The Escherichia coli-derived thymosin ?4 concatemer promotes cell proliferation and healing wound in mice.
ABSTRACT: Thymosin ?4 (T?4) is one of the most promising thymosins for future clinical applications, and it is anticipated that commercial demand for T?4 will increase. In order to develop a new approach to produce recombinant T?4, a 168 bp DNA (termed T?4) was designed based on the T?4 protein sequence and used to express a 4 × T? 4 concatemer (four tandem copies of T?4, termed 4 × T?4) 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 × T?4 protein of the expected size (30.87 kDa) was produced following the induction of the bacterial cultures with isopropyl ?-D-thiogalactoside (IPTG). The E. coli-derived 4 × T?4 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 × T?4 were similar or even better than existing commercially obtained T?4. This production strategy therefore represents a potentially valuable approach for future commercial production of recombinant T?4.
Project description:Thymosin ?4 (T?4) is a peptide that is known to play important roles in protection, regeneration, and remodeling of injured tissues in humans, and that shows great promise in a range of clinical applications. However, current strategies to T?4 are insufficient to meet growing demand and have a number of limitations. In this current study we investigated whether expression of recombinant T?4 in plants, specifically in tobacco (Nicotiana tabacum) leaves, represents an effective approach. To address this question, a 168?bp T?4 gene optimized for tobacco codon usage bias was constitutively expressed in tobacco as a 4-unit repeat concatemer, fused to a polyhistidine tag. Quantitative polymerase chain reaction and Western blot analyses were used to verify 4×T?4 expression in 14 transgenic tobacco lines and enzyme-linked immunosorbent assay analysis indicated 4×T?4 protein concentrations as high as 3??g/g of fresh weight in the leaves. We observed that direct administration of tobacco-derived T?4 was more effective than T?4 either obtained commercially or derived from expression in Escherichia coli at promoting splenocyte proliferation in vitro and wound healing in mice through an endothelial migration assay. This study provides new insights into the development of plant-derived therapeutic proteins and their application by direct administration.
Project description:Two forms of beta-thymosins, designated thymosin beta 11 and thymosin beta 12, were isolated from trout (Salmo gairdneri) spleen. This suggests that the presence of two beta-thymosins, previously thought to be a property of mammalian tissues only, is a more general phenomenon in vertebrate species. Both trout beta-thymosins were found to be N-terminally blocked by a group identified as acetyl by m.s. Automated protein sequencing of tryptic, thermolytic and Staphylococcus aureus in 41-residue V8 proteinase fragments revealed that one of the two beta-thymosins corresponds to the previously reported 41-residue-long sequence of thymosin beta 11 with two substitutions at positions 5 and 7, i.e. Asn instead of Asp, and Glu instead of Gln, whereas the other beta-thymosin, designated thymosin beta 12, was found to be a 42-residue polypeptide closely similar in sequence to thymosin beta 11, with five substitutions (i.e. at positions 5, 7, 10, 11 and 41, with Asp, Ala, Ser, Asn and Thr instead of Asn, Glu, Ala, Ser and Ser respectively) and one addition at position 42 (Ala). Comparison of the known six sequences of beta-thymosins together with the sequences reported here showed that the sequence similarity of the two beta-thymosins in trout (86%) is greater than that of the two beta-thymosins in mammalian species (74%) and that residues at 28 positions are identical in all beta-thymosins, the longer conserved segments located at positions 16-26 and 31-38.
Project description:A cDNA encoding a DNA-binding protein has been isolated by screening a mouse testicular expression cDNA library with a concatemer of a 12-bp putative protein-binding element present in the promoter of the testis-specific gene PGK-2. Sequence analysis of the isolated cDNA indicated the presence of an open reading frame that encodes a protein with two conserved DNA-binding motifs known as the high-mobility-group (HMG) boxes. Northern (RNA) blot analysis demonstrated that expression of the gene is restricted to the postpuberal testis. The DNA-binding activity and sequence specificity of the recombinant HMG protein were confirmed by DNA mobility shift assay using the initial concatemer of the PGK-2 promoter element as a probe as well as the wild-type or mutated versions of the 12-bp element within its natural sequence context. Immunocytochemical staining of adult testis sections with polyclonal antisera recognizing this recombinant HMG protein demonstrated that it is located predominantly in the nuclei of elongated spermatids at steps 9 and 10. These results suggest that this novel HMG box protein gene may be involved in the regulation of gene expression of the haploid male genome. The gene from which the cDNA was derived has been termed testis-specific HMG (tsHMG).
