[Biotechnological production of acetylated thymosin beta4].

Thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin beta4 is produced with solid-phase chemical synthesis. Biotechnological synthesis of this peptide presents difficulties because N-terminal amino acid residue of thymosin beta4 is acetylated. In this study we propose a method for producing the recombinant precursor of thymosin beta4 and its subsequent targeted chemical acetylation. Desacetylthymosin beta4 was synthesized as a part of a hybrid protein with thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for the purification of desacetylthymosin beta4: (i) the biosynthesis of a soluble hybrid protein (HP) in Escherichia coli; (ii) isolation of the HP by ion exchange chromatography; (iii) cleavage of the HP with TEVprotease; (iv) purification of desacetylthymosin beta4 by ultra-filtration. N-terminal acetylation of desacetylthymosin beta4 was performed with acetic anhydride under acidic conditions (pH 3). The reaction yield was 55%. Thymosin beta4 was then purified by reverse-phase high performance liquid chromatography. The proposed synthetic approach to recombinant thymosin beta4 is suitable for scale-up and can provide for the medical use of highly purified preparation with a yield of 20 mg from 1 L of culture.

Thymosin alpha 1: biological activities, applications and genetic engineering production.

Thymosin alpha 1 (Tα1), a 28-amino acid peptide, was first described and characterized from calf thymuses in 1977. This peptide can enhance T-cell, dendritic cell (DC) and antibody responses, modulate cytokines and chemokines production and block steroid-induced apoptosis of thymocytes. Due to its pleiotropic biological activities, Tα1 has gained increasing interest in recent years and has been used for the treatment of various diseases in clinic. Accordingly, there is an increasing need for the production of this peptide. So far, Tα1 used in clinic is synthesized using solid phase peptide synthesis. Here, we summarize the genetic engineering methods to produce Tα1 using prokaryotic or eukaryotic expression systems. The effectiveness of these biological products in increasing the secretion of cytokines and in promoting lymphocyte proliferation were investigated in vitro studies. This opens the possibility for biotechnological production of Tα1 for the research and clinical applications.

Optimized Fmoc solid-phase synthesis of Thymosin alpha1 by side-chain anchoring onto a PEG resin.

Thymosin alpha1 is a 28-amino acid acetylated peptide used for the treatment of hepatitis B and C. This peptide has a difficult sequence because of the presence of consecutive beta-branched amino acids and shows a tendency to form beta-sheet structures, partly as a result of the many protecting groups required to assemble the peptide (up to 20 side-chain protecting groups). Consequently, its synthesis has been generally achieved by convergent solution chemistry. Here we report a straightforward stepwise solid-phase synthesis on a polyethylene glycol solid-support that enables the scaling-up of this key therapeutic peptide.

Expression and hydroxylamine cleavage of thymosin alpha 1 concatemer.

Human thymosin alpha 1 (Talpha1) 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 Talpha1 gene according to the E. coli codon usage preference and constructed a 6xTalpha1 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 Talpha1 monomer. Gly-SDS-PAGE and mass spectrometry confirmed that the recombinant protein was cleaved as intended. The bioactivity of the Talpha1 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 Talpha1, which may prove useful in future biomedical research.

Syntheses of two deacetyl-thymosin beta4 analogs and their anti-inflammatory effect on carrageenin-induced edema in the mouse paw.

Two [Met(0)6]deacetyl-thymosin beta4 analogs containing Phe(4F) or Tyr(Me) at position 12 were synthesized by the manual solid-phase method, and their anti-inflammatory effect on carrageenin-induced edema in the mouse paw was studied. Fluorination of the para-position of Phe12 resulted in a marked antiinflammatory effect on carrageenin-induced edema in the mouse paw compared with that of our synthetic [Met(0)6]deacetyl-thymosin beta4, but the other analog, [Met(0)6, Tyr(Me)12]deacetyl-thymosin beta4, showed a marked reduction of the anti-inflammatory effect.

Effects of synthetic thymosin-alpha 1 and its analogs on fertilizability of human sperm: search for a biologically active, stable epitope.

