Vibrio natriegens: An Alternative Expression System for the High-Yield

Communication Cite This: Biochemistry 2019, 58, 2799−2803

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Vibrio natriegens: An Alternative Expression System for the HighYield Production of Isotopically Labeled Proteins Walter Becker,* Florian Wimberger, and Klaus Zangger* Institute of Chemistry, University of Graz, Graz 8010, Austria

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S Supporting Information *

PichiaPink (Thermo Fisher Scientific Inc., USA), or insect cells (ExpiSf (Thermo Fisher Scientific Inc., USA))19−21 were successfully established. These expression systems pushed organismic boundaries and created new ways to produce eukaryotic proteins in their native or close to native environments including post-translational modifications. Here we present guidelines for the high-yield expression of isotopically labeled recombinant proteins in Vibrio natriegens.22 The Gram-negative, nonpathogenic marine bacterium V. natriegens was first described by Payne et al. in the early 1960s23 and was initially called Pseudomonas natriegens. This organism is known to have a doubling time of about ∼10 min or less, which means a 1.6 to 3.9 times faster growth rate than E. coli in minimal media.24,25 In the work presented here, we worked out an optimized protocol for the commercially available V. natriegens type Vmax Express (Synthetic genomics Inc., USA) and compared the expression levels with BL21 (DE3). We used the commonly utilized laboratory equipment, M9 minimal medium protocol,26,27 and the T7-based E. coli vector pET-1a (EMBL).28 Also, we did not codon optimize the genes and vectors for the V. natriegens expression but rather kept the optimized E. coli codons. To show the ability for high expression yields of isotopically labeled proteins, we chose three well-known proteins, FK506 binding protein (FKBP),29 enhanced yellow fluorescent protein (EYFP),30 and tobacco etch virus protease (TEV).31 The FKBP and EYFP are also well-characterized by NMR. All three genes were inserted into three different pET-1a vectors with different tags. FKBP was expressed with the N-utilization substance tag (NusA, 54.76 kDa)32,33 (FKBP-NusA, 69.07 kDa), EYFP with the Z-domain tag (Z2, 6.64 kDa)34 (EYFP-Z2, 37.14 kDa), and TEV with His6-Tag35,36 (TEV-His6, 26.1 kDa). In the early stages of our expression test, we realized that just applying the E. coli standard M9 medium conditions to V. natriegens did not produce high protein levels. To understand which parameters influence the protein production, we varied different expression conditions (Figures S1−S9). All expression tests and scale-up experiments are based on the high-celldensity M9 minimal medium (M9) protocol27 supplemented with 12C glucose and 15NH4Cl (Tables S1 and S6). Due to the fast-growing rate, V. natriegens can reach a high cell mass in a shorter time. With this logic, when the cell mass was higher, more protein and a higher concentration of the carbon source were needed. The main carbon source in M9 medium is

ABSTRACT: Isotopic labeling of recombinant proteins is crucial for studying proteins by liquid state NMR spectroscopy. Nowadays, conventional E. coli-based expression systems like BL21 (DE3) are typically used to express recombinant proteins. Still, the production of isotopically labeled proteins is often costly and timeconsuming, and yields are not sufficient enough for structural studies. Here, we present Vibrio natriegens (Vmax) as an alternative expression system in M9 minimal medium. Due to our optimized M9 minimal medium and conditions and the early time point of induction, we obtained a 2- to 4-fold higher protein yield for two test proteins, FKBP and EYFP, compared to E. coli BL21 (DE3). Production of proteins in V. natriegens in minimal medium is not only more cost-effective and convenient but also less time-consuming than in E. coli. Comparing 15N HSQC spectra of FKBP and EYFP expressed in Vmax and BL21 (DE3) revealed correct folding during expression.