Project description:It is shown that Methyl Red can be used as an indicator dye that changes color in Escherichia coli culture as a result of time- and cell density-dependent bleaching by azoreductase produced by the bacteria. For cell cultures that are being used to express a recombinant protein, this phenomenon can be exploited to provide a simple visual cue that cell cultures have reached an appropriate growth phase for addition of an agent to induce protein expression, such as isopropyl thiogalactoside.
Project description:BACKGROUND:Purification of peptides offers unique challenges with respect to obtaining the desired process yield and selectivity. Lethal Toxin Neutralizing Factor (LTNF) is a peptide that is known to neutralize snake venom in mice when the peptide is preincubated with the venom prior to intravenous injection. A process for producing highly purified recombinant LTNF has been developed. The process has been modelled in SuperPro designer using laboratory data for a plant capable of producing 10 Kg of purified rLTNF. Economic analysis has been performed for manufacturing 3 ton of purified rLTNF. RESULTS:The process developed produces peptide in the form of concatemer that has been specifically designed to accumulate as insoluble inclusion bodies (IB) during expression in E. coli. A cation exchange chromatography step has been developed to capture the rLTNF concatemer at 140 g/L dynamic binding capacity. Further, the purified concatemer is cleaved completely into monomeric rLTNF using alpha-chymotrypsin enzyme. Finally, a reversed phase high performance liquid chromatography has been designed to purify rLTNF with a recovery of more than 90% and purity greater than 98%. The overall process recovery is 78±2% resulting in 3.36 g of purified product per batch. Techno-economic evaluation of the process has been performed to demonstrate its economic feasibility against currently marketed antivenom products. CONCLUSIONS:The developed process is able to produce purified rLTNF with 78±2% recovery. The study shows that recombinant technology can be used to produce rLTNF cost effectively and shows potential as a substitute for currently available antivenoms against snakebite.
Project description:As the group II LEA (late embryogenesis abundant) proteins, dehydrins (DHNs) play an important role in plant growth and development, as well as in response to abiotic or biotic stress challenges. In this study, a DHN gene named CsLEA11 was identified and characterized from Cucumis sativus. Sequence analysis of CsLEA11 showed that it is a Y3SK2-type DHN protein rich in hydrophilic amino acids. Expression analyses revealed that the transcription of CsLEA11 could be significantly induced by heat and cold stress. The recombinant plasmid was transformed into Escherichia coli BL21 and isopropy-β-D-thiogalactoside (IPTG) was used to induce recombinant E. coli to express CsLEA11 gene. Overexpression of CsLEA11 in E. coli enhanced cell viability and conferred tolerance to heat and cold stress. Furthermore, CsLEA11 protein could protect the activity of lactate dehydrogenase (LDH) under heat stress. Taken together, our data demonstrate that CsLEA11 might function in tolerance of cucumber to heat and cold stress.
Project description:Alicyclobacillus acidoterrestris, an acidophilic and thermophilic bacteria, is an important microbial resource for stress resistance genes screening. In this study, DnaK gene from A. acidoterrestris was subcloned to construct the recombinant plasmid pET28a-DnaK. The successful construction of the plasmid was verified by double-enzyme digestion and sequencing analysis. The recombinant plasmid was transformed into Escherichia coli BL21 and isopropy-?-D-thiogalactoside (IPTG) was used to induce recombinant E. coli to express DnaK gene. A 70 kD fusion protein was identified by SDS-PAGE, which suggested that DnaK gene from A. acidoterrestris was successfully expressed. The recombinant and wild BL21 were treated with high temperatures of 54, 56 and 58 °C at pH values of 5.0-7.0 to compare the effects of heterogeneous expression of the DnaK gene from A. acidoterrestris on the stress resistance. The experimental results showed that survival rate of recombinant BL21-DnaK has been improved considerably under heat and acid stresses in contrast with the wild BL21, and D-values of recombinant BL21 were 14.7-72% higher than that of wild BL21, which demonstrated that heterogeneous expression of DnaK gene from A. acidoterrestris could significantly enhance the resistance of host bacteria E. coli against heat and acid stresses.