Thymosin-alpha 1 (T alpha 1) and six T alpha 1 analogs were synthesized to study structure-function relationships and to search for the biologically active and stable epitope(s) that would have clinical application in the treatment of male infertility. Four of these analogs were prepared by modification/substitution of N- and C-terminal amino acids of T alpha 1 peptide, and the other two analogs were fragments having only N-16 amino acids (N-terminal) or C-14 amino acids (C-terminal), respectively, of the T alpha 1 peptide. T alpha 1 and these six analogs were tested for their effects on human sperm penetration rates in the sperm penetration assay (SPA). T alpha 1 significantly (p < .0001) increased the penetration rates in SPA, with the strongest enhancing effect at 0.5 microgram/100 microL concentration. Of the six analogs tested only two, T alpha 1-Gly-NH2 and T alpha 1-C14, retained the enhancing effects in SPA. None of the analogs decreased the penetration rates or affected sperm motility compared to control. The enhancing activity resides primarily in an epitope, the C-terminal 14 amino acids of T alpha 1. However, for maximal effect both N- and C-terminal amino acids (serine and asparagine, respectively) have to be intact and unmodified. The T alpha 1-Gly-NH2 analog that had its C-terminal protected was as potent as the intact T alpha 1 peptide. T alpha 1 and this analog may have clinical applications in treatment of male-factor-mediated infertility.

Radioimmunoassays for the C-terminus of prothymosin alpha and the N-terminus of parathymosin alpha for the measurement of the levels of alpha-thymosins in human cancer.

A radioimmunoassay specific for the C-terminus of human prothymosin alpha was developed using the synthetic peptide [Cys-Aca degrees]-human prothymosin alpha (90-109)-OH coupled to KLH as antigen and the analogue [Tyr-Aca degrees]-human prothymosin alpha (90-109)-OH labelled with 125I as tracer. The radioimmunoassay measured intact prothymosin alpha, in the range of 2-100 pmol and does not cross-react with the partly homologous polypeptide parathymosin alpha. A major epitope was located in the segment 95-107. A radioimmunoassay specific for the N-terminus of human parathymosin alpha, also measuring intact parathymosin alpha in the range of 1-20 pmol and not cross-reacting with prothymosin alpha, was developed using the synthetic peptide [Cys-Aca degrees]-human parathymosin alpha (1-30)-OH as antigen coupled to KLH and the analogue [Tyr-Aca degrees]-human parathymosin alpha (1-10)-OH labelled with 125I as tracer. A major epitope was located in the segment 1-10. These radioimmunoassays, together with a previously established radioimmunoassay for the N-terminus of prothymosin alpha, permitted the identification of the molecular forms of the cross-reactive materials in both normal and neoplastic breast tissue extracts as intact prothymosin alpha and parathymosin alpha. It was also possible to reveal significantly higher levels of both alpha-thymosins in breast cancer tissue compared to the nearby healthy tissue--the mean of 14 samples was over 14-fold higher--suggesting a role of both prothymosin alpha and parathymosin alpha in cell proliferation. The reported radioimmunoassays are expected to facilitate the search for prognostic and/or diagnostic applications of these polypeptides in human cancer.

Synthesis of deacetyl-thymosin beta 12 and examination of its immunological effects on the impaired T and B lymphocytes in uremic patients.

Deacetyl-thymosin beta 12 was synthesized in a conventional manner by assembling six peptide fragments followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole (molar ratio, 1:1) in trifluoroacetic acid in the presence of m-cresol and dimethyl-selenium. Incubation of peripheral lymphocytes isolated from uremic patients with the synthetic deacetyl-thymosin beta 12 showed an enhancing effect on the reduced beta lymphocytes but had no restoring effect on the impaired blastogenic response of T lymphocytes.

Synthesis of prothymosin alpha deduced from nucleotide sequence of the murine cDNA and its effect on the impaired T lymphocytes of uremic patients.

