (pET-30a)BadBad
Trx (pET-32a)BadBad
NusA (pET-44a)BadGood
MBP (pVFT4S)BadBad
GST (pGEX-4T-1)BadBad
Co-expression with E. coli chaperonesHis-tag (pCold1) + groEL, groES, tig (pG-TF2)NT*Less than one-third of total Grim-19**
His-tag (pCold1) + dnaK, J, grpE, groEL, groES (pG-KJE8)Less than one-third of total Grim-19
GST (pGEX-4T-1) + groEL, groES, tig (pG-TF2)Bad
GST (pGEX-4T-1) + dnaK, J, grpE, groEL, groES (pG-KJE8)Bad

*NT: not tried..

**The amount of protein in pellets or supernatant was estimated by the density of protein bands as shown in Fig. 5..



As described above, only NusA fused Grim-19 could be sufficiently obtained in the soluble fraction. Thus we used this construct to purify and characterize Grim-19 hereafter. The purification was performed with a combination of several chromatographic methods including Ni affinity chromatography, ion exchange chromatography, and size exclusion chromatography. The final yield of Grim-19 was about 5 mg for 1 L culture and with a purity of more than 95% (Fig. 6A).

Figure 6. The purification of NusA fused Grim-19 and its structural characteristic. (A) SDS PAGE results for each purification step. Lane 1; induction in E. coli, Lane 2; pellets after lysis, Lane 3; supernatant after lysis, Lane 4; purified NusA fused Grim-19, Lane 5; enzyme cleavage, Lane 6; purified Grim-19. (B) CD spectroscopy of Grim-19. NusA fused Grim-19 is represented by black dots and cleaved Grim-19 by red dots. (C) Melting of Grim-19. NusA fused Grim-19 is represented by black dots and cleaved Grim-19 by red dots.

Structural characteristic of Grim-19

NusA fused Grim-19 seems to be monomeric in solution, as judged by multi-angle light scattering (MALS) data while NusA cleaved Grim-19 showed time-dependent multimerization and became unstable in the buffer without 10% glycerol. Based on the hydrodynamic molecular weight, cleaved Grim-19 may form a trimeric state (Table 2).

Table 2 . Oligomeric states of Grim-19 constructs by MALS analysis.

ConstructsMw (kDa, theoretical)Mw (kDa, observed)Polydispersity (Mw/Mn)Fraction of mass (% of Total)
NusA fused Grim-197670.4 ± 5.1 (monomer)1.00297
Cleaved Grim-191615.2 ± 3.2 (monomer)1.01167
50.1 ± 7.4 (Trimer)1.00318


To characterize the structural features of Grim-19 fused NusA, the CD spectrum was measured. The structures of NusA fused Grim-19 and cleaved Grim-19 were compared at the same concentration of the proteins. Fig. 6B shows two negative minima around 208 nm and 222 nm, suggesting that all proteins adopt the helical structure, similarly (Park et al. 2021). The secondary structural composition calculated by the software, CDNN revealed more than 90% of the secondary components are α-helices, which is in good agreement with the predicted structure as shown in Fig. 1. The NusA also adopts α-helical structure (Gopal et al. 2001).

The melting temperatures of the two proteins were slightly different from each other. Melting of NusA fused Grim-19 and cleaved Grim-19 similarly began around 37℃ while the 50% denaturation temperatures were 47℃ for cleaved Grim-19 and 51℃ for NusA fused Grim-19 (Fig. 6C).

Discussion

To improve the solubility of the expressed Grim-19 in E. coli, various constructs and expression conditions were investigated in this study. Intact Grim-19 and Grim-19 fused with various proteins showed good expression in E. coli while most of the constructs could not yield soluble Grim-19 except the largest fusion tag, NusA, and co-expression with E. coli chaperones. This result suggested that Grim-19 is easily aggregated during protein synthesis, which may be related to the long helical structure as predicted by Alphofold. The hydrophobic residues such as Ile, Leu, and Val are exposed to a solvent environment on the surface of helices (Fig. 7). Intermolecular hydrophobic interactions between these residues appear to be critical for the aggregation of Grim-19. Resultantly, this aggregation of Grim-19 may be inhibited by fusion with the huge protein, NusA, and binding to chaperone; the soluble NusA tag may prevent the association of Grim-19 by steric hindrance and the chaperones may mask the solvent-exposed hydrophobic residues by making complex with Grim-19 in E. coli. The fusion with NusA was only effective at low-temperature cultivation, which reveals that the expression rate of NusA fused Grim-19 is also important for the proper folding of the protein. The stability of NusA fused Grim-19 is much higher over time than the NusA-free form. Incubation of cleaved Grim-19 at 4℃ overnight leads to the multimerization of the protein.

Figure 7. The location of hydrophobic residues in Grim-19. Ile (blue), Leu (green), and Val (orange) are depicted in the structure of Grim-19. The structure shown is predicted by AlphaFold.

CD analysis showed the α-helical propensity of Grim-19, which corresponds to the structure predicted by AlphaFold. This structural feature may be maintained in NusA fused Grim-19 since both the CD spectra of cleaved Grim-19 and NusA fused Grim-19 were very similar. However, NusA fused Grim-19 appears to be thermodynamically more stable, which may be contributed to the intrinsically high stability of NusA.

Grim-19 may be a potential therapeutic target for the treatment of cancer, viral infections, and neurological disorders. In addition, it is possible to use Grim-19 as a biologic. Developing a biologic based on Grim-19 would require extensive research to determine its efficacy, safety, and potential side effects. To develop a protein-based biologic, mass protein production is typically required. This is because biologics are typically administered at relatively high doses, and therefore require large quantities of the active protein. The process of producing large quantities of protein for use in biologics is often complex and time-consuming. This study established a mass production system and highly pure protein can be obtained as a soluble form. The established system may help study structure as well as for discovering Grim-19-related therapeutic agents at the protein level.

