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Efficient expression of glutathione peroxidase with chimeric tRNA in amber-less Escherichia coli Zhenlin Fan, Jian Song, Tuchen Guan, Xiuxiu Lv, and JingYan Wei ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00290 • Publication Date (Web): 03 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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ACS Synthetic Biology

Efficient expression of glutathione peroxidase with chimeric tRNA in amber-less Escherichia coli 1#

2#

1

1

*

Zhenlin Fan , Jian Song , Tuchen Guan , Xiuxiu Lv and Jingyan Wei 1

College of Pharmaceutical Science, Jilin University, Changchun 130021, P. R. China

2

College of Electronic Science and Engineering, Jilin University, Changchun 130000, P. R. China

Corresponding Author: Jingyan Wei, E-mail: [email protected]

ABSTRACT: The active center of selenium-containing glutathione peroxidase (GPx) is selenocysteine (Sec), which is is biosynthesized on its tRNA in organisms. The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence (SECIS) to suppress the UGA codon. The expression of mammalian GPx is extremely difficult with traditional recombinant UTu

DNA technology. Recently, a chimeric tRNA (tRNA

) that is compatible with elongation factor Tu

(EF-Tu) has made selenoprotein expression easier. In this study, human glutathione peroxidase UTu

(hGPx) was expressed in amber-less Escherichia coli C321.∆A.exp using tRNA chimeric tRNAs that were constructed based on tRNA UTu

substitutes the acceptor stem and T-stem of tRNA

and seven

UTu

UTu2

. We found that chimeric tRNA Sec

with those from tRNA

, which

, enabled the

expression of reactive hGPx with high yields. We also found that chimeric tRNA

UTuT6

, which has a

UTu

single base change (A59C) compared to tRNA

, mediated the highest reactive expression of

hGPx1. The hGPx1 expressed exists as a tetramer and reacts with positive cooperativity. The UTuT6

SDS-PAGE analysis of hGPx2 produced by tRNA

with or without sodium selenite

supplementation showed that the incorporation of Sec is nearly 90%. Our approach enables

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efficient selenoprotein expression in amber-less Escherichia coli and should enable further characterization of selenoproteins in vitro.

KEY WORDS: selenocysteine, tRNA, glutathione peroxidase, positive cooperativity

The glutathione peroxidase (GPx) superfamily is widespread over all of the kingdoms of life. In humans, eight GPxs have been identified, five of which are selenoproteins whose catalysis 1

depends on selenocysteine (Sec) in the active center . Sec is the 21st amino acid and is encoded 2

by UGA, which requires decoding against a termination mechanism . As an important amino acid for selenoproteins, its synthesis and encoding are complicated and inefficient. Most canonical amino acids are specifically recognized by their corresponding aminoacyl-tRNA synthetases (aaRSs) and their cognate tRNAs to form aminoacyl-tRNAs, maintaining the fidelity of protein 3

synthesis . However, there are no Sec-specific aaRSs in organisms. The synthesis of Sec 4

proceeds through a tRNA-dependent, two-step synthesis pathway in prokaryotes . In the first step Sec

of this pathway, the seryl-tRNA synthetase (SerRS) aminoacylates serine onto tRNA Sec

Ser-tRNA

Sec-tRNA

to create Sec

. A second enzyme (selenocysteine synthase, SelA) then converts Ser-tRNA

Sec

5

Sec

with selenophosphate . The products Ser-tRNA

Sec

and Sec-tRNA

to

are Sec

discriminated by SelB, a specialized translation elongation factor that delivers Sec-tRNA

to the

6

ribosome . SelB specifically binds selenocysteine insertion sequence (SECIS) in mRNA to Sec

suppress UGA upstream of SECIS and insert Sec-tRNA

Sec

stem and the first two base pairs in the T-stem of tRNA Sec

that block the interactions between Sec-tRNA

. The last base pair in the acceptor

are thought to be the antideterminants 7

and EF-Tu .


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Despite this detailed knowledge of the factors involved, the mechanism of Sec incorporation in prokaryotes cannot be used for site-specific Sec insertion because the SECIS element must immediately follow the UGA codon. However, there are currently several non-SECIS-dependent UTu

methods for site-directed incorporation of Sec. A chimeric tRNA UTu

and EF-Tu was constructed that enabled Sec-tRNA 8

the ribosomal A-site . tRNA Ser

of tRNA

UTu

Sec

with that of tRNA

Based on tRNA

compatible with SerRS, SelA

to be synthesized in vivo and delivered to Ser

was constructed based on tRNA

by replacing the acceptor stem

, and the anticodon was changed to CUA to encode UAG as Sec.

