Generation of Mature Nα-Terminal Acetylated Thymosin α1 by Cleavage of Recombinant Prothymosin α

Nα-terminal acetylation of peptides plays an important biological role but is rarely observed in prokaryotes. Nα-terminal acetylated thymosin α1 (Tα1), a 28-amino-acid peptide, is an immune modifier that has been used in the clinic to treat hepatitis B and C virus (HBV/HCV) infections. We previously documented Nα-terminal acetylation of recombinant prothymosin α (ProTα) in E. coli. Here we present a method for production of Nα-acetylated Tα1 from recombinant ProTα. The recombinant ProTα was cleaved by human legumain expressed in Pichia pastoris to release Tα1 in vitro. The Nα-acetylated Tα1 peptide was subsequently purified by reverse phase and cation exchange chromatography. Mass spectrometry indicated that the molecular mass of recombinant Nα-acetylated Tα1 was 3108.79 in, which is identical to the mass of Nα-acetylated Tα1 produced by total chemical synthesis. This mass corresponded to the nonacetylated Tα1 mass with a 42 Da increment. The retention time of recombinant Nα-acetylated Tα1 and chemosynthetic Nα-acetylated Tα1 were both 15.4 min in RP-high performance liquid chromatography (HPLC). These data support the use of an E. coli expression system for the production of recombinant human Nα-acetylated Tα1 and also will provide the basis for the preparation of recombinant acetylated peptides in E. coli.


Introduction
Protein acetylation is a common posttranslational modification in eukaryotes but is rarely found in prokaryotes [1][2][3][4].Acetylation has been suggested to play an important biological role in modulating enzymatic activity, protein stability, DNA binding, and peptide-receptor recognition [1].Charbaut proposed that neutralization of the aminoterminal (N-terminal) positive charge by N  -acetylation may influence specific interactions with protein partners [3].For example, nonacetylated rat glycine M-methyltransferase does not exhibit the cooperative behavior of its native counterpart [5].Acetylation can also affect protein stability.The halflife of nonacetylated-MSH (-melanocyte-stimulating hormone) in rabbit plasma appears to be one-third of that of the acetylated form [6].
Although acetylation is not as common in prokaryotes as in eukaryotes, researchers have identified acetylated forms of recombinant peptides and proteins to be expressed in E. coli, examples include interferon  and  [7,8], somatotropin [9], interleukin-10 [10], and chemokine RANTES S24F [11].However, some of these proteins or peptides, such as interferon  and , are not known to be acetylated in their native forms.Thus, the acetylated forms of these proteins are of little clinical utility.

The Scientific World Journal
To date, N  -terminal acetylated T1 has been produced by total chemical synthesis for clinical use.However, with 28 residues, it is expensive to produce the peptide by chemical synthesis.As an alternative to synthesis, prokaryotic expression systems are widely used for producing large quantities of recombinant proteins or peptides.When prothymosin  (ProT) and its N  -terminal peptide, T1, were identified 20 years ago, no acetylation was found when they were expressed in E. coli [19,20].Other groups have expressed recombinant T1 as a fusion protein or in concatemer form in E. coli [21][22][23][24].
We have previously reported that a fraction of recombinant human ProT is N  -terminal acetylated when expressed in E. coli [4].In this study, we expand on this observation and describe a new method for production and purification of N  -terminal acetylated T1 from E. coli.This method may provide a useful alternative to chemical synthesis for producing acetylated peptides for clinical applications.

Plasmids and Strains.
The human ProT expression plasmid pBV220-proT was constructed previously [4].The yeast Pichia pastoris expression plasmid pPIC9, which contains the promoter of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) gene, the -mating factor prepro secretion signal from Saccharomyces cerevisiae, and the HIS4 auxotrophic selection marker, was purchased from Invitrogen (San Diego, CA, USA).The P. pastoris GS115 (his4) host strain was from Invitrogen.HepG2 liver carcinoma cell line was purchased from the Institute of Biochemistry and Cell biology (Shanghai, CAS, China).

Production and Purification of Recombinant Human
Prothymosin  in E. coli .Recombinant human N  -acetylated ProT was produced as described previously [4] with some modification.The ProT gene was cloned in plasmid pBV220, with expression under control of the PLPR promoter.Expression of ProT was induced by increasing the culture temperature from 30 ∘ C to 42 ∘ C for 6 h.After induction, cells were harvested by centrifugation.
The cells were suspended in buffer A (10 mM Tris-HCl, pH 8.0) and incubated at 85 ∘ C for 15 min.Acetic acid was added to adjust the pH to 4.0 and to precipitate E. coli proteins.Precipitated proteins were spun down, and the supernatant containing ProT was applied on a DEAE Sepharose Fast Flow column (Ø1.6 × 20 cm, GE Healthcare, Piscataway, NJ) equilibrated with buffer A. After washing, the bound protein was eluted with a gradient from 0 to 0.4 M NaCl.Since ProT does not contain any aromatic residues, elution of ProT was monitored by UV absorption at 214 nm.The level of protein and nucleic acid contamination was estimated by UV absorption at 280 nm [25].The purity of the ProT in eluate fractions was analyzed by 18% SDS-PAGE.

