Recombinant Expression and Bioactivity Characterization of TAT-Fused Thymosin ß10.

Thymosin beta 10 (TB10) is one of the common members among beta-thymosins. Human TB10 is reported to play a role in anti-angiogenesis and inhibition of cell migration during the tumorigenesis or metastasis of some certain cancers. Thus, it would be a potent clinical agent. In the present study, the coding sequence of TB10 was optimized based on the codon preference of Escherichia coli and cloned to pET28a (+) by chemical synthesis and molecular cloning methods. The recombinant protein was highly expressed employing E. coli expressing system and purified by a simple step of Ni2+ affinity chromatography. The TEV proteinase recognition site was inserted in the His6-tag and the target protein for easy removal of the His6-tag. To improve the biological activity of TB10, the transactivator of transcription (TAT) short peptide, a transduction domain, was added to the N-terminus of TB10. About 14.3 mg of the recombinant TB10 proteins was obtained from 1 L bacterial culture. The functional analyses demonstrated that the recombinant TB10 proteins displayed the distinct inhibition on angiogenesis by chick embryo chorioallantoic membrane assay and endothelial cell migration by wound healing assay. The TAT-fused TB10 even had stronger effects, probably due to the better transduction into the cells.

Anti-Inflammatory Activity of ß-thymosin Peptide Derived from Pacific Oyster (Crassostrea gigas) on NO and PGE2 Production by Down-Regulating NF-κB in LPS-Induced RAW264.7 Macrophage Cells.

ß-thymosin is known for having 43 amino acids, being water-soluble, having a light molecular weight and ubiquitous polypeptide. The biological activities of ß-thymosin are diverse and include the promotion of wound healing, reduction of inflammation, differentiation of T cells and inhibition of apoptosis. Our previous studies showed that oyster ß-thymosin originated from the mantle of the Pacific oyster, Crassostrea gigas and had antimicrobial activity. In this study, we investigated the anti-inflammatory effects of oyster ß-thymosin in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells using human ß-thymosin as a control. Oyster ß-thymosin inhibited the nitric oxide (NO) production as much as human ß-thymosin in LPS-induced RAW264.7 cells. It also showed that oyster ß-thymosin suppressed the expression of prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, oyster ß-thymosin reduced inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Oyster ß-thymosin also suppressed the nuclear translocation of phosphorylated nuclear factor-κB (NF-κB) and degradation of inhibitory κB (IκB) in LPS-induced RAW264.7 cells. These results suggest that oyster ß-thymosin, which is derived from the mantle of the Pacific oyster, has as much anti-inflammatory effects as human ß-thymosin. Additionally, oyster ß-thymosin suppressed NO production, PGE2 production and inflammatory cytokines expression via NF-κB in LPS-induced RAW264.7 cells.

Thymosin fraction 5 re-evaluated after 35 years by high-resolution mass spectrometry.

OBJECTIVES: We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances. METHODS: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5. RESULTS: We detected more than 100 monoisotopic masses corresponding to thymosin ß4 and truncated forms of ubiquitin, prothymosin α, thymosin ß4, and thymosin ß9. Additionally, we discovered a new polypeptide present in thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3. CONCLUSION: In spite of the well-known proteolytic processes inherent to the preparation of thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of thymosin fraction 5 is even nowadays highly promising.

Advances in the basic and clinical applications of thymosin ß4.

INTRODUCTION: Thymosin ß4 (Tß4), a multifunctional peptide, has been used successfully in several clinical trials involving tissue repair and regeneration. The review will first update the current information on the common underlying cellular cascades and pathways that are basic to Tß4's regenerative activity and second, on the current and potential uses of this protein in the clinic. AREAS COVERED: Significant advances in our understanding of the actions of Tß4 have occurred in directing stem cell maturation and in regeneration and repair of injuries. Many of its activities directly affect the repair cascade following injury. Using PubMed, we summarize the discovery and isolation of Tß4 as well as the studies on tissue repair, which have provided the scientific foundation for ongoing and projected trials in the treatment of eye injuries, dermal wounds, repair of the heart following myocardial infarction and healing of the brain following stroke, trauma or neurological diseases. EXPERT OPINION: Based on its multifunctional activities during tissue regeneration in various animal studies, Tß4 has the potential for new clinical applications such kidney and liver disease, as well as repair of spinal cord, bone and ligament damage. In addition, it may be useful in the treatment of a wide range of other applications, including the consequences of aging and viral infections.