UTu

, an improved chimeric tRNA

UTuX

, which is a better substrate for SelA, was

constructed and realized optimal Ser to Sec conversion, leading to nearly 100% incorporation of 9

SecUx

Sec . tRNA

10

, which mediated nearly complete Sec incorporation , was selected using NMC-A Sec

β-lactamase as a reporter protein after evolution of the antideterminant sequence of tRNA

. The

strategy for targeted incorporation of genetically encoded nonstandard amino acids (nsAAs) was 11

used to incorporate synthetic photocaged Sec residue in yeast cells . The adaptation of a chimeric tRNA for the site-specific incorporation of Sec into protein is limited by its binding affinity to SerRS, SelA and EF-Tu. Poor binding of SelA and EF-Tu results in incomplete conversion of Ser-tRNA to Sec-tRNA and the misincorporation of Ser in response to cognate codon. Substantial efforts have been made to enhance the amount of Sec incorporation and to increase the selenoprotein product yield

12, 13

UTu

. Compared with tRNA

UTuX

, tRNA

contains

more critical structural parameters for SelA binding is a good substrate for SelA and increase the 9

yield of Sec conversion . The binding affinity of EF-Tu for an aa-tRNA is modulated by the amino 14-16

acid and the tRNA body

. Tuning the amino acid binding pocket of EF-Tu is commonly



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performed to improve nsAAs incorporation efficiently deliver Sec-tRNA

UTu

17-19

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UTu

, and engineered EF-Sel1 coupled with tRNA

can

13

to the ribosome . Engineering the acceptor stem and T-stem of a 20, 21

tRNA body can also tune the binding affinity of EF-Tu for an aa-tRNA UTu

sequences in the T-stem of tRNA

. Selecting optimized

may facilitate the presentation and decoding of the tRNA in

the ribosome. UTu

In this study, we used tRNA

in amber-less E.coli C321.∆A.exp for the expression and

characterization of hGPx4, hGPx1 and hGPx2. The amber-less E.coli C321.∆A.exp has all its endogenous 321 UAG amber codons replaced by UAA and its UAG-specific release factor 1 22

deleted, thus facilitating amber codon suppression . During the process of tuning the T-stem of UTu

tRNA

Sec

for the improvement of Sec encoding, we found that the entire antideterminant of tRNA UTuT2

could be recognized by EF-Tu in a chimeric tRNA (tRNA UTuT6

hGPx4 with high yields. Another chimeric tRNA

) that mediated expression of reactive

, which has a single base change (A59C)

UTu

compared to tRNA UTuX

tRNA

, could produce hGPx1 with GPx activity as high as that produced by

. The recombined hGPx1 produced exists as a tetramer and reacts with positive

cooperativity. Kinetic analysis demonstrated that it followed a typical ping-pong mechanism similar to natural hGPx1. Finally, the incorporation ratio of Sec was evaluated by SDS-PAGE analysis of UTuT6

hGPx2 produced by tRNA

with or without sodium selenite supplementation.


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RESULTS AND DISCUSSION

UTu

IPTG-induced production of Sec-containing hGPx4 using tRNA

in amber-less E.coli.

UTu

Sec-containing hGPx4 was expressed using the previously reported tRNA

in the host strain

C321.∆A.exp with sodium selenite supplementation. Plasmid pN565, which contains the encoding gene for IPTG-inducible T7 RNA polymerase mutant, was used for the expression of multiple proteins under the control of the T7 promoter. Read-through of the amber stop codon at position 46 in the gene encoding hGPx4 resulted in a full-length hGPx4 (Figure 1a) with GPx activity. The activity of purified hGPx4 with H2O2 as an oxidizing substrate was 18.3 U/mg protein. Treatment with 0.1 mM DTT did not influence the activity, indicating that the hGPx4 was expressed in its reduced form and reactive Sec is incorporated without undesired side reactions inside producing cells. The yield of hGPx4 in C321.∆A.exp was approximately 2.5 mg/L of culture, which is much higher than that in BL21(DE3) (0.2 mg/L). The incorporation of Sec at position 46 was further confirmed by intact protein ESI-MS (Figure 1b) and ESI-MS/MS detection (Figure 1c). The results also show that the N-terminal methionine was removed from the recombinant hGPx4 expressed in 8

C321.∆A.exp. Consistent with previous results , a major peak (21143.5 Da) corresponding to 46Ser

hGPx4

(theoretical mass: 21145.1 Da) was found. Other detected peaks include a 21207.4 Da

corresponding to hGPx4

46Sec

corresponding to hGPx4

46Glu/Lys/Gln

(theoretical masses: 21208.03 Da), and a 21185.2 Da peak (theoretical mass: 21186.17-21187.07 Da). Using C321.∆A.exp

as the host strain can increase the yield of hGPx4; however, besides Ser, the incorporation of canonical amino acids including Glu, Lys and Gln at UAG codon were detected. The



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mis-incorporation is thought to be the result of near-cognate recognition of amber codon by the 23

respective native aminoacyl-tRNAs . The slow recruitment of Ser/Sec-tRNA

UTu

to EF-Tu may

increase the near-cognate recognition of amber codon by the native aminoacyl-tRNAs.