N

MR is a unique tool to study biomacromolecules like proteins, nucleic acids, and complexes of those. Due to the low sensitivity of this technique and the low natural abundance of isotopes with spin 1 nuclei like 13C or 15N, most 2 biomolecular NMR experiments require isotopic labeling (2H, 15 N, 13C) of recombinant proteins. Particularly large amounts of isotopically labeled proteins are needed, e.g., for screening applications of potential drug candidates like SAR by NMR,1 optimization of NMR buffer conditions,2,3 structure determination and assignments,4 heteronuclear spin relaxation methods,5 and structural genomics studies.6 In addition to the costly conventional uniform isotopic labeling, many other specialized labeling strategies were introduced, e.g., MethylTROSY,7−9 segmental,10−12 LEGO-NMR subunit,13 and fluorine labeling,14−16 etc. Most of these approaches, which are carried out in M9 minimal medium, are performed in E. coli.16,17 In the last few decades, intensive genomic research of E. coli made it not only the best-known laboratory bacterium but also the most often used expression system due to low isotope costs and high expression yields. Nowadays, a variety of genetically modified E. coli strains for different purposes are commercially available, e.g., BL21 (DE3) (New England Biolabs, USA), BL21 Star (DE3) or BL21 (DE3) pLysS (Thermo Fisher Scientific Inc., USA), etc. Furthermore, in the last few decades, new expression systems like yeast (Kluyveromyces lactis (New England Biolabs, USA)),18 © 2019 American Chemical Society

Received: May 3, 2019 Revised: June 5, 2019 Published: June 14, 2019 2799

DOI: 10.1021/acs.biochem.9b00403 Biochemistry 2019, 58, 2799−2803

Communication

Biochemistry

However, a low NaCl concentration may cause either a kind of stress reaction, or due to a slower growth, the energy distribution inside the cell can be redirected better toward protein production. Nonetheless, the implementation of the early induction allows the use of lower NaCl concentrations without observation of the growth rate as there is no necessity to wait until a certain OD600 value is reached. Two other important expression adjustment screws are temperature (Figure S7) and shaking frequency (rpm) (Figure S8). In the case of temperature, the highest expression for all 3 proteins was found at 25 °C (Figure S7), and in the case of shaking frequency (Figure S8), lower frequencies like 120 rpm tend to have beneficial effects for expression. Those two values can be used as good guidance to start. Triple resonance NMR experiments are key components for protein structure elucidation, and so, double labeling with 15N and 13C plays an essential role. Comparing the expression levels (Figure S9) of Vmax among non-, single (15N)-, and double-labeled (15N/13C) proteins revealed no difference, as expected due to their insignificant isotope effects. Therefore, early induction along with all parameters discussed above can be applied for even high-yield production of double-labeled proteins. After optimization of all conditions, the expression was scaled up, the proteins were purified, and 15N HSQCs were acquired. FKBP-NusA (Figure 1) and EYFP-Z2 (Figure

glucose. So, increasing the glucose concentration from 0.3%, 0.5%, and 0.8% to 1.0% showed a linear increase in protein production for all three proteins (Figure S1) with the highest yield at 1.0%. In addition, Vmax cells generate not only a higher cell mass but also produce more protein per cell compared to BL21 (DE3) (Figure S2). As higher glucose concentrations result in more production of organic acids,37 a drop in pH to ∼5.5 after less than 2 h after induction was observed and, as a result of that, a high amount of foam formation. To circumvent this situation, the buffer capacity was increased by the addition of M9 buffer components (M9b) up to 8-fold, while all other M9 medium ingredients were kept constant (Table S1). A simple pH adjustment to higher pH values around 8.2 was not efficient enough. There is a strong dependence between expression level and the buffer capacity (Figure S3). The optimum buffer capacity depends on the particular protein. FKBP-NusA is more highly expressed at 3× M9, EYFP-Z2 at 1× M9, and TEV-His6 at 6× M9, but it also shows that a higher M9 concentration like 8× for FKBP-NusA or a lower concentration like 1× for TEV-His6 can lead to no or minor production of protein. Therefore, we recommend testing different buffer concentrations prior to scale up; 3× M9 showed excellent results in all of our cases and can be used as a good starting point. When the pH was stabilized, the foam formation was significantly reduced. Another important issue to consider is the time point of induction. An induction between OD600 of 0.5−1.0 or even higher has been suggested.22,38−40 Due to a poor switch toward protein production of Vmax after induction (cells grow and overexpress at the same time), we found out that induction with IPTG (1 mM) approximately ∼30 min after inoculation of the main culture (OD600 ∼ 0.06) leads to same expression levels (Figure S4) as induction at OD600 0.8. This fact enables us to skip long waiting periods during M9 media protein production before the right OD600 is reached and early induction is possible. In contrast, BL21 (DE3) shows a significantly reduced or no expression if cells did not reach an OD600 of about 0.8 (Figure S4). Applying the early induction to Vmax makes the entire expression (overnight culture (ONC) preparation, reaching the right OD600, induction, expression overnight) of isotopically labeled proteins within 24 h or even less possible. Higher IPTG concentrations, above 1 mM, do not influence the protein production at all (Figure S5). According to our experience, reaching an OD600 of 0.8−1.0 in M9 takes almost the same time as for BL21 (DE3) or even longer if no NaCl (up to 1.7%) is added. NaCl is a crucial component for the halophilic bacterium V. natriegens.25 In complete absence of NaCl, Vmax shows a growing phase up to 12 h in M9 to reach an OD600 of 0.8. It has become apparent that adding 1.7% NaCl to 1× M9 reduces the growing phase to 2 h to reach the same OD600. However, adding up to 1.7% NaCl significantly increases the growth rate but can also negatively influences the protein overexpression (Figure S6). For a more thorough investigation of the salt effect (Figure S6), we kept the NaCl concentration in 3× M9 for FKBPNusA at 0.15%, in 1× M9 for EYFP-Z2 at 0.05%, and in 6× M9 for TEV-His6 at 0.3% NaCl and compared the protein production with the same M9 conditions but added up NaCl to a total amount of 1.7% accordingly. It shows in the case of FKBP-NusA (0.15% NaCl) and TEV-His6 (0.3% NaCl) an increased expression level, whereby EYFP-Z2 remained unchanged under both NaCl concentrations. Still, the real cause for overexpression under low salt conditions is unclear.