The Escherichia coli-derived thymosin ß4 concatemer promotes cell proliferation and healing wound in mice.

Thymosin ß4 (Tß4) is one of the most promising thymosins for future clinical applications, and it is anticipated that commercial demand for Tß4 will increase. In order to develop a new approach to produce recombinant Tß4, a 168 bp DNA (termed Tß4) was designed based on the Tß4 protein sequence and used to express a 4 × Tß 4 concatemer (four tandem copies of Tß4, termed 4 × Tß4) together with a histidine tag (6 × His) in E. coli (strain BL21). SDS-PAGE and western blot analysis were used to confirm that a recombinant 4 × Tß4 protein of the expected size (30.87 kDa) was produced following the induction of the bacterial cultures with isopropyl ß-D-thiogalactoside (IPTG). The E. coli-derived 4 × Tß4 was purified by Ni-NTA resin, and its activities were examined with regard to both stimulating proliferation of the mice spleen cells in vitro and in vivo wound healing. The results demonstrate that these activities of the E. coli-derived recombinant 4 × Tß4 were similar or even better than existing commercially obtained Tß4. This production strategy therefore represents a potentially valuable approach for future commercial production of recombinant Tß4.

Bacterial expression, purification and angiogenesis-promoting activity of human thymosin ß4.

Thymosin ß4 (Tß4) is an actin-binding peptide involved in tissue regeneration and angiogenesis. This 43-amino acid peptide is chemically synthesized for research or clinical trials. To overcome the high costs of solid phase synthesis, we developed a genetic engineering procedure of Tß4 expression in a protease-deficient host strain, Escherichia coli BL21(DE3), transformed with different expression vectors (pRSETA, pET-15b and pEcoli-Cterm6 × HN). The recombinant, non-glycosylated peptide was overexpressed in soluble form and purified by two-step immobilized metal ion affinity chromatography. Use of the pET vector expression system allowed for easy removal of the polyhistidine tag by thrombin. Functional studies revealed that recombinant Tß4 stimulated angiogenesis via activation of the endothelial proteolytic systems, inhibition of endothelial cell adhesion, promotion of migration and capillary tube formation in Matrigel, and that its activity was similar to that observed for the synthetic peptide. The presented study comprises the first evidence that recombinant Tß4 promotes angiogenesis in an in vitro endothelial cell model.

Separation and isolation of fusion protein using a new native preparative PAGE device.

A human serum albumin and Thymosin α1 (HSA-Tα1) fusion protein was designed and over-expressed in Pichia pastoris. To purify the fusion protein, a new native preparative electrophoresis system that involved a modified device with a sample receiving chamber, and an assay method with Coomassie Blue G-250 tracing the collection of the protein of interest. In this device, two gels were run in parallel: native vertical collecting polyacrylamide gel electrophoresis (PAGE) and native vertical tracing PAGE. Samples mixed with or without Coomassie Blue G-250 loading buffer were separately loaded to the two aforementioned gels, and the fractions were collected until the tracing protein band combined with dye reached 1 cm from the sample-receiving chamber at the bottom of the gel. Approximately nine fractions were collected at regular intervals of 15 min. HSA-Tα1 fusion protein with 95% relative homogeneity was harvested and manifested similar immunological activities as synthetic Tα1 after a single-step purification of this preparative PAGE. As a result, this system offers a new, rapid and simple method for the purification of the protein of interest.

Production of Nα-acetylated thymosin α1 in Escherichia coli.

