# Expression and Hydroxylamine Cleavage of Thymosin  Alpha 1 Concatemer

Authors: Liang Zhou; Zong-Teng Lai; Min-Kan Lu; Xing-Guo Gong; Yi Xie
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

Human thymosin alpha 1 (Tα1) is an important peptide in the development and senescence of immunological competence in human, and many studies have reported the expression of this peptide. In this study, we designed and synthesized the Tα1 gene according to the E. coli codon usage preference and constructed a 6×Tα1 concatemer. The latter was inserted into an E. coli expression vector pET-22b (+), and transformed into E. coli BL21 (DE3). After induction with IPTG, the concatemer protein was successfully expressed in E. coli then cleaved by hydroxylamine to release the Tα1 monomer. Gly-SDS-PAGE and mass spectrometry confirmed that the recombinant protein was cleaved as intended. The bioactivity of the Tα1 monomer was analyzed by lymphocyte proliferation and by mitochondrial activity in two different tumor cell lines. This study provides a description of the preparation of a bioactive Tα1, which may prove useful in future biomedical research.


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## Body

## 1. Introduction

Thymosin alpha 1 (Tα1), a 28-amino-acid peptide with molecular weight (MW) of 3108 Da,
was first isolated from calf thymus in 1977 by Goldstein et al. [1, 2]. Tα1 is
mainly produced in vivo by thymic epithelial cells and is stable
at 80–90°C [3]. Tα1 has no aromatic amino acids (see
Figure 1), and N-acetylation is important for protecting towards proteolysis. Tα1
plays a role against viral infection, against tumors, and in
immunomodulation. Upon stimulation of Tα1, T cells
become mature in vitro and express IL-2, IL-2R, and CD2 [4, 5]. Tα1 inhibits viral replication at
least in hepatitis virus B-transfected HepG2 tumor cells [6, 7]. In addition, Tα1
antagonizes T cell apoptosis induced by dexamethasone, CD3 monoclonal antibody treatment
[8], or addition of sera from
tumor-bearing mice [9]. It also downregulates tumor weight
in mice [10], enhances major
histocompatibility complex (MHC) class I antigen expression in tumor
cells and antigen-presenting cells [11–13], and partly restores cellular
immunity [14]. Tα1 is used in combination with
IFN-α for patients with chronic hepatitis virus B or C infection [15, 16]. Tα1
is used to reduce adverse effects from chemotherapy or radiotherapy [17]. Recently, Tα1 has even been
used as an adjuvant for vaccines against influenza and hepatitis B [18].Figure 1
The complete amino acid sequence of Tα1.In the past two decades, although the successful expression of the recombinant Tα1 (r Tα1) and Prothymosin (thymosin comprises the N-terminus of Prothymosin) in Escherichia coli has
been reported [19, 20]. However, Tα1 was
expressed with a fusion protein or in a concatemer form [21, 22]. The expression of the
Tα1 monomer peptide has not been reported, because it is difficult to extract the micromolecular
peptide from the fermentation broth. The fusion protein must be removed because
fusion protein could influence the bioactivity of the peptide. The concatemer
protein has not been processed further. All of these issues made it difficult
to prepare active recombinant Tα1. In this study, we have expressed Tα1 concatemer
protein and cleaved the concatemer protein with hydroxylamine to obtain active Tα1
monomer peptide.


## 2. Materials and Methods

### 2.1. Materials

The pET-22b (+) vector,E. coli BL21
(DE3) (hsdS gal [λcIts857 ind1 Sam7 nin5 lacUV5-T7]), the HepG2 liver carcinoma cell line, and the SPC-A-1
lung carcinoma cell line were kindly provided by Institute of Biochemistry and
Cell biology (Shanghai, CAS, China). E.
coli BL21 cells were maintained in Luria-Bertani (LB). HepG2 and SPC-A-1
cells were cultured in RMPI 1640 medium with 10% fetal bovine serum in
humidified air at 37°C with 5%
CO2. Lymphocytes were isolated from adult mouse spleens (BAL/C) and
cultured in RMPI 1640 medium with 10% fetal bovine serum in humidified air at 37°C with 5% CO2. Synthesized Tα1
(Sinopep Pharmaceutical, Inc., Hangzhou, China.) was supplied as purified, endotoxin-free. The synthesized Tα1 and the
cleaved Tα1 were dissolved in phosphate-buffered solution
(PBS pH 7.2) at 1 mg/mL (approximately 3×10−8 mol/L)
and then sterilized with a 0.22 μm filter.


