Optimizing primary recovery and refolding of human interferon-beta from escherichia coli inclusion bodies

The refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. The purpose of this study was optimization of recombinant human interferon-beta purification in order to achieve higher efficiency, yield, and a product with a better and more suitable biological activity. Triton X-100 and sodium deoxycholate were used to wash the recombinant human interferon-beta inclusion bodies prior to solubilization. The inclusion bodies solubilization process was performed by denaturants and reducing agents; guanidine-hydrochloride, urea, beta-mercaptoethanol and dithiothritol. The best recovery was obtained in the presence of 0.5% TritonX-100 [v/v]. Low concentrations of urea only gave a marginal improvement on the refolding of recombinant human interferon- beta. Successful refolding was achieved by gradient elution [decreasing the guanidine-hydrochloride concentration] in the presence of L-arginine. Partial purification was also achieved continuously, and recombinant human interferon-p was recovered with 93.5% purity. The interferon prepared in this project was biologically active and inhibited the replication of vesicular stomatitis virus in Hela cells, when compared to the standard interferon. In this research, the best recovery of inclusion bodies was found at a concentration 0.5 M of Triton X-100, the maximum efficiency of solubility was found in pH 10.5 and the maximum efficiency of refolding was achieved by final buffer containing 2M urea and 0.6 M L-Arg

Human Bhsp90 as adjuvant in HCV recombinant vaccine

There are more than 350 million individuals with hepatitis C in the world. One of the important problems in vaccine project is development of effective and suitable adjuvant in human vaccines. At present research we applied human BHsp90 protein as an adjuvant in recombinant HCV vaccine design. The thermal vector of pGP1-2 was used for human heat shock protein 90 expression. This protein injected to BalbC mice as an adjuvant together with recombinant protein of HCV core. The combination of these proteins was used and we evaluated the humoral and cellular immunity and the cytokine secretion of inguinal and popliteal lymph nodes lymphocytes were analyzed in vitro and ex vivo conditions. So the combination of Core protein together with hsp90 induced total IgG and IgG2a secretion. The spleen lymphocytes proliferation were increased equal to serum IgG2a level that was constant in second time bleeding with significant different to complexes with freund's adjuvant. At first IL-4 and IL-5 cytokines were increased, after one week it decreased. Production of IL-4 showed there was no hypersensitivity reaction after vaccine injection

Organelle isolation for proteomics: mitochondria from peripheral blood mononuclear cells

Mitochondria play key roles in many cell functions including energy production, fatty acid metabolism, pyrimidine biosynthesis, calcium homeostasis, and aging. They also regulate crucial signaling cascades such as apoptosis and oxidative stress. The proteome is often used to investigate the functional correlations on protein levels. Based upon the human, genome there is estimated 2000 to 2500 associated mitochondrial proteins, however, just over 600-800 have been identified at the protein level. For this reason, mitochondria contain a great number of proteins that have yet to be identified and characterized. The identification of these proteins can help in discovery of biological process. This protocol focuses on step-by-step procedure of mitochondrial proteome extraction from peripheral blood mononuclear cell [PBMC] mitochondria. The isolation and preparation procedures described here require 6 hours approximately

Cloning and expression of gumboro VP2 antigen in Aspergillus niger

Infectious Bursal Disease Virus [IBDV] causes a highly immunosuppressive disease in chickens and is a pathogen of major economic importance to the poultry industry worldwide. The VP2 protein is the major host-protective immunogen of IBDV and has been considered as a potential subunit vaccine against the disease. VP2 coding sequence was cloned in an inducible fungal vector and the protein was expressed in Aspergillus niger [A. niger]. Aiming at a high level of expression, a multicopy AMA1-pyrG-based episomal construct driven by a strong inducible promoter, glaA, was prepared and used in transformation of A. niger pyrG[-] protoplasts. SDS-PAGE and western blot analysis was carried out to confirm the expression of the protein. A number of pyrG[+] positive transformants were isolated and the presence of expression cassette was confirmed. Western blot analysis of one of these recombinant strains using monospecific anti-VP2 antibodies demonstrated the successful expression of the protein. The recombinant protein was also detected by serum obtained from immunized chicken. In the present study, we have generated a recombinant A. niger strain expressing VP2 protein intracellulary. This recombinant strain of A. niger may have potential applications in oral vaccination against IBDV in poultry industry