BACKGROUND: Thymosin α1 (Tα1), a 28-amino acid Nα-acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining Nα-acetylation. In this study, we describe a novel production process for Nα-acetylated Tα1 in Escherichia coli. RESULTS: To obtain recombinant Nα-acetylated Tα1 efficiently, a fusion protein, Tα1-Intein, was constructed, in which Tα1 was fused to the N-terminus of the smallest mini-intein, Spl DnaX (136 amino acids long, from Spirulina platensis), and a His tag was added at the C-terminus. Because Tα1 was placed at the N-terminus of the Tα1-Intein fusion protein, Tα1 could be fully acetylated when the Tα1-Intein fusion protein was co-expressed with RimJ (a known prokaryotic Nα-acetyltransferase) in Escherichia coli. After purification by Ni-Sepharose affinity chromatography, the Tα1-Intein fusion protein was induced by the thiols ß-mercaptoethanol or d,l-dithiothreitol, or by increasing the temperature, to release Tα1 through intein-mediated N-terminal cleavage. Under the optimal conditions, more than 90% of the Tα1-Intein fusion protein was thiolyzed, and 24.5 mg Tα1 was obtained from 1 L of culture media. The purity was 98% after a series of chromatographic purification steps. The molecular weight of recombinant Tα1 was determined to be 3107.44 Da by mass spectrometry, which was nearly identical to that of the synthetic version (3107.42 Da). The whole sequence of recombinant Tα1 was identified by tandem mass spectrometry and its N-terminal serine residue was shown to be acetylated. CONCLUSIONS: The present data demonstrate that Nα-acetylated Tα1 can be efficiently produced in recombinant E. coli. This bioprocess could be used as an alternative to chemosynthesis for the production of Tα1. The described methodologies may also be helpful for the biosynthesis of similar peptides.

Separation and purification of Escherichia coli-expressed human thymosin-α1 using affinity chromatography and high-performance liquid chromatography.

In this study, a human thymosin-α1 (hTα1) fusion protein was overexpressed in Escherichia coli (E. coli). The hexahistidine-tagged hTα1 fusion protein was obtained in soluble form in cells of the engineered E. coli strain BL21 (DE3)/pET-28a-hTα1 that had been induced with isopropyl -D-1-thiogalactopyranoside (IPTG). The recombinant protein accounted for approximately 50-60% of the total protein. We then developed and validated a separation method for hTα1 from E. coli cells based on thermal denaturation, nickel-resin affinity chromatography and high-performance liquid chromatography. The purification method showed good reproducibility and was easy to operate. Purified recombinant hTα1 of high homogeneity was characterized and found to be of high purity (over 99%), as determined by high-voltage electrophoresis and high-performance liquid chromatography analysis. Isoelectric focusing analysis indicated a pI of approximately 4.0, and full wavelength screening showed an optimal absorbance wavelength at around 214nm.

[Purification of recombinant proteins with an example of tumor necrosis factor thymosin-alpha1].

Hybrid protein, cancer necrosis factor thymosin-alpha1 (CNF-T), when synthesizing in strain-producer of Escherichia coli SG200-50 with plasmid pThy315, was a part of "inclusion bodies" mostly in the form of a high-molecular complex with other proteins due to the S-S bonds formation. An approach of purification of CNF-T has been proposed, which is based on the destruction of the complex in the presence of sodium dodecylsulfate (DDS-NA) and dithiotreitol (DDT) followed by gel-filtration on Sephadex G-100 and renaturation by ultrafiltration on hollow fibers. The method allows the isolation of electrophoretically homogeneous CNF-T containing no DDS-Na and having high cytotoxic activity against cancer cells of mouse adenocarcinome L-929. The yield of CNF-T achieved 80% relative its content in biomass and 30% relative the total protein.

Production of thymosin alpha1 via non-enzymatic acetylation of the recombinant precursor.

Human thymosin alpha1 is an effective immune system enhancer for the treatment of cancer and viral diseases. Therefore the development of new methods for its synthesis is an urgent problem. In the present work, we propose an efficient scalable scheme for the production of recombinant thymosin alpha1. We used an expression system based on the pET32b+ plasmid and Escherichia coli strain ER2566 to obtain a fusion protein consisting of thymosin alpha1 and thioredoxin separated by a TEV (tobacco etch virus) protease cleavage site. The fusion protein was overexpressed in soluble form and purified by ion-exchange chromatography. After proteolytic cleavage of the fusion protein with TEV protease, recombinant desacetylthymosin alpha1 was isolated by ultrafiltration. Acetic anhydride was used for selective N-terminal acetylation of the obtained peptide (yield=62%). The resultant thymosin alpha1 was purified by RP-HPLC (reversed-phase HPLC). The distinctive feature of this technology is that it is a combination of different approaches: the biotechnological production of recombinant fusion protein, its enzymatic cleavage, and chemical acetylation of desacetylthymosin alpha1. Each stage of the process was optimized to increase the yield of the target peptide, which averaged 29 mg/litre of bacterial culture. The proposed method is simple and cost-effective and is suitable for large-scale production of recombinant thymosin alpha1.