### 2.2. Synthesis of the Tα1 Concatemer

Two single strand DNA oligomers encoding the entire
thymosin alpha peptide (synthesized by Songon, Shanghai, China.), partially complementary to
each other (as indicated by the underlined sequences) were synthesized. We used
codons that were preferred inE. coli.THY-F: GTCGACATGGGATCCAACGGTTCTGATGCTGCTGTAGATACTTCTTCTGAGATTACTACTAAAGACCTAATHY-R: CTCGAGTCAAGATCTCCCGTTCTCAGCCTCTTCGACAACTTCCTTCTTCTCCTTTAGGTCTTTAGTAGTAThe 2 oligonucleotides were mixed and denatured by incubation at 94°C  for 10 minutes,
and subsequently annealed at 60°C for 10 minutes in presence of TaqPlus (BBI, Toronto, Canada).
The desired double-strand DNA was treated with T4 polynucleotide kinase (BBI, Toronto,
Canada) and cloned into the cloning vector pUCm-T (BBI, Toronto, Canada).The plasmid (pUCm-T-Tα1, pT-Tα1) containing the Tα1 gene was digested with BamH I/Xho I or Bgl II/Xho I, respectively. The digested fragments containing
the coding sequence of Tα1 were ligated with T4 DNA Ligase for 16 hours at 4°C to produce the concatemer Tα1 gene of 2 repeats (pT-Tα1×2). When
the plasmid was digested with isocaudamer BamHI (G∗CTAG C) and Bgl II (A∗GATC
T), the 2 fragments had similar termini. When the 2 fragments were catalyzed
with T4 DNA Ligase the new sequence formed was GGATCT, and this new site could
not be digested by BamH I or Bgl II (see Figure 2).Figure 2
Construction of the concatemer Tα1 gene. A T-vector containing the Tα1 gene was digested with BamH I/Xho I
and Bgl II/Xho I. When digested with Bgl II and BamH
I, the 2 fragments have identical termini. The two fragments containing the Tα1 gene were ligated with T4 DNA Ligase.
A plasmid containing double Tα1 genes
was constructed. When the 2 fragments were catalyzed with T4 DNA Ligase, the
new sequence formed was GGATCT, and this new site could not be digested by BamH
I or Bgl II. Therefore, the new 2× concatemer gene could not be destroyed when
the 4× concatemer gene was constructed. Thus, the plasmid containing 4×, 6×Tα1 genes could also be constructed.Using a pT-Tα1×2 plasmid, plasmids containing concatemer 4
repeats (pT-Tα1×4) of the Tα1 gene were
prepared as described above. Using a pT-Tα1×2 plasmid and a pT-Tα1×4 plasmid,
the preparation of plasmids containing concatemer 6 repeats (pT-Tα1×6) was the
same as pT-Tα1×2 plasmid.Finally, a fragment containing the concatemerTα1 gene of 6 repeats was constructed and
inserted into expression vector pET-22b (+),
between the restriction endonuclease recognition sites Hind III
and Nde I.


### 2.3. Expression and Purification of Tα1  Concatemer Protein

A single bacterial colony containing pET-22b (+)-Tα1 was grown
overnight at 37°C in 5 mL
LB medium with 50 μg/mL Ampicillin (BBI, Toronto, Canada). The overnight culture was
inoculated into 250 mL LB medium in a 1 L flask with 50 μg/ml Ampicillin.
After 2 hours of incubation, isopropyl-1-thio-beta-D-galactoside (IPTG; BBI, Canada)
was added to the culture at a final concentration of 1.0 mM, when the cell density had
reached OD600nm= 0.6. After incubation at 37°C for 8 hours, the bacterial cells were harvested by centrifugation (5000 ×g, 15 minutes) at 4°C. The inducibility of the concatemer gene
was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (data not shown).The cell pellet was resuspended in 100 mL buffer A (50 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0], and 0.1 M NaCl) and then sonicated (800 W, 20 minutes).
After sonication, the sedimented material (containing the concatemer protein) was
washed once first with 100 mL buffer B (50 mM Tris-HCl [pH 8.0], 1 mM EDTA (pH 8.0), and 0.5% Triton-X 100) and then with 100 mL buffer
C (50 mM Tris-HCl [pH 8.0], 1 mM EDTA (pH 8.0), and 2 M urea) by centrifugation. The sedimented
material was then dissolved in 100 mL buffer D (50 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0], and 8 M urea) with stirring for 1 hour at 25°C. The
soluble protein was then dialyzed (Dialysis Membrane MWCO 10 000, SPECTRUM, Tex, USA) with double distilled H2O and cryodesiccated.


### 2.4. Hydroxylamine Cleavage Reaction and Analysis of Cleavage Products

The concatemer protein was then cleaved with hydroxylamine.
The cleavage sites are shown in Figure3. The cleavage buffer [21] contained 0.1 M trizma base and 2.0 M hydroxylamine-HCl. The pH of the
cleavage buffer was adjusted with 5 M NaOH solution to a pH of 9.0, and the reaction was performed at 45°C for 4 hours. The reaction was terminated
by adjusting the pH to 4.0 with concentrated formic acid and cooling to 4°C [21]. Using Sephadex-G25 (GE) column
chromatography, the
products of cleavage were desalted. Column chromatography revealed 2
peaks; one peak corresponded to the Tα1 monomer and the other contained the incompletely
cleaved concatemer protein. The molecular mass of the Tα1 monomer was analyzed
by Gly-SDS-PAGE and mass spectrometry.Figure 3
The sequence and hydroxylamine cleavage sites of the concatemer
protein. The concatemer protein contained a total
of 200 amino acids, and the molecular weight of the concatemer protein was approximately
23 kD. The 6× Tα1 sequence is underlined, and the cleavage sites Asn-Gly are
indicated with a vertical arrow.