Periplasmic expression of Bacillus thermocatenulatus lipase in Escherichia coli in presence of different signal sequences

Efforts to express lipase in the periplasmic space of Escherichia coli have so far been unsuccessful and most of the expressed recombinant lipases accumulate in the insoluble cell fraction. To evaluate the role of native and heterologous signal peptides in translocation of the lipase across the inner membrane of E. coli, the lipase gene [btl2] was cloned downstream of the native Bacillus signal peptide and also in fusion with the pelB, ansB and ansB/asp signal peptides. For this purpose, four recombinant expression vectors [pYRKP.P, pYRKP.N, pYRKP. A and pYRKP.AA] were constructed and expressed in E. coli. Osmotic shock analysis showed that recombinant lipase was overexpressed as inclusion bodies in E. coli. The lipase inclusion bodies were subsequently solublized, refolded and purified using single column ion-exchange chromatography. To evaluate localization of lipase in the cell, the purified lipases were subjected to capillary isoelectric focusing and tandem mass spectrometry. Results showed that all signal peptides were able to direct the lipase from the cytoplasm into the periplasmic space of E. coli, because the periplasmic space of E. coli is not suitable for lipase folding, the translocated lipase aggregates in this space as inclusion bodies

Bacterial expression and purification of C1C2 domain of human factor VIII

The cDNA from the mycoparasitic fungus Trichoderma atroviride PTCC5220 encoding a 42 kDa chitinase [Chit42] was isolated. The nucleotide sequence of the cDNA fragment as having a 1263 bp open reading frame that encodes a 421 amino acid polypeptide, and a high homology was found with other reported Chit42 belonging to the Trichoderma sp. The 22 amino acid N-terminal sequence is a putative signal pep-tide for the possible secretion of the protein. The protein has been expressed and secreted as a mature form in Escherichia coll BL21[DE3] using the pelB leader sequence. The E. coll strain expressed Chit42 in an active form and secreted the protein into the medium. This recombinant chitinase has been shown to have inhibitory activity on mycelial growth and also, lytic activity on the cell wall of Rhizoctonia solani [AG2-2], causal agent of root rot in sugar beet in vitro. Expressed chitinase was optimally active at pH 5 and at 40°C. It is thermally stable at 60°C for more than 120 min at pH5

Transient expression of human growth hormone in potato [Solanum tuberosum], tobacco [Nicotiana tobacum] and lettuce [Lactuca sativa] leaves by agroinfiltration

The use of polymorphic markers in breeding programmes could make selection more accurate and efficient. A total of 324 individuals from six Iranian cattle populations [Sarabi, Golpayegani, Sistani, Taleshi, Mazandarani, Dashtiyari], F1 Golpayegani Brown Swiss and Iranian buffalo populations were genotyped for the Pit-1 HinfI and leptin Sau3AI polymorphisms by the polymerase chain reaction and restriction fragment length polymorphism [PCR-RFLP]. The genotype and gene frequencies for each breed were determined and shown to be quite variable among the breeds. The highest frequencies of allele B for the leptin gene and allele A for the Pit-1 gene were found in Dashtiyari and Sistani cattle, respectively. According to our results, the highest AB genotype frequencies were found in the Taleshi and F1 Golpayegani x Brown Swiss cross for the leptin and Pit-1 genes, respectively. These allele frequencies were comparable to previously published data on exotic breeds. The highest and lowest heterozygosities were found in Taleshi and Dashtiyari cattle for the leptin gene and in F1 Golpayegani x Brown Swiss cross and Sistani cattle for the Pit-1 gene, respectively. These values indicated the presence of low variation for these genes in the studied populations. The possible association between molecular polymorphisms within these candidate genes and economic traits for the studied populations should be further investigated