Crucial role of non-hydrophobic residues in H-region signal peptide on secretory production of l-asparaginase II in Escherichia coli.

Protein secretion into the periplasmic space requires several interactions between the amino-terminal signal sequence of the protein and secretion machinery components. Therefore, modification of the components of amino-terminal sequence can be used as a powerful strategy to improve secretion efficiency. The hydrophobic region is an important domain for signal peptide function due to interaction with different components of the secretion apparatus. In this study, to evaluate the effect of hydrophobicity level and secondary structure of the h-domain signal peptide on the secretion efficiency, a series of missense mutations were constructed in the hydrophobic domain of the l-asparaginase II signal peptide. The h-region hydrophobicity level of mutants G8L, T16L, and G8L/T16L was increased compared with the wild-type. In addition, the amino acid glycine as a helix-breaker residue was substituted with leucine (G8L), forming a stable and extended α-helix structure in the h-domain. The effect of introducing an aromatic residue in this region was also investigated by mutant G8F. Our mutagenesis studies showed that increasing the hydrophobicity levels, extending the α-helical conformation, and the introduction of an aromatic residue within the h-region signal sequence reduced the secretion level of asparaginase. These results imply a vital role of non-hydrophobic residues in the H-region.

Optimizing the Expression and Solubilization of an E. coli-Produced Leukemia Inhibitory Factor for Anti-LIF Antibody Production and Use Thereof for Contraception in Mice.

Leukemia inhibitory factor (LIF) is an essential cytokine for blastocyst implantation. This study evaluated the effect of LIF inhibition on the blockage of embryo implantation. A truncated mouse LIF (tmLIF) was designed and expressed in E. coli. The protein expression was optimized using different culture media and inducers. To block pregnancy, the mice were immunized by the purified protein via maternal injection of the protein or in utero injection of the anti-LIF serum. The expression of implantation-relevant genes was quantified in the uterine tissue. The results showed that the protein was expressed in aggregated form in E. coli. The highest yield of protein was produced in the M9 medium. The insoluble protein was completely dissociated by SDS and 2-ME combination, but not by urea. The maternal immunization reduced the number of offspring, but not significantly. Instead, in utero injection of the anti-LIF serum prevented the blastocyst implantation. Gene expression analyses showed decrease of Jam2, Msx1and HB-EGF genes and increase of Muc1 gene as the result of intrauterine administration of the anti-LIF serums. In conclusion, SDS-mediated solubilization of inclusion bodies was compatible with in vivo studies. The intrauterine administration of anti-LIF serum could prevent mouse pregnancy. This indicates that in utero application of LIF antibodies might be used as a contraceptive.

Cohnella amylopullulanases: Biochemical characterization of two recombinant thermophilic enzymes.

Some industries require newer, more efficient recombinant enzymes to accelerate their ongoing biochemical reactions in harsh environments with less replenishment. Thus, the search for native enzymes from extremophiles that are suitable for use under industrial conditions is a permanent challenge for R & D departments. Here and toward such discoveries, two sequences homologous to amylopullulanases (EC 3.2.1.41, GH57) from an endogenous Cohnella sp., [Coh00831 (KP335161; 1998 bp) and Coh01133 (KP335160: 3678 bp)] were identified. The genes were heterologously expressed in E. coli to both determine their type and further characterize their properties. The isolated DNA was PCR amplified with gene specific primers and cloned in pET28a, and the recombinant proteins were expressed in E. coli BL21 (DE3). The temperatures and pH optima of purified recombinants Coh 01133 and Coh 00831 enzymes were 70°C and 8, and 60°C and 6, respectively. These enzymes are stable more than 90% in 60°C and 50°C for 90 min respectively. The major reactions released sugars which could be fractionated by HPLC analysis, from soluble starch were mainly maltose (G2), maltotriose (G3) and maltotetraose (G4). The enzymes hydrolyzed pullulan to maltotriose (G3) only. Enzyme activities for both proteins were improved in the availability of Mn2+, Ba2+, Ca2+, and Mg2+ and reduced in the presence of Fe2+, Li2+, Na2+, Triton X100 and urea. Moreover, Co2+, K+, and Cu2+ had a negative effect only on Coh 01133 enzyme.

Enzymatic production of biodiesel from microalgal oil using ethyl acetate as an acyl acceptor.