### 2.5. Assay for Proliferation of Mice  Splenic Lymphocytes

The proliferation response of splenocytes was determined
by MTT assay [23]. Spleens were removed from mice and dispersed through
nylon mesh to generate a single-cell suspension. Cells were pelleted (1500 ×g, 6 minutes), and the cell pellet was
resuspended in erythrocyte lysis solution (0.15 M NH4Cl, 0.02 M Tris, sterilized with a 0.22 μm filter) for 5 minutes to remove the red cells.
After lysis, the cell suspension was incubated in a culture flask for 2 hours,
and then the suspension was transferred to a new flask. Splenic macrophages
adhered to the flask; this procedure enriched for lymphocytes.Splenic lymphocytes were incubated in RPMI
1640 medium with 5μg/mL concanavalin A (ConA; BBI, Canada) for 12 hours, and
then harvested by centrifugation (1500 ×g, 6 minutes). The cell pellet
was resuspended in RPMI 1640 medium at a concentration of 1×106 cells/mL. A 96-well
culture plate (Costar, Inc.,
Calif, USA) was used for incubation of
180 μL lymphocytes in RPMI 1640 medium. 50 μg/mL of the proteins
(cleaved Tα1 and
synthesized Tα1) were added to the wells. After 24-hour or 48-hour incubation
at 37°C, 20 μL
of MTT (0.5 mg/mL), solution was added to each well, and the microplates were
incubated for 4 hours at 37°C.
After incubation, the plates were centrifuged (1400 ×g, 18°C, 10 minutes). The supernatants were
removed, and 200 μL of dimethyl sulfoxide (DMSO) was added to
each well and incubated for 15 minutes at room temperature. After
incubation, the solubilized reduced MTT was measured colorimetrically at
570 nm using a Benchmark microculture plate reader (Bio-Rad, Calif, USA).


### 2.6. Assay for Mitochondrial Activity of HepG2 and SPC-A-1 Cells

The influence of Tα1 on mitochondrial activity was measured using MTT reduction, a
common experimental method to quantify mitochondrial activity. The cells were
seeded in a 96-well culture plate at 1×106 cells/mL.
After 24-hour, 48-hour, and 72-hour exposures to Tα1 at a concentration of 0.1 mg/mL,
the cells were incubated with MTT (0.5 mg/mL) for 4 hours at 37°C. The formazan precipitate
was then dissolved in 200 μL DMSO, and absorbance at 570 nm was measured using the Benchmark microculture plate reader (Bio-Rad, Calif, USA).


### 2.7. Statistic Analysis

The data are presented as mean ±SEM, for at least 3 replicate
experiments and evaluated using the Student'st-test. P-values below 0.05 were regarded as statistically
significance (two-tailed test).


## 2.1. Materials

The pET-22b (+) vector,E. coli BL21
(DE3) (hsdS gal [λcIts857 ind1 Sam7 nin5 lacUV5-T7]), the HepG2 liver carcinoma cell line, and the SPC-A-1
lung carcinoma cell line were kindly provided by Institute of Biochemistry and
Cell biology (Shanghai, CAS, China). E.
coli BL21 cells were maintained in Luria-Bertani (LB). HepG2 and SPC-A-1
cells were cultured in RMPI 1640 medium with 10% fetal bovine serum in
humidified air at 37°C with 5%
CO2. Lymphocytes were isolated from adult mouse spleens (BAL/C) and
cultured in RMPI 1640 medium with 10% fetal bovine serum in humidified air at 37°C with 5% CO2. Synthesized Tα1
(Sinopep Pharmaceutical, Inc., Hangzhou, China.) was supplied as purified, endotoxin-free. The synthesized Tα1 and the
cleaved Tα1 were dissolved in phosphate-buffered solution
(PBS pH 7.2) at 1 mg/mL (approximately 3×10−8 mol/L)
and then sterilized with a 0.22 μm filter.


## 2.2. Synthesis of the Tα1 Concatemer

Two single strand DNA oligomers encoding the entire
thymosin alpha peptide (synthesized by Songon, Shanghai, China.), partially complementary to
each other (as indicated by the underlined sequences) were synthesized. We used
codons that were preferred inE. coli.THY-F: GTCGACATGGGATCCAACGGTTCTGATGCTGCTGTAGATACTTCTTCTGAGATTACTACTAAAGACCTAATHY-R: CTCGAGTCAAGATCTCCCGTTCTCAGCCTCTTCGACAACTTCCTTCTTCTCCTTTAGGTCTTTAGTAGTAThe 2 oligonucleotides were mixed and denatured by incubation at 94°C  for 10 minutes,
and subsequently annealed at 60°C for 10 minutes in presence of TaqPlus (BBI, Toronto, Canada).
The desired double-strand DNA was treated with T4 polynucleotide kinase (BBI, Toronto,
Canada) and cloned into the cloning vector pUCm-T (BBI, Toronto, Canada).The plasmid (pUCm-T-Tα1, pT-Tα1) containing the Tα1 gene was digested with BamH I/Xho I or Bgl II/Xho I, respectively. The digested fragments containing
the coding sequence of Tα1 were ligated with T4 DNA Ligase for 16 hours at 4°C to produce the concatemer Tα1 gene of 2 repeats (pT-Tα1×2). When
the plasmid was digested with isocaudamer BamHI (G∗CTAG C) and Bgl II (A∗GATC
T), the 2 fragments had similar termini. When the 2 fragments were catalyzed
with T4 DNA Ligase the new sequence formed was GGATCT, and this new site could
not be digested by BamH I or Bgl II (see Figure 2).Figure 2
Construction of the concatemer Tα1 gene. A T-vector containing the Tα1 gene was digested with BamH I/Xho I
and Bgl II/Xho I. When digested with Bgl II and BamH
I, the 2 fragments have identical termini. The two fragments containing the Tα1 gene were ligated with T4 DNA Ligase.
A plasmid containing double Tα1 genes
was constructed. When the 2 fragments were catalyzed with T4 DNA Ligase, the
new sequence formed was GGATCT, and this new site could not be digested by BamH
I or Bgl II. Therefore, the new 2× concatemer gene could not be destroyed when
the 4× concatemer gene was constructed. Thus, the plasmid containing 4×, 6×Tα1 genes could also be constructed.Using a pT-Tα1×2 plasmid, plasmids containing concatemer 4
repeats (pT-Tα1×4) of the Tα1 gene were
prepared as described above. Using a pT-Tα1×2 plasmid and a pT-Tα1×4 plasmid,
the preparation of plasmids containing concatemer 6 repeats (pT-Tα1×6) was the
same as pT-Tα1×2 plasmid.Finally, a fragment containing the concatemerTα1 gene of 6 repeats was constructed and
inserted into expression vector pET-22b (+),
between the restriction endonuclease recognition sites Hind III
and Nde I.