Cloning, Expression and Purification of Recombinant
Human Prolegumain in Pichia pastoris.Total mRNA was isolated from HepG2 cell line and converted to single-stranded cDNA using MMLV Reverse Transcriptase as described by the manufacturer (TIANGEN BIOTECH Co., Beijing, China).The human legumain gene without signal peptide was amplified by PCR using forward primer legu4 (5  -AGACTCGAGAAAAGAGTTCCTATA-GATGATCCTGAA G-3  ) and reverse primer legu5 (5  -TATGCGGCCGCTTAGTAGTGACCAAGGCACACGTG-3  ); primers were designed based on the ORF of legumain reported previously [26].Amplification reactions were performed with Pyrobest polymerase (TaKaRa BIOTECH Co., Dalian, China) at 95 ∘ C for 2 min, followed by 30 cycles of 94 ∘ C for 30 s, 55 ∘ C for 40 s, 72 ∘ C for 2 min, and a final extension step at 72 ∘ C for 10 min.PCR products were cloned into the XhoI and NotI sites (both underlined in primers) of Pichia pastoris expression vector pPIC9 to generate the legumain expression vector pPIC9-legumain.The sequence of legumain was verified by sequencing.
Culture supernatant containing prolegumain was diluted with 5 volumes of buffer C (20 mM sodium acetate, pH 5.5) and applied to SP Fast flow column (Ø1.0 × 16 mm, GE Healthcare, Piscataway, NJ).After washing with buffer C, bound protein was eluted with a step gradient of 25%, 45%, and 100% buffer D (20 mM sodium acetate, 1 M NaCl, and pH 5.5).Eluate fractions were analyzed by 12% SDS-PAGE.Fractions containing recombinant prolegumain were dispensed into aliquots and stored at −70 ∘ C until use.

Autoactivation of Recombinant Human Legumain.
To determine the optimum time for autocatalytic activation, 500 L aliquots of the recombinant prolegumain (0.2 mg/mL) were incubated in 1 mM dithiothreitol (DTT), 1 mM EDTA, and 0.1 M sodium citrate pH 4.0.Samples were incubated for 0 h, 0.25 h, 1 h, and 4 h at 37 ∘ C. Digest products were analyzed by SDS-PAGE (12%) and stained with Coomassie Blue.

Cleavage of ProT𝛼 by Recombinant Human Legumain
In Vitro.To optimize digestion of ProT by recombinant autoactivated legumain, 250 L aliquots of recombinant ProT (2 mg/mL) were mixed with 50 L of legumain in buffer containing 1 mM DTT, 1 mM EDTA, and 0.1 M sodium citrate (pH 4.0).Samples were incubated for 0, 2, 4, 6, 8, and 16 h at 37 ∘ C. Digest products were assayed by SDS-PAGE (18%) and stained with Coomassie Blue.
T1-containing fractions were loaded onto an SP High performance column (Ø1.6 × 20 mm, ABI, Foster City, CA, USA) and washed with equilibration buffer M (10 mM NaCl).Bound protein was eluted with 0.2 M NaCl, 0.3 M NaCl, and 0.5 M NaCl.Eluate fractions were assayed by reverse phasehigh performance liquid chromatography (HPLC).

RP-HPLC Analysis of N
-Terminal Acetylated T1.RP-HPLC was performed using the HP HPLC 1090 system.A C18 column (5 m, Ø4.6×250 mm, Johnson Technologies Co. Dalian, China) was equilibrated at a flow rate of 1.0 mL/min in solvent A (0.1% trifluoroacetic acid (TFA) in water).Solvent B was 0.1% TFA in acetonitrile.Samples (25 L) were injected on the column, and peptides were eluted with a linear solvent gradient (100% A : 0% B to 85% A : 15% B for 5 min; 85% A : 15% B to 75% A : 25% B for 15 min; 75% A : 25% B to 0% A : 100% B for 5 min).Peptide elution was detected by UVabsorption at 214 nm.The column was reequilibrated with solvent A for 20 min between runs.