The complete murine prothymosin alpha molecule (110 residues) except for the N-terminal methionine deduced from the cloned cDNA has been synthesized by a solid-phase method. Peptide synthesis was performed manually by the stepwise solid-phase method using the base-labile Fmoc group for protecting the alpha-amino group. The peptide was assembled on a p-alkoxybenzyl alcohol resin. After the last coupling step, the Fmoc group was removed with 50% piperidine in DMF. The peptide resin was treated with thioanisole-o-cresol in TFA, and then purified by gel filtration, ion-exchange column chromatography and high-performance liquid chromatography. A 2.9-mg sample of a highly purified peptide was finally obtained. The overall yield of the synthesis was less than 1%, based on the amino acid content of the starting Fmoc-Asp (OtBu)-resin. The synthetic peptide was found to have a restoring activity on low-E-rosette-forming lymphocytes after incubation of peripheral blood from uremic patients with the synthetic peptide. This peptide exhibited far stronger restoring effect than that of our synthetic thymosin alpha 1.

Cleavage and deprotection of peptides on MBHA-resin with hydrogen bromide.

Dilute hydrogen bromide in trifluoroacetic acid containing pentamethylbenzene and thioanisole was used in the cleavage and deprotection of peptides on MBHA-resin. Particular attention was paid to potential applicability of the method to kilogram scale synthesis of thymosin alpha 1. In the HPLC purification of the peptides, acetonitrile was replaced by relatively nontoxic isopropanol. The change should be economically and environmentally very attractive.

Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells.

Thymosin beta 4 (beta 4) is a 5-kDa polypeptide originally identified in calf thymus. Although numerous activities have been attributed to beta 4, its physiological role remains elusive. Recently, beta 4 was found to bind actin in human platelet extracts and to inhibit actin polymerization in vitro, raising the possibility that it may be a physiological regulator of actin assembly. To examine this potential function, we have increased the cellular beta 4 concentration by microinjecting synthetic beta 4 into living epithelial cells and fibroblasts. The injection induced a diminution of stress fibers and a dose-dependent depolymerization of actin filaments as indicated by quantitative image analysis of phalloidin binding. Our results show that beta 4 is a potent regulator of actin assembly in living cells.

Solid-phase syntheses of two deacetyl-thymosin alpha 1 analogues with substitution at position 21 and their effects on low E-rosette-forming lymphocytes of uremic patients.

Two deacetyl-thymosin alpha 1 analogues containing Phe or Phe(4F) at position 21 were synthesized by the manual solid-phase method and their immunological effects on the low E-rosette-forming lymphocytes of uremic patients were studied. Fluorination of the p-position of Phe21 resulted in a marked restorative effect on the low E-rosette-forming lymphocytes of uremic patients compared with that of [Phe21]deacetyl-thymosin alpha 1. The synthetic [Phe21]deacetyl-thymosin alpha 1 was approximately equal in potency to our synthetic deacetyl-thymosin alpha 1 in uremic patients.

Synthesis of rat parathymosin alpha fragment 1-28 and examination of its inhibitory activity towards the restoring activity of thymosin alpha 1 on the impaired T-lymphocytes of uremic patients.

A fragment corresponding to N-terminal octaeicosapeptide of rat parathymosin alpha was synthesized by assembling 5 peptide fragments, followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole (molar ratio 1:1) in trifluoroacetic acid in the presence of dimethylselenium. Incubation of impaired T-lymphocytes isolated from uremic patients with the synthetic parathymosin alpha fragment 1-28 showed no immunological restoring effect, but when it was administered together with thymosin alpha 1, it appeared to suppress the restoring effect of the thymosin alpha 1 on the impaired T-lymphocytes of uremic patients.

Solid-phase synthesis of biologically active fragment 29-111 of rat prothymosin alpha.

Rat prothymosin alpha fragment 29-111, an 83-residue polypeptide corresponding to desthymosin alpha 1-prothymosin alpha, has been synthesized by a solid-phase method. Hydrogen fluoride was used to deprotect and cleave the peptide from the resin. The crude product was purified by gel-filtration, ion-exchange chromatography and high-performance liquid chromatography. A 3.2-mg sample of a ca. 96% pure peptide was finally obtained. The overall yield of the synthesis was less than 1%. An increase of E-rosette-forming lymphocytes was obtained after incubation of peripheral blood from uremic patients with the synthetic prothymosin alpha fragment 29-111. The restoring effect of the synthetic prothymosin alpha fragment 29-111 was greater than that of our synthetic thymosin alpha 1.

Synthesis of an immunologically active fragment analog of prothymosin alpha with enhanced enzymatic stability.