## 2.3. Expression and Purification of Tα1  Concatemer Protein

A single bacterial colony containing pET-22b (+)-Tα1 was grown
overnight at 37°C in 5 mL
LB medium with 50 μg/mL Ampicillin (BBI, Toronto, Canada). The overnight culture was
inoculated into 250 mL LB medium in a 1 L flask with 50 μg/ml Ampicillin.
After 2 hours of incubation, isopropyl-1-thio-beta-D-galactoside (IPTG; BBI, Canada)
was added to the culture at a final concentration of 1.0 mM, when the cell density had
reached OD600nm= 0.6. After incubation at 37°C for 8 hours, the bacterial cells were harvested by centrifugation (5000 ×g, 15 minutes) at 4°C. The inducibility of the concatemer gene
was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (data not shown).The cell pellet was resuspended in 100 mL buffer A (50 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0], and 0.1 M NaCl) and then sonicated (800 W, 20 minutes).
After sonication, the sedimented material (containing the concatemer protein) was
washed once first with 100 mL buffer B (50 mM Tris-HCl [pH 8.0], 1 mM EDTA (pH 8.0), and 0.5% Triton-X 100) and then with 100 mL buffer
C (50 mM Tris-HCl [pH 8.0], 1 mM EDTA (pH 8.0), and 2 M urea) by centrifugation. The sedimented
material was then dissolved in 100 mL buffer D (50 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0], and 8 M urea) with stirring for 1 hour at 25°C. The
soluble protein was then dialyzed (Dialysis Membrane MWCO 10 000, SPECTRUM, Tex, USA) with double distilled H2O and cryodesiccated.


## 2.4. Hydroxylamine Cleavage Reaction and Analysis of Cleavage Products

The concatemer protein was then cleaved with hydroxylamine.
The cleavage sites are shown in Figure3. The cleavage buffer [21] contained 0.1 M trizma base and 2.0 M hydroxylamine-HCl. The pH of the
cleavage buffer was adjusted with 5 M NaOH solution to a pH of 9.0, and the reaction was performed at 45°C for 4 hours. The reaction was terminated
by adjusting the pH to 4.0 with concentrated formic acid and cooling to 4°C [21]. Using Sephadex-G25 (GE) column
chromatography, the
products of cleavage were desalted. Column chromatography revealed 2
peaks; one peak corresponded to the Tα1 monomer and the other contained the incompletely
cleaved concatemer protein. The molecular mass of the Tα1 monomer was analyzed
by Gly-SDS-PAGE and mass spectrometry.Figure 3
The sequence and hydroxylamine cleavage sites of the concatemer
protein. The concatemer protein contained a total
of 200 amino acids, and the molecular weight of the concatemer protein was approximately
23 kD. The 6× Tα1 sequence is underlined, and the cleavage sites Asn-Gly are
indicated with a vertical arrow.


## 2.5. Assay for Proliferation of Mice  Splenic Lymphocytes

The proliferation response of splenocytes was determined
by MTT assay [23]. Spleens were removed from mice and dispersed through
nylon mesh to generate a single-cell suspension. Cells were pelleted (1500 ×g, 6 minutes), and the cell pellet was
resuspended in erythrocyte lysis solution (0.15 M NH4Cl, 0.02 M Tris, sterilized with a 0.22 μm filter) for 5 minutes to remove the red cells.
After lysis, the cell suspension was incubated in a culture flask for 2 hours,
and then the suspension was transferred to a new flask. Splenic macrophages
adhered to the flask; this procedure enriched for lymphocytes.Splenic lymphocytes were incubated in RPMI
1640 medium with 5μg/mL concanavalin A (ConA; BBI, Canada) for 12 hours, and
then harvested by centrifugation (1500 ×g, 6 minutes). The cell pellet
was resuspended in RPMI 1640 medium at a concentration of 1×106 cells/mL. A 96-well
culture plate (Costar, Inc.,
Calif, USA) was used for incubation of
180 μL lymphocytes in RPMI 1640 medium. 50 μg/mL of the proteins
(cleaved Tα1 and
synthesized Tα1) were added to the wells. After 24-hour or 48-hour incubation
at 37°C, 20 μL
of MTT (0.5 mg/mL), solution was added to each well, and the microplates were
incubated for 4 hours at 37°C.
After incubation, the plates were centrifuged (1400 ×g, 18°C, 10 minutes). The supernatants were
removed, and 200 μL of dimethyl sulfoxide (DMSO) was added to
each well and incubated for 15 minutes at room temperature. After
incubation, the solubilized reduced MTT was measured colorimetrically at
570 nm using a Benchmark microculture plate reader (Bio-Rad, Calif, USA).


