Iranian Journal of Biotechnology
Volume:16 Issue: 1, Winter 2018

Rabies virus is a neurotropic virus that causes fatal, but, a preventable disease in mammals. Administration of rabies immunoglobulin (RIG) is essential for the post-exposure of the prophylaxis to prevent the disease. However, replacement of polyclonal RIGs with alternative monoclonal antibodies (MAbs) that are capable of neutralizing rabies virus has been recommended.
Here, we have investigated the transient expression of the full-size human MAb against rabies virus glycoprotein; the MAb SO57 in the tobacco plants using vacuum agro-infi ltration. Previously, stably transformed plants expressing the MAb have been reported.
In this study three vectors carrying the codon-optimized genes for the heavy or light chain and p19 silencing-suppressor were constructed. These vectors were co-infi ltrated into Nicotiana tabacum leaves and the transgenes were expressed.
Dot blot, Western blotting, ELISA, and in vitro neutralization assays of the plant extracts showed that the human MAb could assemble in tobacco leaves and was able to neutralize rabies virus.
This study is the fi rst report of transient expression of human MAb SO57 gene in tobacco plant within a few days after vacuum agro-infiltration.

The use of the desirability function approach combined with the response surface methodology (RSM), also called Desirability Optimization Methodology (DOM), has been successfully applied to solve medical, chemical, and technological questions. It is particularly effi cient for the determination of the optimal conditions in natural or industrial processes involving diff erent factors leading to the antagonist responses.
Surprisingly, DOM has never been applied to the research programs devoted to the study of plant responses to the complex environmental changes, and thus to biotechnological questions.
In this article, DOM is used to study the response of Datura stramonium hairy roots (HRs), obtained by genetic transformation with Agrobacterium rhizogenes A4 strain, subjected to the jasmonate treatments.
Antagonist eff ects on the growth and tropane alkaloid biosynthesis are confi rmed. With a limited number of experimental conditions, it is shown that 0.06 mM jasmonic acid (JA) applied for 24 h leads to an optimal compromise. Hyoscyamine levels increase by up to 290% after 24 h and this treatment does not significantly inhibit biomass growth.
It is thus demonstrated that the use of DOM can efficiently - with a minimized number of replicates - leads to the optimization of the biotechnological processes.

One of the main sunfl ower diseases is the white mold Sclerotinia sclerotiorum. The oxalic acid (OA), which is one of the main pathogenicity factors of this fungus, beside the direct toxicity on the host, has other functions such as the disruption of the cell wall and chelating out the calcium ions.
Regarding the importance of this disease, it is important to study the reactions of the plant against OA which is a nonspecifi c toxin of many other necrotrophic fungi.
In this study, two susceptible and moderately resistant sunfl ower lines were inoculated with OA and samples at the fi rst leaf stage were collected within the intervals of 2, 6, 12 and 24 hours post inoculation. The expression of five genes related to tricarboxylic acid cycle, including citrate synthase, fumarase, iso-citrate lyase, malate synthase and malate dehydrogenase was studied under OA treatment.
Two hours after the inoculation, no signifi cant change was observed in the expression of the fi ve studied genes in the moderately resistant line. The iso-citrate lyase gene, which is related to glyoxylate cycle (a variation of the tricarboxylic acid cycle), showed no change in the moderately resistant line; however, it showed an increase in the susceptible line. The increase in fumarase gene expression in moderately resistant line was higher than the susceptible line. The result showed the activation of glyoxylate cycle and destruction of fatty acids in the susceptible line.
Activation of glyoxylate cycle indicated induction of senescent symptoms by OA in susceptible line. Increasing in H2O2 leads to oxidative burst and cell death. Cell death has an apparent benefi t for development and growth of necrotrophic pathogens in the plant cells. The study of resistance mechanisms in response to the pathogen can be useful for breeding programs to provide lines with higher resistance to this pathogen.