Figure 1. Expression, purification and characterization of hGPx4. (a) Coomassie-stained SDS-PAGE gel of purified hGPx4. (b) Deconvoluted ESI-Q-TOF mass spectrum of hGPx4 produced in amber-less E. coli. The purified hGPx4 was analyzed with Q-TOF-MS and the intact masses were deconvoluted from the peak detected with charge 25+ in the raw spectrum. The product without Met1 contained a mixture of hGPx4 (21185.2 Da, peak2) and hGPx4

46Sec

46Ser

46Glu/Lys/Gln

(21143.5 Da, peak 1), hGPx4

(21207.4 Da, peak 3). The raw spectrum is provided in

Supplementary Figure S1. (c) ESI-MS/MS spectrum of the Sec-containing peptide from hGPx4


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digested with trypsin. The matched b and y ions are labeled by black and the unmatched b and y ions are labeled by light gray. Sec was identified by MASCOT as a Se-IAN-Cys modification of Cys. The m/z values for the peptide fragments are listed in Supplementary Table S3. The unit m/z describes the mass-to-charge ratio. ESI-MS/MS spectra and the m/z values of Ser/Glu/Lys/Gln-containing peptides are provided in Supplementary Figure S2 and Supplementary Tables S4, S5, S6 and S7.

UTu

Tuning the acceptor stem and T-stem and the T-loop of tRNA

results in variants that

mediate the expression of hGPx4 with different yields and activities. To improve the ratio of Sec incorporation, we focused on evolving tRNA

UTu

to obtain a tRNA that binds EF-Tu tighter

when esterified with Sec than Ser. The affinity of EF-Tu for an aminoacyl-tRNA is supposed to be determined by the esterified amino acid as well as the acceptor stem and T-stem of the tRNA 14

body . Tuning the amino acid binding pocket of EF-Tu or acceptor stem and T-stem of the tRNA body could improve the ratio of Sec incorporation at the UAG codon. During the process of tuning UTu

the T-stem of the tRNA UTuT1

tRNA

for optimal binding affinity of EF-Tu to chimeric tRNA, we found that Sec

(Figure 2a), whose T-stem originates from tRNA

, was able to express hGPx4 (Figure

2b) with GPx activity (30.2 U/mg protein, Figure 2c). Based on this result, we further examined Sec

whether the entire antideterminant of tRNA UTuT2

tRNA

UTu

could be recognized by EF-Tu in tRNA

. Thus,

was constructed, leading to the production of hGPx4 with a purified yield of 2.1 mg/L of

culture and GPx activity of 28.1 U/mg protein (Figure 2b, c). Only the base 59 in the T-loop of UTuT2

tRNA

Sec

is different from tRNA

in the whole acceptor stem and T-stem and the T-loop. The



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24

genomic tRNA database indicates that the T-loop is a conserved region in tRNAs from E. coli . Furthermore, the unique tertiary base pair formed between base 59 in the T-loop and base 16 in 25

the D-loop participate in the D-loopT-loop interactions , which play the key role in maintenance of 26

the tRNA L-shape . The L-shaped conformation of tRNA is essential for the elongation cycle

27, 28

.

Next, the last base pair in the acceptor stem and T-stem and base 59 were tested independently UTuT3-7

or collectively for binding to EF-Tu for Sec encoding. Five tRNA variants tRNA

(Figure 2a)

were constructed and used for hGPx4 expression to validate the Sec incorporation efficiency. No UTuT3

obvious recombinant hGPx4 was purified from the culture mediated by tRNA

with or without

sodium selenite supplementation (SDS-PAGE analysis, data not shown). The results showed that the remaining 4 tRNA variants were recognized by EF-Tu, and hGPx4 with GPx activity was expressed (Figure 2b, c). The GPx activity of the hGPx4 produced by tRNA UTuT1

improved compared with tRNA

UTuT5

. The tRNA

UTuT7

and tRNA

UTuT4

was further

mediated expression yielded UTu

hGPx4 with slightly higher GPx activity compared with that produced by tRNA

UTuT6

. The tRNA

UTuT6

contributed the highest GPx activity (38.1 U/mg protein). The hGPx4 expressed using tRNA

was further investigated by ESI-MS (Figure 2d). A major peak at a mass of 21207.9 Da was 46Sec

observed, corresponding to the calculated mass for hGPx4

(theoretical mass: 21208.03 Da).