## 2.6. Assay for Mitochondrial Activity of HepG2 and SPC-A-1 Cells

The influence of Tα1 on mitochondrial activity was measured using MTT reduction, a
common experimental method to quantify mitochondrial activity. The cells were
seeded in a 96-well culture plate at 1×106 cells/mL.
After 24-hour, 48-hour, and 72-hour exposures to Tα1 at a concentration of 0.1 mg/mL,
the cells were incubated with MTT (0.5 mg/mL) for 4 hours at 37°C. The formazan precipitate
was then dissolved in 200 μL DMSO, and absorbance at 570 nm was measured using the Benchmark microculture plate reader (Bio-Rad, Calif, USA).


## 2.7. Statistic Analysis

The data are presented as mean ±SEM, for at least 3 replicate
experiments and evaluated using the Student'st-test. P-values below 0.05 were regarded as statistically
significance (two-tailed test).


## 3. Results

Hydroxylamine-induced cleavage
at the Asp-Gly dipeptide site inserted between the 2 moieties of recombinant
fusion proteins had been used for both analysis and preparation of mature
protein. Cleavage occurs at the Asn-Gly bonds and results from
the tendency of the Asp side chain to cyclize the formation of a substituted
succinimide that is susceptible to nucleophilic attack by hydroxylamine. The
infrequency of Asn-Gly bonds in most proteins results in the production of very
large fragments that may overlap CNBr-produced fragments [24]. In the complete amino acid
sequence of Tα1 [1] (see Figure 1), only 1 Asp was
found at the C-terminus. However, the hydroxylamine-induced
cleavage process was performed conveniently. Hydroxylamine-induced
cleavage is high specificity and safe, however, the efficiency of
hydroxylamine cleavage is low. So we expressed a concatemer protein of 6
repeats of Tα1
to increase the yield of Tα1 after cleavage and to decrease the cleavage sites.


### 3.1. Characterization of Recombinant  Concatemer Protein

The optimal conditions for inducing production of recombinant concatemer protein were
at an IPTG concentration of 1.0 mM 37°C for 8 hours. Under these
conditions, the recombinant protein (~23 kD) comprised 31% of the total
bacterial protein (see Figure 4(a)). The concatemer protein was identified
primarily in the inclusion bodies (see Figure 4(b)).Overexpression of the recombinant concatemer protein. (a) Lane 1: prestained
molecular mass markers. Lanes 2 and 3: full bacterial protein from 100μL
transformed cells after 1.0 mM and 0.5 mM IPTG induction
for 8 hours at 37°C. Lane 4: full bacterial protein from 100 μL transformed cells without induction
of IPTG for 8 hours at 37°C. (b) Lane 1: prestained molecular mass
markers. Lane 2: full bacterial protein from 100 μL transformed cells after 1.0 mM IPTG induction for 8 hours. Lane
3: sedimented material from Sonication
of 100 μL transformed cells after induction with IPTG. Lane 4: suspended material
from Sonication of 100 μL transformed cells after induction with IPTG.
(a)(b)


### 3.2. Analysis of the Products of Hydroxylamine Cleavage

G-25 column chromatography was used to not only desalt but
also remove the incompletely cleaved concatemer protein. Because the molecular weight of the Tα1
monomer was ~3.1 kD, it could enter the G-25 gel. However, the incompletely cleaved concatemer
proteins, including 2×Tα1 (~7 kD), 3×Tα1 (~11 kD), 4×Tα1 (~15 kD),
5×Tα1 (~19 kD), and 6×Tα1 (~23 kD), could not enter the G-25 gel because
the gel only allows the entry of molecules <5 kD. The Gly-SDS-PAGE could satisfactorily
separate polypeptides with molecular weights ranging from 3.5 to 68.0 kD with
good linearity, and demonstrated the successful hydroxylamine-induced cleavage
of the concatemer proteins (see Figure 5). However, not all the concatemer proteins were cleaved, and the
incompletely cleaved proteins could be completely cleaved by another reaction (data
not shown).Figure 5
Analysis of hydroxylamine cleavage
products. Lane 1: peak 1 of G-25 column chromatography revealed the protein
with a molecular weight of more than 5 kD that was eluted early. Lane 2: peak 2
of G-25 column chromatography containing the protein with a molecular weight of
less than 5 kD that was eluted later. Lane 3: the Tα1 synthesized as a standard
protein.MS was performed to analyze the exact
molecular weights of the products and the synthesized Tα1. The result showed that
the products had only 1 peak of m/z =1042.19, while the synthesized Tα1 had 2
peaks of m/z = 1037.80 and 1555.06 (see Figure 6). The molecular weight of the
synthesized Tα1 was found to be approximately 3108 Da, it was assumed that the
peptide might attach 2 or 3 protons [(1037.80−1) × 3 = 3110.4;
(1555.06−1) × 2 = 3108.12].
Therefore, the cleaved Tα1 was believed to attach 3 protons, and the molecular
weight of the products was approximately 3123.57 [(1042.19-1) × 3 = 3123.57]. The
difference in the molecular weights between the cleaved Tα1 and the synthesized
Tα1 was approximately 15. According to a previous research [24], the structural difference
between the products and the synthesized Tα1 was a –NH added to the
N-terminus
of the cleaved peptide (see Figure 9).(a) Mass spectrum of the cleaved Tα1. The attachment of 3 protons per peptide molecule leads to
only 1 of m/z peak; m/z = 1042.19. (b) Mass spectrum of the synthesized Tα1. The attachment of 2 and 3 protons
per peptide molecule leads to 2 peaks of m/z = 1037.80 and 1555.06.
(a)(b)