Figure 1. Vmax vs BL21 (DE3) upscaled expression test of 15N FKBP-NusA in 500 mL of M9 minimal media. All purification steps are applied and run on 20% SDS-PAGE gel. (Lanes 1 and 8) Pellet. (Lanes 2 and 9) Supernatant. (Lanes 3 and 10) Flow-through. (Lanes 4 and 11) Eluate after Ni2+-NTA column. (Lanes 5 and 12) After TEV digest, pure FKBP at ∼12.08 kDa. (Lanes 6 and 13) Second Ni2+-NTA column run to remove the NusA-tag. (Lanes 7 and 14) Pooled fraction after SEC of pure FKBP.

2) were expressed in 500 mL of M9 (Table S1) and compared with the same protein expressed in BL21 (DE3). In order to estimate the protein concentrations from the lysed cell pellet to the purified protein, the total and specific protein concentrations were recorded during every step (Tables 1 and 2). Vmax shows, for both proteins, a 2 to 4 times higher expression yield and better solubility qualities (see pellet fraction in Figures 1 and 2) than BL21 (DE3). Since isotopes like deuterium and 13C may are costly, it is important to use more cost-effective isotopic labeling strategies. Therefore, we applied the common and cost-efficient pellet exchange protocol.26 First, Vmax was grown in 1 L of enhanced 2YT medium (Table S5) until the cells reached OD600 ∼ 0.5; then the cells were spun down, and the pellet was transferred into M9 medium. Here, it is important to notice that all steps after the cells reach the right OD600 in rich medium must be performed at room temperature. Vmax as part of the Vibrio family is sensitive to stress factors like cold41−43 and lose viability as 2800


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Table 3. Protein Concentration Measurements during Purification of 15N FKBP-NusA in 300 mL of M9 Minimal Medium after Pellet Exchange (Enhanced 2xYT Media)a

Figure 2. Vmax vs BL21 (DE3) upscaled expression test of 15N EYFP-Z2 in 500 mL of M9 minimal media. All purification steps are applied and run on 20% SDS-PAGE gel. (Lanes 1 and 8) Pellet. (Lanes 2 and 9) Supernatant. (Lanes 3 and 10) Flow-through. (Lanes 4 and 11) Eluate after Ni2+-NTA column. (Lanes 5 and 12) After TEV digest, pure EYFP at ∼27.24 kDa. (Lanes 6 and 13) Second Ni2+-NTA column run to remove the Z2-tag. (Lanes 7 and 14) Pooled fraction after SEC of pure EYFP.

protein concentration measurements FKBP (pellet exchange)

V. natriegens Vmax Express

E.coli BL21 DE3

factor

TP: supernatant SP: eluate (before TEV cleavage) TP: eluate (after TEV cleavage) SP: eluate (after TEV cleavage) SP: after SEC