Two medium peaks at 21144.7 Da and 21185.2 Da, corresponding to hGPx4 mass: 21145.1 Da) and hGPx4

46Glu/Lys/Gln

46Ser

(theoretical

(theoretical mass: 21186.17-21187.07 Da), respectively,

were also detected. The possibility of incorporation of 20 natural amino acids at position 46 was further detected by ESI-MS/MS, while only peptide containing Lys at position 46 was detected


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(Supplementary Figure S4). This result indicates that the mis-incorporation of Glu and Gln may be UTuT6

and decreased.

outcompeted by incorporation of Sec mediated by tRNA

Sec

The last base pair in the acceptor stem and the first two base pairs in the T-stem of tRNA Sec

are antideterminants that hinder the recognition of Sec-tRNA

7

by EF-Tu . The corresponding

region in canonical tRNAs directly interacts with EF-Tu and contributes to the overall binding affinity to maintain the fidelity of protein synthesis UTuT2

29-31

. The expression of hGPx4 in vivo shows that

can be recognized by EF-Tu. According to a study

the entirety of the antideterminants in tRNA

31

on the EF-Tu binding affinities of acceptor stem and T-stem mutations , the base pairs G49-U65 UTuT1

and C50-G64 in the T-stem of tRNA

UTuT2

tRNA

UTuT4

and tRNA

-1

could bind EF-Tu (1.0 kcalmol ) UTu

more tightly than the base pairs C49-G65 and A50-U64 in the T-stem of tRNA UTuT1

the predicted increase in binding energy between EF-Tu and tRNA

. It is possible that

UTuT2

/tRNA

UTuT4

/ tRNA

compensates for weaker binding of Sec in the EF-Tu negatively charged binding pocket, and that UTuT1

this compensation is the reason for increases in the activity of hGPx4 mediated by tRNA UTuT2

tRNA

UTuT4

and tRNA

UTuT3

. The failure to express hGPx4 using tRNA

with or without sodium UTuT3

selenite supplementation possibly occurred because the formation of Ser-tRNA

catalyzed by

Sec

SerRS was blocked. The X-ray crystal structure of human SerRS bound with tRNA Sec

the variable arm, the D-loop and T-loop of tRNA

,

reveals that

32

interact with SerRS . Combined mutations in

the acceptor-stem and T-arm might influence the tertiary structure of the D-loop and T-loop in UTuT3

tRNA

and cause the failure in expression hGPx.



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UTu

Figure 2. Substitution of the acceptor stem, T-stem and T-loop of tRNA

Sec

with that of tRNA UTuTX

affected the activities of hGPx4 produced by each chimeric tRNA. (a) tRNA UTu

tRNA

variants based on

selected for efficient hGPx4 expression in response to the amber stop codon. The Ser

anticodon sequence is shown in gray, and the bases from tRNA

Sec

and tRNA

are shown in black

and blue, respectively. (b) Coomassie-stained SDS-PAGE gel of purified hGPx4 produced by each chimeric tRNA. M, marker. (c) Comparison of the GPx activities of hGPx4 produced by each tRNA variant. Student’s t-test was used for statistical analysis. The data are represented as the


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mean ± SD (n = 3); *p < 0.05; **p < 0.005. (d) Deconvoluted ESI-Q-TOF mass spectrum of hGPx4 UTuT6

produced by tRNA

. The purified hGPx4 was analyzed with Q-TOF-MS and the intact masses

were deconvoluted from the peak detected with charge 26+ in the raw spectrum. The product without Met1 contained a mixture of hGPx4 46Sec

Da, peak 2) and hGPx4

46Ser

46Glu/Lys/Gln

(21144.7 Da, peak 1), hGPx4

(21185.2

(21207.9 Da, peak 3). The raw spectrum is provided in

Supplementary Figure S3.

Expression and characterization of hGPx1. To further validate the Sec incorporation efficiency of the tRNA variants, we expressed hGPx1 with Sec at position 49. hGPx1 is a more efficient antioxidant enzyme compared with hGPx4. The recently reported hybrid tRNA UTuX

(designated tRNA

9

) that mediates close to 100% Sec incorporation was used to express

hGPx1 for comparison in our hands. The successful expression and purification of hGPx1 was detected by SDS-PAGE (Figure 3a). The GPx activity of hGPx1 produced by each chimeric tRNA variant was examined (Figure 3b). Consistent with the results of the GPx activity assay for hGPx4, UTuT6

tRNA

mediated the expression of hGPx1 with the highest activity among the tRNA variants, UTu

notably higher than tRNA Sec

T-stem from tRNA

UTuX

and as high as tRNA

UTuT2

. tRNA

, which has the acceptor stem and

, produced hGPx1 with activity higher than tRNA

UTu

UTuX

but lower than tRNA

Compared with the 2-fold increase in GPx activity of hGPx4, the GPx activity of hGPx1 produced UTuT6

by tRNA

UTu

was three times higher than that produced by tRNA

. Natural hGPx1 is known to

33

exist as a tetramer , and the additional increase in GPx activity of hGPx1 indicated that the hGPx1 expressed may react with positive cooperativity. Semi-native SDS-PAGE gel analysis of


.