Microalgae have become an important source of biomass for biodiesel production. In enzymatic transesterification reaction, the enzyme activity is decreased in presence of alcohols. The use of different acyl acceptors such as methyl/ethyl acetate is suggested as an alternative and effective way to overcome this problem. In this study, ethyl acetate was used for the first time in the enzymatic production of biodiesel by using microalga, Chlorella vulgaris, as a triglyceride source. Enzymatic conversion of such fatty acids to biodiesel was catalyzed by Novozym 435 as an efficient immobilized lipase which is extensively used in biodiesel production. The best conversion yield of 66.71% was obtained at the ethyl acetate to oil molar ratio of 13:1 and Novozym 435 concentration of 40%, based on the amount of oil, and a time period of 72 h at 40℃. The results showed that ethyl acetate have no adverse effect on lipase activity and the biodiesel amount was not decreased even after seven transesterification cycles, so ethyl acetate has a great potential to be substituted for short-chain alcohols in transesterification reaction.

Esophageal cancer alters the expression of nuclear pore complex binding protein Hsc70 and eIF5A-1.

Nuclear pore complex (NPC) is the only corridor for macromolecules exchange between nucleus and cytoplasm. NPC and its components, nucleoporins, play important role in the diverse physiological processes including macromolecule exchange, chromosome segregation, apoptosis and gene expression. Recent reports also suggest involvement of nucleoporins in carcinogenesis. Applying proteomics, we analyzed expression pattern of the NPC components in a newly established esophageal cancer cell line from Persia (Iran), the high-risk region for esophageal cancer. Our results indicate overexpression of Hsc70 and downregulation of subunit alpha type-3 of proteasome, calpain small subunit 1, and eIF5A-1. Among these proteins, Hsc70 and eIF5A-1 are in direct interaction with NPC and involved in the nucleocytoplasmic exchange. Hsc70 plays a critical role as a chaperone in the formation of a cargo-receptor complex in nucleocytoplasmic transport. On the other hand, it is an NPC-associated protein that binds to nucleoporins and contributes in recycling of the nucleocytoplasmic transport receptors in mammals and affects transport of proteins between nucleus and cytoplasm. The other nuclear pore interacting protein: eIF5A-1 binds to the several nucleoporins and participates in nucleocytoplasmic transport. Altered expression of Hsc70 and eIF5A-1 may cause defects in nucleocytoplasmic transport and play a role in esophageal carcinogenesis.

Proteomics of a new esophageal cancer cell line established from Persian patient.

Although the highest incidence of esophageal squamous cell carcinoma (ESCC) has repeatedly been reported from Persia (Iran), nevertheless the so far proteomic published reports were limited to one study on tissue specimens. Here we report the proteome of a newly established cell line from Persian ESCC patients and compare it with the normal primary cell proteome. Among polypeptides, whose expression was different in cell line sixteen polypeptides were identified by MALDI/TOF/TOF spectrometry. S100-A8 protein, annexin A1, annexin A2, regulatory subunit of calpain, subunit alpha type-3 of proteasome and glutamate dehydrogenase 1 were proteins down-regulated in cell line while peroxiredoxin-5, non-muscle myosin light polypeptide 6, keratin 1, annexin A4, keratin 8, tropomyosin 3, stress-induced-phosphoprotein 1 and albumin were found to be subject of up-regulation in cell line compared to the primary normal cells. The proteomic results were further verified by western blotting and RT-PCR on annexin A1 and keratin 8. In addition, among the aforementioned proteins, glutamate dehydrogenase 1, regulatory subunit of calpain, subunit alpha of type-3 proteasome and annexin A4 are proteins whose deregulation in ESCC is reported for the first time by this study.

Identification of squamous cell carcinoma associated proteins by proteomics and loss of beta tropomyosin expression in esophageal cancer.

AIM: To assess the proteome of normal versus tumor tissue in squamous cell carcinoma of the esophagus (SCCE) in Iranian patients and compare our results with former reports by using proteomics. METHODS: Protein was extracted from normal and tumor tissues. Two dimensional electrophoresis was carried out and spots with differential expression were identified with mass spectrometry. RNA extraction and RT-PCR along with immunodetection were performed. RESULTS: Fourteen proteins were found whose expression levels differed in tumor compared to normal tissues. Mass spectrometric analysis resulted in the identification of beta-tropomyosin (TMbeta), myosin light chain 2 (and its isoform), myosin regulatory light chain 2, peroxyredoxin 2, annexin I and an unknown polypeptide as the down regulated polypeptides in tumor tissue. Heat shock protein 70 (HSP70), TPM4-ALK fusion oncoprotein 2, myosin light polypeptide 6, keratin I, GH16431p and calreticulin were the up-regulated polypeptides found in tumor tissue. Several of these proteins, such as TMbeta, HSP70, annexin I, calreticulin, TPM4-ALK and isoforms of myosins, have been well recognized in tumorigenesis of esophageal or other types of cancers. CONCLUSION: Our study not only supports the involvement of some of the formerly reported proteins in SCCE but also introduces additional proteins found to be lost in SCCE, including TMbeta.