### 3.3. Tα1 Cleaved by Hydroxylamine Stimulated the Proliferation of Mice Splenic Lymphocytes

To determine the bioactivity of the Tα1 monomer on lymphocyte
proliferation, the mitochondrial activity was compared in the presence with the
absence of the Tα1. The bioactivity of Tα1 preparations was analyzed by MTT assay. Preliminary results
showed that the peptides could stimulate the mice spleen lymphocyte
proliferation [22]. After a 24-hour treatment with Tα1 cleaved by hydroxylamine,
mitochondrial activity was upgraded to 119.1% and after 48 hours, it was 110.4%.The
numbers of the synthesized Tα1 were 117.5% and 116.0% at 24 hours and 48 hours,
respectively (see Figure 7). Both of the peptide preparations could stimulate the
proliferation of mice spleen lymphocytes to a similar degree.Figure 7
Tα1 (cleaved and synthesized)
stimulated the proliferation of mice spleen leukomonocytes. The mice spleen leukomonocytes were treated with the indicated concentrations of Tα1 for 24 hours, and 48 hours. MTT assay
was used to measure the mitochondrial activity. Significant stimulation of the
mitochondrial activity, as compared to the control, occurred after 48 hours of
Tα1 treatment. All the results are expressed as the mean ±SEM of data obtained in 3
independent experiments (P<.05,
relative to control).


### 3.4. Tα1 Cleaved by Hydroxylamine Exhibits Decreased Mitochondrial Activity in SPC-A-1 and HepG2 Cells

In this study, we used human liver tumor cells HepG2
and human lung tumor cells SPC-A-1 as model, because the liver tumor and lung
tumor are the most general tumor. To determine the role of mitochondria during incubation,
the mitochondrial activity was examined. After the treatment with the cleaved Tα1
for 24 hours, 48 hours, and 72 hours, the mitochondrial activity of the SPC-A-1
cells decreased to 82.4%, 27.1%, 11.6%, respectively, and the corresponding
activities with synthesized Tα1 were 62.0%, 15.1%, and 9.8%, respectively. On
the other hand, after the treatment for 24 hours, 48 hours, and 72 hours, the
mitochondrial activity of HepG2 cells decreased to 86.6%, 18.4%, and 10.4%,
respectively, and corresponding activities with the synthesized Tα1 were 59.2%,
13.1%, and 10.0%, respectively (see Figure 8). Both of the peptide preparations
decreased the mitochondrial activity remarkably, as compared to the control.
The difference in the activity between the 2 peptides was not significant;
however, the process of synthesizing Tα1 was rather intricate.Tα1 (cleaved and synthesized) induced a decrease in the
mitochondrial activity in the SPC-A 1 and HepG2 cells. (a) The SPC-A1 cells were treated with the indicated concentrations
of Tα1 for 24 hours, 48 hours, and 72 hours. MTT assay was used to measure the mitochondrial
activity. Significant decreases in the mitochondrial activity, as compared to
the control, occurred after 48 hours of Tα1 treatment. (b) The HepG2 cells were treated with the indicated concentrations
of Tα1 for 24 hours, 48 hours, and 72 hours. MTT assay was used to measure the mitochondrial
activity. Significant decreases in the mitochondrial activity, as compared to the
control, occurred after 48 hours of Tα1 treatment. All the results are expressed as the mean ±SEM of data
obtained in 3 independent experiments (P<.05,
relative to control).
(a)(b)Differences in the structures of the synthesized Tα1 and the cleaved Tα1.
(a)
Cleaved Tα1(b)
Synthesized Tα1


## 3.1. Characterization of Recombinant  Concatemer Protein

The optimal conditions for inducing production of recombinant concatemer protein were
at an IPTG concentration of 1.0 mM 37°C for 8 hours. Under these
conditions, the recombinant protein (~23 kD) comprised 31% of the total
bacterial protein (see Figure 4(a)). The concatemer protein was identified
primarily in the inclusion bodies (see Figure 4(b)).Overexpression of the recombinant concatemer protein. (a) Lane 1: prestained
molecular mass markers. Lanes 2 and 3: full bacterial protein from 100μL
transformed cells after 1.0 mM and 0.5 mM IPTG induction
for 8 hours at 37°C. Lane 4: full bacterial protein from 100 μL transformed cells without induction
of IPTG for 8 hours at 37°C. (b) Lane 1: prestained molecular mass
markers. Lane 2: full bacterial protein from 100 μL transformed cells after 1.0 mM IPTG induction for 8 hours. Lane
3: sedimented material from Sonication
of 100 μL transformed cells after induction with IPTG. Lane 4: suspended material
from Sonication of 100 μL transformed cells after induction with IPTG.
(a)(b)