Acknowledgements

This work was supported by grants from the National Research Foundation of Korea (NRF-2021R1F1A1061607 and 2020R1A6A1A0304370812). This work was also supported by the Gachon University research fund of 2022 (GCU-202209030001).

Conflict of interest

The authors declare that they have no conflict of interest.

Fig 1.

Figure 1.The structure of the mitochondrial respiratory system. (A) The megacomplex consists of 18 chains of respiratory proteins including a part of Grim-19 (red). (B) The predicted structure of Grim-19 obtained by AlphaFold. The binding site of Grim-19 is supposed to be the N-terminal loop (red) of the predicted structure.
Drug Targets and Therapeutics 2023; 2: 71-79https://doi.org/10.58502/DTT.23.0013

Fig 2.

Figure 2.The seven constructs of Grim-19 used in this study. GB1, domain B1 of Immunoglobulin G-binding protein G; Trx, thioredoxin; NusA, N-utilization substance A; MBP, E. coli maltose binding protein; GST, glutathione-S-transferase.
Drug Targets and Therapeutics 2023; 2: 71-79https://doi.org/10.58502/DTT.23.0013

Fig 3.

Figure 3.Expression results of recombinant Grim-19 at 37℃. The expressed proteins are depicted with rectangles. Lane M; molecular weight marker, Lane 1; before induction, Land 2; expression of His-tag Grim-19 (pET-15b), Lane 3; expression of His-tag Grim-19 (pCold1), Lane 4; expression of GB1 tagged Grim-19, Lane 5; expression of Trx tagged Grim-19, Lane 6; expression of NusA tagged Grim-19, Lane 7; expression of MBP tagged Grim-19, Lane 8; expression of GST tagged Grim-19.
Drug Targets and Therapeutics 2023; 2: 71-79https://doi.org/10.58502/DTT.23.0013

Fig 4.

Figure 4.Refolding results of His-tagged Grim-19 at 4℃. The pellets (Lane 1, 3, 5, and 7) and supernatants (Lane 2, 4, 6, and 8) are shown after the final step. All Grim-19 was found in the pellets. The final dialysis buffer contains various solubilizing agents such as proline (Lane 1 and 2), Glycine (Lane 3 and 4), Triton X-100 (Lane 5 and 6), and Tween 80 (Lane 7 and 8).
Drug Targets and Therapeutics 2023; 2: 71-79https://doi.org/10.58502/DTT.23.0013

Fig 5.

Figure 5.Co-expression with E. coli chaperone. His-tagged (Lane 1 and 2) or GST fused (Lane 3 and 4) Grim-19 was co-expressed with GroEL, GroES, and tig encoded in pG-TF2. In the supernatant fraction after lysis, a moderate level of His-tagged Grim-19 was found (Lane 2) while that of GST-fused Grim-19 was very low (Lane 4). The pellets after lysis are shown in Lane 1 (His-tagged Grim-19) and Lane 3 (GST-fused Grim-19).
Drug Targets and Therapeutics 2023; 2: 71-79https://doi.org/10.58502/DTT.23.0013

Fig 6.

Figure 6.The purification of NusA fused Grim-19 and its structural characteristic. (A) SDS PAGE results for each purification step. Lane 1; induction in E. coli, Lane 2; pellets after lysis, Lane 3; supernatant after lysis, Lane 4; purified NusA fused Grim-19, Lane 5; enzyme cleavage, Lane 6; purified Grim-19. (B) CD spectroscopy of Grim-19. NusA fused Grim-19 is represented by black dots and cleaved Grim-19 by red dots. (C) Melting of Grim-19. NusA fused Grim-19 is represented by black dots and cleaved Grim-19 by red dots.
Drug Targets and Therapeutics 2023; 2: 71-79https://doi.org/10.58502/DTT.23.0013

Fig 7.

Figure 7.The location of hydrophobic residues in Grim-19. Ile (blue), Leu (green), and Val (orange) are depicted in the structure of Grim-19. The structure shown is predicted by AlphaFold.
Drug Targets and Therapeutics 2023; 2: 71-79https://doi.org/10.58502/DTT.23.0013

Table 1 Solubility of Grim-19 in various expression conditions

Protein tags (vectors)Solubility (expression temperature)
37℃15℃
Single expressionHis-tag (pET-15b)BadBad
His-tag (pCold1)BadBad
GB1 (pET-30a)BadBad
Trx (pET-32a)BadBad
NusA (pET-44a)BadGood
MBP (pVFT4S)BadBad
GST (pGEX-4T-1)BadBad
Co-expression with E. coli chaperonesHis-tag (pCold1) + groEL, groES, tig (pG-TF2)NT*Less than one-third of total Grim-19**
His-tag (pCold1) + dnaK, J, grpE, groEL, groES (pG-KJE8)Less than one-third of total Grim-19
GST (pGEX-4T-1) + groEL, groES, tig (pG-TF2)Bad
GST (pGEX-4T-1) + dnaK, J, grpE, groEL, groES (pG-KJE8)Bad

*NT: not tried.

**The amount of protein in pellets or supernatant was estimated by the density of protein bands as shown in Fig. 5.


Table 2 Oligomeric states of Grim-19 constructs by MALS analysis

ConstructsMw (kDa, theoretical)Mw (kDa, observed)Polydispersity (Mw/Mn)Fraction of mass (% of Total)
NusA fused Grim-197670.4 ± 5.1 (monomer)1.00297
Cleaved Grim-191615.2 ± 3.2 (monomer)1.01167
50.1 ± 7.4 (Trimer)1.00318

References

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