A fragment analog, [D-Arg30]prothymosin alpha fragment 1-30, containing D-arginine in place of arginine residue at position 30 was synthesized by the liquid phase procedure and studied for immunological effect on the impaired blastogenic response of T-lymphocytes isolated from uremic patients after treatment of human serum. Deacetyl-thymosin alpha 1, a synthetic octaeicosapeptide corresponding to deacetyl-prothymosin alpha fragment 1-28, has restoration ability for the impaired blastogenic response of T-lymphocytes of uremic patients but is susceptible to proteolytic digestion. On the other hand, the fragment analog, [D-Arg30]prothymosin alpha fragment 1-30 retained activity and was shown to exhibit a high degree of stability when incubated in human serum. These results indicate that N-terminal acetylation and the introduction of D-residue into the C-terminal residue of prothymosin alpha fragment 1-30 increase resistance to proteolytic degradation by exopeptidases.

Synthesis of a thymosin beta 4-like peptide, thymosin beta Met9, and its effect on low E-rosette-forming lymphocytes of lupus nephritis patients.

A thymosin beta 4-like peptide, thymosin beta Met9 isolated from pork spleen, was synthesized using a conventional solution method. The deprotection of the protected thymosin beta Met9 was achieved by treatment with Zn-90% AcOH to remove C-terminal p-nitrobenzyl ester and then with 1 M trifluoromethanesulfonic acid-thioanisole (molar ratio, 1:1) in trifluoroacetic acid in the presence of dimethylselenium. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulfoxide on the methionine side chain. The increase of the E-rosette-forming lymphocytes was obtained after incubation of peripheral blood from lupus nephritis patients with the synthetic thymosin beta Met9. The synthetic thymosin beta Met9 was approximately equal in potency to that of our synthetic calf thymosin beta 9.

Thymosin-like peptides as potential immunostimulants. Synthesis via the polymeric-reagent method.

This paper reports our attempt at designing new immunostimulating peptides which are chemically related to the bioactive peptides thymosin alpha 1 and thymopentin. Three peptides were synthesized, Asp-Leu-Lys-Glu-Arg-Lys-Asp-Val-Tyr (3), Arg-Lys-Asp-Val-Tyr-Glu-Glu-Ala-Glu-Asn (2), and Asp-Leu-Lys-Glu-Arg-Lys-Asp-Val-Tyr-Glu-Glu-Ala-Glu-Asn (1), each of which contains the thymopentin sequence and portions of the bioactive sequence of thymosin alpha 1. Peptides 1-3 were assembled from selected blocked fragments that were synthesized by the polymeric-reagent method, using PHBT (polystyrene-bound 1-hydroxybenzotriazole) as the activating polymer. The ability of peptides 1-3 to enhance the activation (RNA synthesis) and proliferation (DNA synthesis) of human T lymphocytes was determined. In comparison to thymosin alpha 1, thymosin alpha 1 (15-28), and thymopentin, peptides 1-3 did not show significant enhancement of these processes.

Synthesis of a thymosin beta 4-like peptide, deacetyl-thymosin beta Xen4, and its restorative effect on depressed lymphocyte blastogenic response to phytohemagglutinin (PHA) in uremic patients.

An analog of thymosin beta Xen4 isolated from oocytes of Xenopus laevis, deacetyl-thymosin beta Xen4, was synthesized by assembling 6 peptide fragments, followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole (molar ratio, 1:1) in trifluoroacetic acid in the presence of dimethylselenium. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulfoxide on the methionine side chain. The synthetic tritetracontapeptide was found to have a restoring effect on the impaired blastogenic response of T-lymphocytes isolated from uremic patients.

Synthesis of deacetylthymosin beta 11 and its effect on the impaired T-lymphocytes of a uremic patient with common variable immunodeficiency.

The untetracontapeptide corresponding to the entire amino acid sequence of deacetylthymosin beta 11 was synthesized by assembling six peptide fragments via the azide followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole in trifluoroacetic acid in the presence of dimethylselenide. The synthetic peptide was tested for its effect on the impaired blastogenic response of phytohemagglutinin-stimulated T-lymphocytes of a uremic patient with common variable immunodeficiency. The synthetic peptide had some restoring activity on the impaired blastogenic response of T-lymphocytes in the one patient tested.