## 3.2. Analysis of the Products of Hydroxylamine Cleavage

G-25 column chromatography was used to not only desalt but
also remove the incompletely cleaved concatemer protein. Because the molecular weight of the Tα1
monomer was ~3.1 kD, it could enter the G-25 gel. However, the incompletely cleaved concatemer
proteins, including 2×Tα1 (~7 kD), 3×Tα1 (~11 kD), 4×Tα1 (~15 kD),
5×Tα1 (~19 kD), and 6×Tα1 (~23 kD), could not enter the G-25 gel because
the gel only allows the entry of molecules <5 kD. The Gly-SDS-PAGE could satisfactorily
separate polypeptides with molecular weights ranging from 3.5 to 68.0 kD with
good linearity, and demonstrated the successful hydroxylamine-induced cleavage
of the concatemer proteins (see Figure 5). However, not all the concatemer proteins were cleaved, and the
incompletely cleaved proteins could be completely cleaved by another reaction (data
not shown).Figure 5
Analysis of hydroxylamine cleavage
products. Lane 1: peak 1 of G-25 column chromatography revealed the protein
with a molecular weight of more than 5 kD that was eluted early. Lane 2: peak 2
of G-25 column chromatography containing the protein with a molecular weight of
less than 5 kD that was eluted later. Lane 3: the Tα1 synthesized as a standard
protein.MS was performed to analyze the exact
molecular weights of the products and the synthesized Tα1. The result showed that
the products had only 1 peak of m/z =1042.19, while the synthesized Tα1 had 2
peaks of m/z = 1037.80 and 1555.06 (see Figure 6). The molecular weight of the
synthesized Tα1 was found to be approximately 3108 Da, it was assumed that the
peptide might attach 2 or 3 protons [(1037.80−1) × 3 = 3110.4;
(1555.06−1) × 2 = 3108.12].
Therefore, the cleaved Tα1 was believed to attach 3 protons, and the molecular
weight of the products was approximately 3123.57 [(1042.19-1) × 3 = 3123.57]. The
difference in the molecular weights between the cleaved Tα1 and the synthesized
Tα1 was approximately 15. According to a previous research [24], the structural difference
between the products and the synthesized Tα1 was a –NH added to the
N-terminus
of the cleaved peptide (see Figure 9).(a) Mass spectrum of the cleaved Tα1. The attachment of 3 protons per peptide molecule leads to
only 1 of m/z peak; m/z = 1042.19. (b) Mass spectrum of the synthesized Tα1. The attachment of 2 and 3 protons
per peptide molecule leads to 2 peaks of m/z = 1037.80 and 1555.06.
(a)(b)


## 3.3. Tα1 Cleaved by Hydroxylamine Stimulated the Proliferation of Mice Splenic Lymphocytes

To determine the bioactivity of the Tα1 monomer on lymphocyte
proliferation, the mitochondrial activity was compared in the presence with the
absence of the Tα1. The bioactivity of Tα1 preparations was analyzed by MTT assay. Preliminary results
showed that the peptides could stimulate the mice spleen lymphocyte
proliferation [22]. After a 24-hour treatment with Tα1 cleaved by hydroxylamine,
mitochondrial activity was upgraded to 119.1% and after 48 hours, it was 110.4%.The
numbers of the synthesized Tα1 were 117.5% and 116.0% at 24 hours and 48 hours,
respectively (see Figure 7). Both of the peptide preparations could stimulate the
proliferation of mice spleen lymphocytes to a similar degree.Figure 7
Tα1 (cleaved and synthesized)
stimulated the proliferation of mice spleen leukomonocytes. The mice spleen leukomonocytes were treated with the indicated concentrations of Tα1 for 24 hours, and 48 hours. MTT assay
was used to measure the mitochondrial activity. Significant stimulation of the
mitochondrial activity, as compared to the control, occurred after 48 hours of
Tα1 treatment. All the results are expressed as the mean ±SEM of data obtained in 3
independent experiments (P<.05,
relative to control).


## 3.4. Tα1 Cleaved by Hydroxylamine Exhibits Decreased Mitochondrial Activity in SPC-A-1 and HepG2 Cells

In this study, we used human liver tumor cells HepG2
and human lung tumor cells SPC-A-1 as model, because the liver tumor and lung
tumor are the most general tumor. To determine the role of mitochondria during incubation,
the mitochondrial activity was examined. After the treatment with the cleaved Tα1
for 24 hours, 48 hours, and 72 hours, the mitochondrial activity of the SPC-A-1
cells decreased to 82.4%, 27.1%, 11.6%, respectively, and the corresponding
activities with synthesized Tα1 were 62.0%, 15.1%, and 9.8%, respectively. On
the other hand, after the treatment for 24 hours, 48 hours, and 72 hours, the
mitochondrial activity of HepG2 cells decreased to 86.6%, 18.4%, and 10.4%,
respectively, and corresponding activities with the synthesized Tα1 were 59.2%,
13.1%, and 10.0%, respectively (see Figure 8). Both of the peptide preparations
decreased the mitochondrial activity remarkably, as compared to the control.
The difference in the activity between the 2 peptides was not significant;
however, the process of synthesizing Tα1 was rather intricate.Tα1 (cleaved and synthesized) induced a decrease in the
mitochondrial activity in the SPC-A 1 and HepG2 cells. (a) The SPC-A1 cells were treated with the indicated concentrations
of Tα1 for 24 hours, 48 hours, and 72 hours. MTT assay was used to measure the mitochondrial
activity. Significant decreases in the mitochondrial activity, as compared to
the control, occurred after 48 hours of Tα1 treatment. (b) The HepG2 cells were treated with the indicated concentrations
of Tα1 for 24 hours, 48 hours, and 72 hours. MTT assay was used to measure the mitochondrial
activity. Significant decreases in the mitochondrial activity, as compared to the
control, occurred after 48 hours of Tα1 treatment. All the results are expressed as the mean ±SEM of data
obtained in 3 independent experiments (P<.05,
relative to control).
(a)(b)Differences in the structures of the synthesized Tα1 and the cleaved Tα1.
(a)
Cleaved Tα1(b)
Synthesized Tα1