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Page 12 of 32

hGPx1 showed that the non-denatured band migrated as a single band of ~97 kDa (Figure 3c), indicating that the hGPx1 expressed exists as a tetramer similar to natural hGPx1. The kinetic UTuT6

analysis of hGPx1 produced by tRNA

showed that the slope measured was clearly steeper at

concentrations of tBuOOH below 25 µM when the concentration of GSH was fixed at 2 mM (Figure 3d). This finding suggests that the deviation from the straight line at low tBuOOH concentrations 34

results from positive cooperativity between reaction centers within the oligomeric enzyme . Data points above 50 µM tBuOOH were used to construct a series double-reciprocal plots (Figure 3e) while the concentration of GSH was kept at 0.5 mM, 1 mM, 2.5 mM and 5 mM respectively. Double-reciprocal plots of the initial velocity versus reciprocal concentration of the tBuOOH resulted in a plot with parallel lines (Figure 3e). The results indicate that the hGPx1 expressed 35

follows a “Ping-Pong” mechanism, similar to natural GPx1 . The apparent kinetic parameters ′

6

were calculated from Equation 1. The rate constants k+1 and k+2 of the hGPx1 were (3.2±0.4)×10 −1 −1

5

−1 −1

M s and (2.4±0.3)×10 M s , respectively.

[E]0 V0

=

1 1 + ' k+1 [ tBuOOH] k+2 [GSH]

36

Equation 1


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Figure 3. Expression and purification of active hGPx1 produced by each tRNA variant and UTuT6

characterization of hGPx1 produced by tRNA

. (a) Coomassie-stained SDS-PAGE gel of

purified hGPx1 produced by each chimeric tRNA. M, marker. (b) Comparison of the GPx activities of hGPx1 produced by each tRNA variant. Student’s t-test was used for statistical analysis. The data are represented as the mean ± SD (n = 3); *p < 0.05; **p < 0.005. (c) Semi-native UTuT6

SDS-PAGE gel analysis of hGPx1 produced by tRNA

. Lane 1, denatured hGPx1; Lane 2, UTuT6

nondenatured hGPx1. (d) Steady-state kinetics of hGPx1 produced by tRNA

at 37 °C and pH

7.4 with varied concentrations of tBuOOH and 2 mM GSH as co-substrate. Data are presented as UTuT6

the mean ± SD (n=3); **p < 0.005. (e) Steady-state kinetics of hGPx1 produced by tRNA

at

37 °C and pH 7.4 with varied concentrations of tBuOOH when concentrations of GSH were fixed at 0.5 mM (square), 1.0 mM (circle), 2.5 mM (triangle) and 5.0 mM (down triangle).



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Page 14 of 32

Evaluated the Sec incorporation ratio by SDS-PAGE analysis of purified hGPx2. Another UTuT6

selenoprotein hGPx2 with Sec at position 40 was expressed using tRNA

with or without

sodium selenite supplementation. The resulting products Sec-hGPx2 and Ser-GPx2 were analyzed by SDS-PAGE. As shown in Figure 4, the quaternary structure analysis of Sec-GPx2 showed that it migrated as a single band at the expected molecular weight of 25 kDa for monomer, whether with or without boiling treatment before loading. In contrast, Ser-GPx2 migrated as a single band at 25 kDa with boiling treatment before loading, while it migrated as a single band at about 100 kDa without boiling treatment before loading. The natural hGPx2 exists in a tetramer like natural hGPx1. However, the result showed that the recombinant Sec-hGPx2 we expressed exists as a monomer and the non-Sec-containing Ser-hGPx2 exists as a tetramer. The reason for the different results of tetramer formation for Sec-hGPx2 and Ser-hGPx2 is complicated, and we could not give a reasonable explanation. Almost no visible band for Ser-hGPx2 was observed in Sec-hGPx2 and only slightly visible band for hGPx2 Sec

of hGPx2

40Glu/Lys/Gln

was observed in Ser-hGPx2. Ratio

in Sec-hGPx2 to Sec-hGPx2 was measured indirectly using computer-assisted image

analysis software (ImageJ) and calculated to be 89.9%, indicated nearly 90% Sec incorporation UTuT6

using tRNA

in response to the amber codon.