Purification of Recombinant Prothymosin 𝛼 Expressed in E.
coli.Recombinant human prothymosin  was overexpressed in E. coli [4] and purified by thermal denaturation and anion exchange chromatography.Purified protein appeared as a single homogeneous band with a molecular mass of 12 kDa in 18% SDS-PAGE (Figure 1).

Preparation of Recombinant Legumain in Pichia pastoris and Autoactivation.
We overexpressed human legumain proenzyme, which is reported to cleave ProT at asparaginylglycine sites and release peptide products T1 and T11, in Pichia pastoris, and purified it by cation exchange chromatography.The molecular mass of recombinant prolegumain was 56 kDa.When incubated at an acidic pH, the recombinant proenzyme was autocleavaged to a 46 kDa form in a time dependent manner (Figure 2(a), lane 2-4).

Prothymosin 𝛼 is Processed to Thymosin 𝛼1 by Recombinant Legumain
In Vitro.To process ProT by recombinant legumain in vitro, we systematically studied the proteolysis of ProT as a function of pH, temperature, time, and amount of legumain.After incubation with the autoactivated legumain, the band corresponding to ProT decreased in intensity and a band with similar molecular mass of T1 increased in intensity in a time-dependent manner.When 500 g ProT was incubated with 10 g legumain, the majority of ProT was cleaved in 2 h, and the cleavage was complete in 6-8 h (Figure 2(b), lane 4-5).Decreasing the amount of legumain also resulted in complete cleavage but required longer incubation times (data not shown).
Legumains have previously been extracted from mammalian and plant cells and tissues [13,27,28].However, these preparations typically have low concentrations and are easily contaminated by other enzymes.This study established a method with which to overexpress the recombinant enzyme in yeast and confirmed its activity in the enzymatic processing of ProT.The yeast expression system is easy and economical to grow on a large scale, and it has been used as an efficient system for production of heterologous proteins [29,30].The recombinant prolegumain (56 kDa) incubated at an acidic pH was autocleavaged to a 46 kDa form.This result is consistent with reports that the 56 kDa proenzyme cleaves off the Cterminal 110 residues to form the 46 kDa active enzyme [13].

Purification of the N 𝛼 -Terminal Acetylated Thymosin 𝛼1.
To purify the N  -acetylated T1 from the ProT cleavage products, the proteolytic products were separated by RP chromatography in PoRos R50 media.Peptide fractions eluted The Scientific World Journal with 10%, 20%, and 30% ethanol were assayed by RP-HPLC, and N  -acetylated T1 (Figure 3(a), peak II) and nonacetylated T1 (Figure 3(a), peak I) were both identified in 20% ethanol fractions.The acetylated and nonacetylated forms were further separated by cation exchange chromatography.The N  -acetylated T1 eluted as a single peak (as monitored by UV absorption at 214 nm) in the 0.2 M NaCl elution fraction (Figure 3(b), peak II).Other peptides were found in 0.3 M NaCl and 0.5 M NaCl eluted fractions, including the nonacetylated T1 that eluted with 0.5 M NaCl (Figure 3(d), peak I).Thus, the N  -acetylated and nonacetylated forms of T1 were completely separated by cation exchange chromatography.
Although ProT and T1 have been overexpressed in E. coli previously [19,22], it was often not determined if the N  -termianl residue was acetylated.In some cases, this may be due to the difficulty associated with separating N  -acetylated forms from nonacetylated forms.In this study, we were able to completely separate N  -acetylated T1 and nonacetylated T1 by HPLC (Figures 3 and 4).The N  -acetylated T1 was confirmed by MS and MS/MS sequencing.