Expression of thymosin alpha1 concatemer in transgenic tomato (Solanum lycopersicum) fruits.

Talpha1 (thymosin alpha1), an immune booster, plays an important role in the maturation, differentiation and function of T-cells. It can also activate the production of cytokines in dendritic cells. Talpha1 is one of two thymosin proteins that have potential future clinical applications. In order to express Talpha1 protein in plants, we designed and synthesized the Talpha1 gene according to the plant codon usage bias and created a novel 4 x Talpha1 concatemer (four copies of the Talpha1 gene arranged end-to-end in tandem, designated 4 x Talpha1). Subsequently, a plant binary expression vector, PG-pRD12-4 x Talpha1, was constructed and introduced into tomato via Agrobacterium tumefaciens-mediated transformation. Through selection, 54 regenerated tomato plants resistant to kanamycin were obtained, and four transgenic tomato plants were further confirmed by PCR and Southern blotting. RT-PCR (reverse transcription-PCR) analysis showed that the 4 x Talpha1 gene was transcribed specifically in tomato [Solanum lycopersicum (formerly Lycopersicon esculentum)] fruits. ELISA analysis showed that the content of the 4 x Talpha1 protein reached a maximum of 6.098 microg/g fresh weight in mature tomato fruit. Western-blot analysis further confirmed the expression of 4xTalpha1 protein in transgenic tomato fruits. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay showed the 4 x Talpha1 protein derived from transgenic tomatoes exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes in vitro, and the specific activity of Talpha1 protein from the artificial system was higher than that from the synthetic Escherichia coli system. This study is the first to report successful expression of bioactive Talpha1 in plants, and also it will provide the basis for further development of the plant system to produce Talpha1.

A new protocol for high-yield purification of recombinant human prothymosin alpha expressed in Escherichia coli for NMR studies.

Human prothymosin alpha (ProTalpha) is a small acidic protein (12.1 kDa; pI approximately 3.5) ubiquitously expressed in a wide variety of tissues. The amino acid composition of this protein is highly unusual. While close to half of its sequence is composed of acidic amino acids, the protein does not contain any aromatic residues. ProTalpha has been shown to play crucial roles in different biological processes including cell proliferation, transcriptional regulation and apoptosis. Despite the multiple functions this protein has, it does not adopt a stable tertiary fold under physiological conditions. In order to understand how ProTalpha functions, detailed structural characterization of this protein is essential. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for elucidating the protein structure and dynamics at the atomic level. However, milligrams of isotopically labeled protein with high purity are usually required for the studies. In this work, we developed a high-yield protocol for purifying recombinant ProTalpha expressed in Escherichia coli by exploiting the intrinsically disordered and acidic natures of this protein. By combining the heat-cooling extraction, ammonium sulfate precipitation, and anion exchange chromatography, we were able to obtain over 20 mg of ProTalpha with >97% purity from 1L of M9 minimal media culture. The new purification protocol provides a cost effective and an efficient way to produce large quantities of high purity recombinant human ProTalpha in various isotopically labeled forms, which will greatly facilitate the structural studies of this protein by NMR and other biophysical methods.

Influence of prothymosin-alpha on HIV-1 target cells.