Page 15 of 32

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UTuT6

Figure 4. SDS-PAGE analysis of hGPx2 produced by tRNA

with or without sodium selenite

supplementation. M, Marker. Denatured (Lane 1) and nondenatured (Lane 2) Sec-hGPx2 UTuT6

produced by tRNA

with sodium selenite supplementation. Denatured (Lane 3) and

nondenatured (Lane 4) Ser-hGPx2 produced by tRNA

UTuT6

without sodium selenite

supplementation. ImageJ software was used to analyze the ratio of densitometry of nondenatured 40Glu/Lys/Gln

to nondenatured hGPx2

40Glu/Lys/Gln

in lane 4.

hGPx2

hGPx2

40Ser

in lane 4 and the ratio of Sec-hGPx2 in lane 2 to

UTuT6

Potential mechanisms for the improvement of tRNA

for hGPx1 expression. As the

expression of selenoprotein in vivo using chimeric tRNA depends on multiple factors such as SerRS, SelA, and EF-Tu, it is difficult to get an evolved tRNA that compatible to all these factors. The tertiary structure as well as the base sequence of the chimeric tRNA should be considered. Sec

The crystal structure of tRNA

from different species was well studied and there is a common

feather that the formation of tertiary base pair between base 16 and base 59 (Figure 4)

25, 37-39

. The

specific tertiary interaction of base 16 in the D-loop with base 59 in the T-loop contributes to the Sec

stability of the L-shape of tRNA

25

and maintains the overall geometry of the D-stem . The D-arm



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Sec

participates in the recognition of tRNA

37

structure of the Aquifex aeolicus SelA Sec

tRNA

Page 16 of 32

32, 37, 40

by SerRS, SelA and PSTK

. From the X-ray crystal

in complex with Thermoanaerobacter tengcongensis Sec

, the N-terminal domain of SelA binds the D-arm and the T-loop of tRNA Sec

base 59 participates in building the tertiary core of tRNA

. In addition, the

, which is important for tRNA stability in

28

the translation elongation cycle . UTuT6

Considering the tRNA

UTu

differs from tRNA

in only one base and the base 59 does not

participate in the direct interaction with SerRS, SelA, and EF-Tu, the increment in GPx activity of UTuT6

hGPx1 produced by tRNA UTuT6

tRNA

might due to the tertiary structure optimization. Base 59C in

might participate in the overall geometric formation of the T-helix and D-helix and

contribute to the binding affinity of Sec-tRNA

UTuT6

to SerRS, SelA, and EF-Tu.


Page 17 of 32

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Figure 5. Overall structure of the tRNA backbone (purple). The D-loop and T-loop are shown in green and blue, respectively. The base 16 and base 59 are shown as sticks. The red dots represent the hydrogen bond between base 16 and base 59. Crystal structure of (a) E. coli Sec

tRNA

37

(PDB entry 5LZC) , (b) A. aeolicus tRNA

(PDB entry 3A3A)

38

Sec

Sec

and (d) T. tengcongensis tRNA

25

Sec

(PDB entry 3W3S) , (c) Human tRNA (PDB entry 3W1K)

prepared using PyMOL (DeLano Scientific LLC).


39

were visualized and


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Page 18 of 32

UTu

In conclusion, we have utilized amber-less E.coli and tRNA

to express hGPx4 with high UTu

yields. By tuning the acceptor stem and T-stem and the T-loop of tRNA

, chimeric tRNA variants

were found that mediate enhanced expression of hGPx4 and hGPx1 with high activities, especially UTuT6

tRNA

UTuT6

. SDS-PAGE analysis of recombinant hGPx2 produced by tRNA

indicated nearly 90%

Sec incorporation. We also found that the antideterminants that hinder the recognition of Sec-tRNA

Sec

by EF-Tu can be recognized in the chimeric tRNAs by EF-Tu, which in some cases

dependent on the combined mutations at other sites such as base 59 in the T-loop. The base 59 in the T-loop might tune the affinity of the chimeric tRNAs to SerRS, SelA and EF-Tu by affecting the tertiary core of the chimeric tRNAs. The construction of the chimeric tRNAs in this study provide an example for evolution of tRNA for nsAAs incorporation. Engineering the T-loop as well as the T-stem might be useful to harmonize the contributions of the tRNA and the unnatural amino acids to the affinity of binding to EF-Tu and thus improve the yield and fidelity of nsAAs incorporation in E.coli. METHODS

Strains and plasmids. E. coli strain DH5α was used for plasmid propagation. Amber-less E. 22

coli strain C321.∆A.exp Plasmid pN565

41

was obtained from Addgene (49018) and used for protein expression.

was a gift from Christopher Voigt (Addgene plasmid # 49990) and was

transformed into C321.∆A.exp for T7 RNA polymerase expression under Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Plasmids pACYC-[E. coli selA+M. jannaschii pstk] UTu am

(pACYC-selA/pstk) and pGFIB-tRNA

8

were generous gifts from Dieter Söll .