Project description:Approaches based on genetic modification have been invaluable for investigating a wide array of biological processes, with gain- and loss-of-function approaches frequently used to investigate gene function. However, the presence of paralogues, and hence possible genetic compensation, for many genes necessitates the knockout (KO) of all paralogous genes in order to observe clear phenotypic change. CRISPR technology, the most recently described tool for gene editing, can generate KOs with unprecedented ease and speed and has been used in adult stem cell-derived organoids for single gene knockout, gene knock-in and gene correction. However, the simultaneous targeting of multiple genes in organoids by CRISPR technology has not previously been described. Here we describe a rapid, scalable and cost effective method for generating double knockouts in organoids. By concatemerizing multiple gRNA expression cassettes, we generated a 'gRNA concatemer vector'. Our method allows the rapid assembly of annealed synthetic DNA oligos into the final vector in a single step. This approach facilitates simultaneous delivery of multiple gRNAs to allow up to 4 gene KO in one step, or potentially to increase the efficiency of gene knockout by providing multiple gRNAs targeting one gene. As a proof of concept, we knocked out negative regulators of the Wnt pathway in small intestinal organoids, thereby removing their growth dependence on the exogenous Wnt enhancer, R-spondin1.
Project description:N(?)-terminal acetylation of peptides plays an important biological role but is rarely observed in prokaryotes. N(?)-terminal acetylated thymosin ?1 (T?1), 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 N(?)-terminal acetylation of recombinant prothymosin ? (ProT?) in E. coli. Here we present a method for production of N(?)-acetylated T?1 from recombinant ProT?. The recombinant ProT? was cleaved by human legumain expressed in Pichia pastoris to release T?1 in vitro. The N(?)-acetylated T?1 peptide was subsequently purified by reverse phase and cation exchange chromatography. Mass spectrometry indicated that the molecular mass of recombinant N(?)-acetylated T?1 was 3108.79 in, which is identical to the mass of N(?)-acetylated T?1 produced by total chemical synthesis. This mass corresponded to the nonacetylated T?1 mass with a 42 Da increment. The retention time of recombinant N(?)-acetylated T?1 and chemosynthetic N(?)-acetylated T?1 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 N(?)-acetylated T?1 and also will provide the basis for the preparation of recombinant acetylated peptides in E. coli.
Project description:Thymosin beta(4), its sulfoxide, and thymosin beta(10) were detected in whole saliva of human pre-term newborns by reversed-phase high performance chromatography coupled to electrospray ion-trap mass spectrometry.Despite high inter-individual variability, concentration of beta-thymosins increases with an inversely proportional trend to postmenstrual age (PMA: gestational age plus chronological age after birth) reaching a value more than twenty times higher than in adult whole saliva at 190 days (27 weeks) of PMA (thymosin beta(4) concentration: more than 2.0 micromol/L versus 0.1 micromol/L). On the other hand, the ratio between thymosin beta(4) and thymosin beta(10) exhibits a constant value of about 4 along all the range of PMA (190-550 days of PMA) examined. In order to investigate thymosin beta(4) origin and to better establish the trend of its production as a function of gestational age (GA), immunohistochemical analysis of major and minor salivary glands of different pre-term fetuses were carried out, starting from 84 days (12 weeks) of gestational age. Reactive granules were seen in all glands with a maximum of expression around 140-150 days of GA, even though with high inter- and intra-individual variability. In infants and adults reactive granules in acinar cells were not observed, but just a diffuse cytoplasmatic staining in ductal cells.This study outlines for the first time that salivary glands during foetal life express and secrete peptides such as beta-thymosins probably involved in the development of the oral cavity and its annexes. The secretion increases from about 12 weeks till to about 21 weeks of GA, subsequently it decreases, almost disappearing in the period of expected date of delivery, when the gland switches towards the secretion of adult specific salivary peptides. The switch observed may be an example of further secretion switches involving other exocrine and endocrine glands during foetal development.