The important role of CD8(+) T cells in controlling HIV-1 infection through the innate as well as the adaptive immune system is well established. In addition to the major histocompatibility complex (MHC)-dependent cytotoxic activity of CD8(+) T cells, they produce soluble factors that suppress HIV-1 replication in an MHC-independent manner. Several of those factors have been identified, including beta-chemokines, Rantes, MIP-1alpha, MIP-1beta, and MDC. We previously identified that prothymosin alpha (ProTalpha) in the conditioned medium of HVS transformed CD8(+) T cells was a potent inhibitor of HIV-1 replication following proviral integration. In this report we further characterize the anti-HIV-1 activity of ProTalpha by demonstrating its target-cell specificity, distinction from additional inhibitors of HIV-1 transcription in CD8(+) T cell supernatants, as well as the differential regulation of host cell antiviral genes that could impact HIV-1 replication. These genes include a number of transcription factors as well IFN-alpha-inducible genes including PKR, IRF1, and Rantes, in the absence of induction of IFN-alpha. These data suggest that the anti-HIV-1 activity of ProTalpha is mediated through the modulation of a number of genes that have been reported to suppress HIV-1 replication including the dysregulation of transcription factors and the induction of PKR and Rantes mRNA.

Thymosin fraction-5 possesses antiproliferative properties in HL-60 human promyelocytic leukemia cells: characterization of an active peptide.

Thymosin fraction-5 (TF5) is a protein preparation of the bovine thymus. TF5 stimulates many assays of T cell-mediated immunity. We found that TF5 substantially suppressed proliferation of the rat C6 glioma and MMQ pituitary adenoma cell lines. Our current research using the promyelocytic cell line HL-60 suggests that TF5 also prevents proliferation of human myeloid leukemia cells. Our objective is the purification and chemical characterization of TF5 peptide components responsible for inhibition of HL-60 proliferative capacity. Using the inhibition of HL-60 cell proliferation, we have chemically characterized TF5 using fast protein liquid chromatography (FPLC), reversed-phase high-performance liquid chromatography (RP-HPLC), and high-performance capillary electrophoresis (HPCE). Vital dye-exclusion, oxidative metabolism of chromogenic dyes, and clonogenic growth profiles were used to determine rates of HL-60 proliferation. Our results identified an approximately 6000 Da component of TF5 capable of inducing HL-60 growth arrest. Synchronized HL-60 cells exposed to TF5 and its various constituents were subjected to cytometric analysis by flow cytometry. TF5-treated HL-60 cells had an increased subdiploid faction (i.e., sub-G1) compared to control cells. TF5 also increased Annexin V staining in randomly cycling HL-60 cells. Thus, a TF5 subfraction possesses growth-suppressive activity for human myeloid neoplasms. Our results indicate that this effect is characterized by at least one hallmark of apoptosis. Future clinical management strategies for certain leukemias may involve the use of thymic peptides.

In vivo sampling of extracellular beta-thymosin by ultrafiltration probes.

In vivo detection and monitoring of extracellular beta-thymosin will facilitate the understanding of their biological function and association with disease progression. A novel technique using capillary ultrafiltration (CUF) probes linked to mass spectrometry is capable of sensing extracellular thymosin beta-4 and/or thymosin beta-10 in vivo in wounded skin and other tissue microenvironments. In this review, we highlight the association of extracellular beta-thymosin with skin wound healing and the potential adjuvant effects on vaccination. The fabrication and biological application of CUF probes are also described. Data from CUF probe-captured beta-thymosin may guide future exploration of extracellular beta-thymosin.

Identification of prothymosin-alpha1, the necrosis-apoptosis switch molecule in cortical neuronal cultures.

We initially identified a nuclear protein, prothymosin-alpha1 (ProTalpha), as a key protein inhibiting necrosis by subjecting conditioned media from serum-free cultures of cortical neurons to a few chromatography steps. ProTalpha inhibited necrosis of cultured neurons by preventing rapid loss of cellular adenosine triphosphate levels by reversing the decreased membrane localization of glucose transporters but caused apoptosis through up-regulation of proapoptotic Bcl(2)-family proteins. The apoptosis caused by ProTalpha was further inhibited by growth factors, including brain-derived neurotrophic factor. The ProTalpha-induced cell death mode switch from necrosis to apoptosis was also reproduced in experimental ischemia-reperfusion culture experiments, although the apoptosis level was markedly reduced, possibly because of the presence of growth factors in the reperfused serum. Knock down of PKCbeta(II) expression prevented this cell death mode switch. Collectively, these results suggest that ProTalpha is an extracellular signal protein that acts as a cell death mode switch and could be a promising candidate for preventing brain strokes with the help of known apoptosis inhibitors.