Page 19 of 32

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Construction of the hGPx4 and hGPx1 expression plasmids. The gene encoding hGPx4 was amplified from a plasmid we used in one of our previous studies

42

with primers hGPx4-F and

hGPx4-R. After digestion with EcoRI and HindIII, the PCR product was ligated into pRSFDuet-1, yielding pRSF-hGPx4. Then, pRSF-hGPx4 was used as a PCR template for the mutation of the Sec codon TGA at position 46 of hGPx4 to TAG by site directed mutagenesis with primers 46TAG-F and 46TAG-R, finally resulting in pRSF-hGPx4TAG. DNA sequencing (Sangon Biotech, Shanghai, China) verified the mutation. Primer sequences are listed in Supplementary Table S1. The gene encoding hGPx1 was amplified from a plasmid we used in one of our previous studies

43

with primers hGPx1-F and hGPx1-R. Construction of pRSF-hGPx1TAG was carried out as

outlined for hGPx4, except that hGPx1 was ligated into the BamHI and HindIII sites of pRSFDuet-1, and the Sec codon TGA mutated to TAG was located at position 49 using primers 49TAG-F and 49TAG-R. UTuTx

Construction of variant chimeric tRNA

and expression plasmids. The genes

UTuTx

sequences encoding for variant chimeric tRNA

UTuX

and tRNA

were synthesized and

subcloned into the EcoRI and BamHI sites of pUC57 by Sangon Biotech, yielding UTuTx

pUC57-tRNA

UTuX

and pUC57-tRNA

UTuTx

encoding variant chimeric tRNA UTuTx

from pUC57-tRNA UTuX

pGFIB-tRNA

UTuTx

, respectively. The expression plasmid pGFIB-tRNA

was constructed by subcloning the EcoRI/BamHI fragment UTu

into the EcoRI and BamHI sites of pGFIB-tRNA

. Construction of

was performed using the same method. The constructs were verified by DNA UTuTx

sequencing. The sequence for each tRNA

is listed in Supplementary Table S2.



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Protein expression and purification. GPx variants were overexpressed from the plasmids pRSF-hGPx1TAG and pRSF-hGPx4TAG in E. coli strain C321.∆A.exp that had been co-transformed with the plasmids pN565 and pGFIB-tRNAs, and with or without pACYC-selA/pstk. Starting from a single colony after transformation, cells were grown at 37 °C in 1 ml of LB selective medium supplemented with ampicillin (100 µg/ml), kanamycin (25 µg/ml), chloramphenicol (34.4 µg/ml), and spectinomycin (90 µg/ml) on a shaker. Then, 200 µL of overnight culture was added to 200 ml of LB medium supplemented with ampicillin (100 µg/ml), kanamycin (25 µg/ml), chloramphenicol (34.4 µg/ml), spectinomycin (90 µg/ml) and with or without sodium selenite (50 µM), followed by incubation at 37 °C for several hours. When the optical density at 600 nm of the culture reached 1.2-1.5, the temperature was decreased to 20 °C, and 4 h later, protein expression was induced with 100 µM IPTG, followed by continued incubation for 18 h at 20 °C. Cells were collected by centrifugation (5000 g for 5 min at 20 °C), and the cell pellet was resuspended in 10 ml of lysis buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7.4, and 30 mM imidazole). After lysis by sonication, the lysate was clarified by centrifugation (20000 g, 10 min), passed through a 0.2 µm filter and purified using an immobilized metal-affinity chromatography purification system with Ni– NTA resin. Protein concentration was determined by the Bradford method using bovine serum albumin as a standard. SDS-PAGE analysis. For purity analyses, 1-2 µg of purified GPx samples were separated on 12% SDS-PAGE gels after boiling in SDS Loading buffer (10 mM Tris-HCl, 4% SDS, 5% β-mercaptoethanol, 15% glycerol and 0.002% bromophenol blue at pH 6.8) for 5 min. The protein bands were visualized by staining with Coomassie Brilliant Blue R-250. To prepare non-denatured