## 4. Discussion

The requirements of Tα1 for clinical applications are increasing. For
clinical use, Tα1 is mainly obtained by chemical synthesis and animal thymus
extraction. However, in fact, Tα1 from an animal is often impure and
may therefore cause heterogeneous allergy. Although chemically synthesized Tα1 can reach a high level of purity, it is necessary to eliminate the side products at each step, including incorrect joining and dextral compounds. The
longer the peptide is, the more intricate the chemical synthesis process becomes. Tα1
comprises 28 amino acid residues, and its chemical synthesis incurs a high production cost [19]. Prokaryotic
expression systems allow for high expression of foreign genes and are widely used for producing large
amounts of proteins or peptides. In this research, using a prokaryotic
expression system BL21 (DE3)/pET-22b (+), we have established a new method to
obtain relatively pure and large quantities of Tα1.When the plasmid was digested with isocaudamer BamH I (G*CTAG
C) and Bgl II (A*GATC T), the 2 fragments had similar termini. When the 2 fragments
were catalyzed with T4 DNA Ligase, the new sequence formed was GGATCT, and this
new site could not be digested by BamH I or Bgl II (see Figure2). Therefore, the
new 2 × concatemer gene could not be destroyed when the 4 × concatemer gene was
constructed. The new site GGATCT corresponded to the dipeptide Gly-Ser, located at the downstream of the Tα1 gene. When concatemer protein was cleaved with hydroxylamine,
the downstream amino acid of Tα1 peptide was removed from this Gly site. When
the cleavage reaction proceeded, the upstream amino acid of Tα1 peptide was
removed from another Gly site, which was added in front of the Tα1 peptide.It is common knowledge that extraction of the micromolecular recombinant peptide
from fermentation broth is difficult, when Tα1 is expressed alone. Generally, a
fusion protein is used under these circumstances, for example, a DsbA fusion protein [25]. However, the fusion protein must be
removed because the peptide might be inactive, and this process incurs a high production cost. The concatemer protein of 6
repeats—6×Tα1—was prepared in
this research to avoid the use of a fusion protein when expressing the recombinant
protein, but the number of the repeats was not unique. Proteins with 4, 8, 12, and
so forth repeats could also be constructed. The longer the concatemer protein
is, the greater the yield of the Tα1 monomer is. On the other hand, when a concatemer protein
with more repeats is cleaved, the amount of the incompletely cleaved protein also
increases (see Figure 5). However, these incompletely cleaved proteins can be
cleaved by other 1 or 2 rounds of hydroxylamine cleavage.Hydroxylamine
was used to cleave the Asn-Gly peptide bond between the fusion protein and
target protein [26]. In this study, hydroxylamine was used to cleave the concatemer
protein of 6×Tα1 to release the Tα1 monomer. While chemical cleavage methods
use relatively inexpensive and widely available reagents, cleavage conditions
are often relatively harsh and amino acid side-chain modifications may occur.
This is especially true when hydroxylamine is used to cleave the Asn-Gly
peptide linkages as other Asn or Gln residues can be modified to their
hydroxamic acid forms [26]. Because the Tα1 peptide does not contain Gln, Met, or other Asn, chemical
heterogeneity dose not occur by conversion of the Gln or Asn to their
hydroxamate forms. Moreover, there is no possibility of chemical heterogeneity by
conversion of Met to its oxidized forms [27].After hydroxylamine cleavage, a Gly remained at the N-terminus of Tα1. However, the natural Tα1
had an acetylated NH-terminal (see Figure 9). The molecular weight
difference between the 2 groups was 15 Da, but there was no significant difference
between the 2 bands revealed by the Gly-SDS-PAGE (see Figure 5). Hence, we used
an MS to analyze the exact molecular weights of the 2 peptides. The result
showed a difference was 15 Da (3123−3108 Da) (see Figure
6), indicating that the concatemer protein was cleaved satisfactorily.To investigate the influence of the extra
imino group on the Tα1, we compared the antitumor and immunomodulation activities between
the synthesized Tα1 and the cleaved Tα1. The results showed no
significant difference between the 2 peptides. Furthermore, we found that Tα1
was able to depress the prolife ration
of HepG2 cells by downregulating the oxidative stress (data not shown). Jörnvall
[28] had analyzed 40 known
α-amino-acetylated polypeptide chains and postulated that acetylation occurs
during an early posttranslational stage and may have particular importance in
this regard. However, the structural or functional significance of the N-terminal
acetylation of Tα1 is not
known, and a protective function of acetylation with regard to proteolysis is
possible [1]. The change in the N-terminus of
cleaved peptides might not influence the bioactivity of Tα1; however, it might
influence the stability in vivo.In summary, Tα1
with a novel structure was prepared via the expression and hydroxylamine
cleavage of a Tα1 concatemer protein. This novel Tα1 could perform proliferation
of mice splenic lymphocytes activities, identical to the natural Tα1. This
study would provide the basis for the preparation of active Tα1 and other
peptides.



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*Source: 736060-2008-07-15.xml*