Identification of the N 𝛼 -Terminal Acetylated Thymosin
1.Purified, recombinant N  -acetylated T1, and chemically synthesized N  -acetylated T1 were analyzed by RP-HPLC.The elution time of recombinant N  -acetylated T1 was 15.386 min (Figure 4(a)), which was the same as the N acetylated T1 produced by chemical synthesis (15.392 min, Figure 4(b)).When the recombinant and chemosynthetic forms of N  -acetylated T1 were mixed and subjected to RP-HPLC, a single peak eluted with a retention time of 15.386 min (Figure 4(c)).The molecular mass of the recombinant N  -acetylated T1 as determined by Q-TOF mass spectrometry was 3108.79Da (Figure 5); a mass which is the same as the chemosynthetic peptide and corresponds to the theoretical molecular mass of nonacetylated T1 (3065 Da) plus an additional 42 Da increment for the acetyl group.
The sequence of the recombinant N  -acetylated T1 was determined by tandem MS.It was found to be "Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN" (Figure S1a), which is the same as the native and chemosynthetic N acetylated T1.N  -terminal acetylation was confirmed by a m/z of 130.05 for the N  -terminal amino acid residue (Figure S1b), which corresponds to an acetylated serine residue.
T1 had been expressed as a fusion protein or in a concatemer form [22,31,32]. Zhou et al. reported that 6×T1 concatemer was successfully expressed in E. coli and cleaved by hydroxylamine to release the T1 monomer.However, this version of T1 was not acetylated on the N  -terminal serine residue [22].In contrast, this study established an efficient method for preparation of the N  -acetylated T1 following expression of ProT in E. coli.A number of factors make this method easy to scale up for commercial production.Firstly, E. coli can be grown to high-density and is capable of producing large quantities of heterologous protein.ZADAXIN, N terminal acetylated T1 produced by total chemical synthesis energizes the immune response, helping patients to battle invasive cancers and secondary infections.It has been tested and proven safe, effective, and easy to tolerate in numerous patient populations, including the elderly, previous treatment failures, and patients with depressed immune systems.In this study, we provide an alternative way to produce N terminal acetylated thymosin 1 by cleaving the recombinant protein with legumain, which should be cheaper for largescale production; as E. coli is the most efficient and economic host for recombinant polypeptide production.
In addition, recombinant human legumain is easily expressed as the proenzyme in yeast P. pastoris after which it can be autocleaved to yield the active enzyme.Only 2 g of recombinant legumain is needed to process 400 g of ProT (data not shown).Finally, the extraction and purification of ProT were based on thermal denaturation [19] and the use of chromatographic media that are chemically stable (can be regenerated repeatedly), commercially available, and relatively inexpensive.
The isolation of N  -acetylated T1 from recombinant ProT also confirmed our previous findings that ProT   is N  -terminally acetylated in E. coli [4].Acetylation is a common modification in eukaryotic cells, and it plays an important role in protein function and stability.Acetylation, like phosphorylation, can regulate essential cellular processes, such as transcription, nuclear import, microtubule function, and hormonal response [33].Before this report, a few reports have demonstrated acetylation of proteins expressed in E. coli.However, with the exception of N acetylated T1 described here, many of these acetylated proteins are of little clinical utility.N  -acetylation of proteins is catalyzed by N  -acetyltransferases (NATs).Three E. coli NATs, namely, RimL, RimJ, and RimI, have already been identified through mutant analysis and are responsible for the acetylation of ribosomal proteins L12, S5, and S18, respectively [34,35].Fang et al. recently revealed that N  -acetylation of recombinant fusion proteins of T1 and the ribosomal protein L12 is catalyzed by RimJ in E. coli [31].Johnson et al. reported a applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins by coexpressing the fission yeast NatB complex with the target protein in E. coli [36].These results can help reveal the function of acetylation in prokaryotes and find additional mechanisms for producing acetylated proteins in E. coli.

Conclusion
This study established a method for production of recombinant N  -acetylated T1 in E. coli, which is a clinical useful peptide, and was produced by chemosynthesis previously.The product consistent with the chemosynthesis of N acetylated T1, which was confirmed as N  -acetylated T1 by the elution time in HPLC assay (Figure 3(b)), molecular mass in MS assay (Figure 3(c)), and in MS/MS sequencing (Figure 4).The RP-HPLC showed a single peak in 15.4 min also demonstrated the produced recombinant T1 was a uniform component, with nonacetylated T1 free.

Figure 1 :
Figure 1: SDS-PAGE of recombinant human prothymosin  produced by E. coli.The purified recombinant human prothymosin  was separated with 18% SDS-PAGE and stained with Coomassie blue G-250.Molecular weight markers were loaded in the same gel (lane M).

Figure 2 :
Figure 2: (a) Expression and purification of legumain in P. pastoris and its autoactivation.SDS-PAGE of autocatalytic legumain after the prolegumain was incubated in buffer as a function of time (0-4 h) at 37 ∘ C. Aliquots of each reaction were analyzed by SDS-PAGE (12%) and stained with Coomassie blue.Each time point is marked on the bottom of its corresponding lane.(b) SDS-PAGE analysis of Prothymosin  proteolysis by recombinant legumain in vitro.Aliquots of ProT were incubated with recombinant legumain at 37 ∘ C in buffer containing 1 mM DTT, 1 mM EDTA, and 0.1 M sodium citrate (pH 4.0).Each reaction mixture was analyzed by SDS-PAGE under the conditions indicated under "Experimental Section." Each time point is marked on the bottom of its corresponding lane 1-6.Lane 7 is the chemically synthesized N  -acetylated T1.