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samples, β-mercaptoethanol was omitted from the SDS Loading buffer, and the samples were loaded without boiling. Densitometric analysis was performed on the SDS-PAGE images using ImageJ (nih.gov) to quantitate the amount of protein levels. GPx activity assays. The GPx activities of recombinant hGPx1 and hGPx4 were determined 44

using a previously described method . Sodium phosphate (50 mM pH 7.4), EDTA (1 mM), GSH (1 mM), NADPH (0.25 mM), glutathione reductase (1 U) and samples were mixed in a cuvette at 37 °C. The reagent mixture was incubated for 3 min at 37 °C, and the reaction was initiated by addition of 500 µM H2O2 (final concentration), in a total volume of 0.5 ml. GPx activity was determined by measuring the decrease in NADPH absorption at 340 nm over time. Activity units (U) were defined as the amount of enzyme necessary to oxidize 1 µM NADPH per min at 37 °C. The specific activity is expressed in U/mg protein. Samples were each run in triplicate, and the values were averaged. The t test was used for statistical analysis of the enzyme levels. Differences observed were considered to be significant at P values < 0.01. Steady-state kinetics of hGPx1. Steady-state kinetics analysis was carried out using the 35

method described above . For the positive cooperativity study of hGPx1, a range between 5.5-300 µM tert-Butyl hydroperoxide (tBuOOH) was used while the concentration of GSH was kept at 2 mM. The steady-state parameters for hGPx1 were determined using the same assay with varying concentrations of tBuOOH between 50-300 µM while the concentration of GSH was kept at 0.5 mM, 1 mM, 2.5 mM and 5 mM respectively. GPx activities were measured using the same method as described above at 37 °C and pH 7.4. Kinetic data values were calculated from the 36

Dalziel coefficients as described previously .



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Page 22 of 32

Electrospray Ionization mass spectrometry. Mass spectra for the protein samples were acquired on an Agilent 1290 LC-MS (Bruker Daltonics, USA) system equipped with a 6530 Quadrupole spectrometer. The purified protein samples were dialyzed against water before analysis. The solvent system used for liquid chromatography (LC) was 0.2% formic acid in H2O as buffer A and 0.2% formic acid in acetonitrile (MeCN) as buffer B. The capillary voltage of the mass spectrometer was set to 4500 V. The drying gas flow rate and the temperature were 6.0 L/min and 200 °C, respectively. High-resolution mass spectra of the protein products were acquired on an Agilent Q-TOF 6530 mass analyzer (50–3000 m/z range, positive ionization mode). Data were processed via Bruker Data Analysis software version 4.0. Theoretical mass values were obtained using an online tool (http://web.expasy.org/peptide_mass/) with manual adjustments for the removal of N-terminal methionines and the unexpected incorporation of natural amino acids at the UAG codon. Electrospray ionization tandem mass spectrometry. Electrospray ionization tandem mass 45

spectrometry was carried out using a previously described method . Polyacrylamide gel slices (1– 2 mm) containing the purified proteins were prepared for mass spectrometric analysis by manual in situ enzymatic digestion. Briefly, the excised protein gel pieces were washed twice using deionized water and destained with 50% v/v acetonitrile and 50 mM ammonium bicarbonate, reduced with 10 mM DTT at 56 °C for 1 h, and alkylated with 55 mM iodoacetamide (IAM) at 25 °C for 45 min in dark. After alkylation, the gel pieces were washed twice using 25 mM ammonium bicarbonate for 10 min. Proteins were digested with 70 ng/µl trypsin (Promega, UK) overnight at 37 °C. The resulting peptides were extracted in 2% v/v formic acid, 2% v/v acetonitrile. 10 µl of the


Page 23 of 32

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digest was analyzed by nanoscale capillary LC-MS/MS using a prominence nano 2D (shimazhu, Japan) to deliver a flow of approximately 400 nl/min. The peptides were separated on a C18 5 µm, 150A (Eprogen, USA) and eluted with a gradient of acetonitrile. The analytical column outlet was detected by mass spectrometer (MicrOTOF-QII, BrukerDaltonics, USA). MS spectra were collected over an m/z range of 50–2,200. LC-MS/MS data were then searched against protein sequence database containing the protein constructs specific to the experiment, using the Mascot search engine program (Matrix Science, UK). Database search parameters were set with a fragment ion mass tolerance of 0.1 Da. One missed enzyme cleavage was allowed and variable modifications for oxidized methionine, carbamidomethyl cysteine, carbamidomethyl selenocysteine, and pyroglutamic acid were included.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Author Contributions # Zhenlin Fan and Jian Song contributed equally to this article. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We are grateful to Markus Bröcker and Dieter Söll (Yale University) for gifts of materials. We thank Christopher Voigt for technical providing plasmid pN565; and Marc Lajoie and George Church for



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providing strain C321.∆A.exp. This work was supported by the National Natural ScienceFunds, China (No. 31270851).

ASSOCIATED CONTENT Supporting Information Supplementary figures and tables (PDF)

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For Table of Contents Use Only



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81x44mm (300 x 300 DPI)


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