13.8 9.6 5.9 3.5 7.8

5.9 2.3 4.0 1.5 2.5

2.3 4.2 1.5 2.4 3.1

All measurements except “after SEC” are referred to a 12.5 mL volume. The last measurement point (after SEC) is referred to a 1 mL volume. TP = total protein concentration; SP = specific protein concentration; values in mg/mL

a

Table 1. Protein Concentration Measurements during Purification of 15N FKBP-NusA in 500 mL of M9 Minimal Mediuma protein concentration measurements FKBP


E. coli BL21 DE3

factor


16.2 12.1

7.1 2.4

2.3 5.0

7.4

3.6

2.0

5.2 9.8

1.5 2.5

3.5 4.0

All measurements except “after SEC” are referred to a 12.5 mL volume. The last measurement point (After SEC) is referred to a 1 mL volume. TP = total protein concentration; SP = specific protein concentration; values in mg/mL.

a

Table 2. Protein Concentration Measurements during Purification of 15N EYFP-Z2 in 500 mL of M9 Minimal Mediuma protein concentration measurements EYFP


E.coli BL21 DE3

factor


8.0 3.0

3.2 1.9

2.5 1.6

1.8 0.9 4.6

1.1 0.2 0.9

1.7 4.3 5.3

All measurements except “after SEC” are referred to a 12.5 mL volume. The last measurement point (After SEC) is referred to a 1 mL volume. TP = total protein concentration; SP = specific protein concentration; values in mg/mL

a

soon as they are exposed to ice. Also, the pellet exchange method shows a 3−4 times higher expression yield for Vmax than for BL21 (DE3) (Table 3). To check if Vmax correctly folds the protein during protein production, 15N HSQC spectra were acquired (Figures 3 and 4). The overlay of both spectra (BL21 (DE3) vs Vmax) reveals no differences and indicates a correct fold. In conclusion, V. natriegens can be definitely seen as an alternative expression system for the production of isotopically labeled proteins. Using the early time point of induction along with the optimized M9 protocol (glucose concentration, buffer

Figure 3. 15N HSQC of 15N FKBP-NusA expressed in Vmax and BL21 (DE3). (A) The blue spectrum represents the protein expressed in BL21 (DE3) only. (B) Spectra overlay expressed in BL21 (DE3) (blue) and Vmax (red). (C) The red spectrum represents the protein expressed in Vmax only.

capacity, NaCl concentration) and conditions (temperature, rpm) can make the production of isotopically or nonlabeled 2801


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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Walter Becker: 0000-0002-4511-0698 Klaus Zangger: 0000-0003-1682-1594 Author Contributions

W.B. and F.W. contributed equally to this work. W.B. designed the experiments. W.B. and F.W. performed experiments, analyzed data, and wrote the manuscript. W.B. and K.Z. supervised the research. Funding

Financial support to K.Z. by the Austrian Science Foundation (FWF) through the Doktoratskolleg Molecular Enzymology (W901) is gratefully acknowledged. We also thank the interuniversity program in natural sciences, NAWI Graz, for financial support. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Helge Meyer (University of Oldenburg) for providing the pET-1a vector. Bacteria cartoons were designed by brgfx/Freepik.

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ABBREVIATIONS

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REFERENCES

HSQC, heteronuclear single quantum coherence spectroscopy; ONC, overnight culture; SEC, size exclusion chromatography

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Figure 4. 15N HSQC of 15N EYFP-Z2 expressed in Vmax and BL21 (DE3). (A) The blue spectrum represents the protein expressed in BL21 (DE3) only. (B) Spectra overlay expressed in BL21 (DE3) (blue) and Vmax (red). (C) The red spectrum represents the protein expressed in Vmax only.

proteins not only more cost-efficient, due to a higher protein yield per volume medium, but also convenient and less timeconsuming in everyday laboratory work. Nonetheless, we do not see V. natriegens as a competitor to E. coli but rather as an additional tool in any lab that works with minimal media.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.9b00403. Detailed description of preparation and procedure of tests and scale-up expression; determination of the total and specific protein concentration; NMR measurement conditions; media and stock components; expression test SDS-gel with the corresponding pH and OD600 values (PDF) 2802


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Vibrio natriegens: An Alternative Expression System for the High-Yield

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