Purification and characterization of the central segment of prothymosin-alpha: methodology for handling highly acidic peptides.

Prothymosin-alpha is a highly acidic protein consisting of 110 amino acids. The central segment of this protein, residues 51-89, is thought to be involved in metal binding which may be necessary for its physiological function. To carry out studies of this peptide, this central segment was synthesized in a linear fashion using Fmoc-based methods on rink amide MBHA resin. However, this peptide could not be purified with the typical straightforward approach of RP HPLC followed by negative mode electrospray ionization mass spectrometry (ESI-MS). This was attributed to the high proportion of acidic residues: 26 out of the 39 residues are aspartic and glutamic acids. The acidity of the peptide prevented retention on the RP HPLC column. Additionally, the ability of the highly negatively charged peptide to retain sodium ions prevented molecular weight determination with ESI-MS. A systematic approach to the purification of this highly acidic peptide was undertaken. Ultimately, strong anion exchange chromatography was used to purify the peptide. Extensive desalting using dialysis was required prior to ESI-MS, and the choice of the buffer proved to be critical. In the end, a purification method was devised that yielded a highly purified peptide and is readily compatible with analysis by ESI-MS.

Construction and application of a yeast expression system for thymosin a1

We want to construct a yeast expression system for thymosin a1 (Ta1) to make the orally administered Ta1 preparation possible. The whole Ta1 DNA fragment was obtained by PCR. After being digested with restriction enzymes, it was cloned into pYES2 vector. Sequencing was performed to identify the recombinant. The sequence of Ta1 in recombinant coincided with the original one reported in Genbank. When pYES2-Ta1 plasmid was transformed into yeast, galactose instead of glucose was used to induce Ta1 expression. Western blot was performed to identify the quality of the expressed Ta1. Dried yeast containing pYEST2-Ta1 was fed to Balb/c mice whose immunities were inhibited by cyclophosphamide in advance. Synthesized Ta1 peptide was used as positive control and empty yeast was used as negative control. Compared with the negative control group, both dried yeast containing pYEST2-Ta1 and synthesized Ta1 peptide can significantly increase the CD8+ level (22.74±1.09 and 18.77±4.72 vs 7.49±2.14, p<0.01), while both of them had little effect on the CD4+ lymphocytes (61.86±6.94 and 65.91±4.78 vs 57.93±10.40, p>0.05). We concluded that a high effective yeast expression system for Ta1 was constructed successfully and the Ta1 protein expressed by this system can improve CD8+ level in immune inhibited mice.

Construction and application of a yeast expression system for thymosin alpha1.

We want to construct a yeast expression system for thymosin alpha1 (Talpha1) to make the orally administered Talpha1 preparation possible. The whole Talpha1 DNA fragment was obtained by PCR. After being digested with restriction enzymes, it was cloned into pYES2 vector. Sequencing was performed to identify the recombinant. The sequence of Talpha1 in recombinant coincided with the original one reported in Genbank. When pYES2-Talpha1 plasmid was transformed into yeast, galactose instead of glucose was used to induce Talpha1 expression. Western blot was performed to identify the quality of the expressed Talpha1. Dried yeast containing pYEST2-Talpha1 was fed to Balb/c mice whose immunities were inhibited by cyclophosphamide in advance. Synthesized Talpha1 peptide was used as positive control and empty yeast was used as negative control. Compared with the negative control group, both dried yeast containing pYEST2-Talpha1 and synthesized Talpha1 peptide can significantly increase the CD8+ level (22.74 +/- 1.09 and 18.77 +/- 4.72 vs 7.49 +/- 2.14, p < 0.01), while both of them had little effect on the CD4+ lymphocytes (61.86 +/- 6.94 and 65.91 +/- 4.78 vs 57.93 +/- 10.40,p > 0.05). We concluded that a high effective yeast expression system for Talpha1 was constructed successfully and the Talpha1 protein expressed by this system can improve CD8+ level in immune inhibited mice.