Overexpression of known genes encoding key phosphate (Pi)-metabolizing enzymes, such as acid phosphatases (APases), is presumed to help plants with Pi availability and absorption as they are mostly exposed to suboptimal environmental conditions for this vital element.
In this study, the overexpression effect of AtPAP26, one of the main contributors in retrieving Pi from intracellular and extracellular compounds, was evaluated from various viewes in tobacco plant.
As a heterologous expression system, the encoding cDNA sequence of AtPAP26 was transferred into tobacco plants.
A high growth rate of the transgenic lines was observed which could be due to an increased APase activity, leading to the high total phosphorus as well as the free Pi content of the transgenic plants. Interestingly, a significant increased activity of the other APases was also noticed, indicating a networking among them. These were accompanied by less branched and short primary roots and a decreased lateral root numbers grown in Pi-starvation condition compared to the wild type seedlings. Besides, a delayed germination and dwarf phenotype indicates the possible reduction in gibberellic acid biosynthesis in the transgenic lines.
Such transgenic plants are of interest not only for increased yield but also for the reduced need for chemical fertilizers and removal of excessive Pi accumulation in soils as a consequence of fertilizers’ or poultry wastes’ over-usage.

Corynebacterium glutamicum (C. glutamicum) is a potential host for the secretory production of the heterologous proteins. However, to this date few secretion-type gene expression systems in C. glutamicum have been developed, which limit applications of C. glutamicum in a secretory production of the heterologous proteins.
In this study, a novel and efficient general secretory (Sec) pathway-dependent type gene expression system for the production of heterologous proteins was developed in C. glutamicum.
The synthesized cloning/expression cassette C was assembled into the basic E. coli-C. glutamicum shuttle vector pAU2, generating the Sec-dependent type gene expression vector pAU5. Subsequently, the applicability of the C. glutamicum/pAU5 system was tested using the α-amylase AmyE from Bacillus subtilis as a reporter protein.
The vector pAU5 was successfully constructed. The SDS-PAGE experiment showed the AmyE protein band could be observed in the original culture supernatant of the 14067/pAU5-amyE. The Western blotting experiment showed that the AmyE polypeptide could be detected in the culture supernatant of the 14067/pAU5-amyE, not in the cell lysate of 14067/pAU5-amyE. The α-amylase specific activity of the culture supernatant of 14067/pAU5-amyE was 103.24±7.14 U.mg-1 protein, while no α-amylase activity was detected in the cell homogenate supernatant of 14067/pAU5-amyE. These results demonstrate that the recombinant AmyE was efficiently expressed and completely secreted into the extracellular environmentin an active form in C. glutamicum/pAU5 system.
A novel efficient Sec-dependent type gene expression vector pAU5 was constructed in the C. glutamicum. The vector pAU5 employs the strong promoter tac-M for controlling a constitutive transcription of the target gene, the consensus ribosome binding site (RBS) sequence of C. glutamicum to ensure protein translation, and the efficient Sec-type cgR_2070 signal sequence to mediate protein secretion in the C. glutamicum. The C. glutamicum/pAU5 system is an efficient expression system for the secretory production of the heterologous proteins.

The increase of the protein expression via ribosomal manipulation is one of the suggested cellular mechanisms involved in EnBase fed-batch mode of cultivation. However, this system has not been implemented for cytotoxic proteins.
Here, the expression pattern of α-Luffin, a ribosome inactivation protein (RIP) with an innate toxicity, was investigated in EnBase system and the effect of low temperature cultivation on the increase of α-Luffin solubility was determined.
The encoding cDNA for mature α-Luffin was synthesized and subcloned into pET28a plasmid under the control of T7 promoter. The E. coli expression yield in EnBase® Flo fed-batch system was compared with traditional batch mode at two temperatures: 25 °C and 30 °C. Sampling was performed at several time intervals and solubility of recombinant-protein was checked on SDS-PAGE in pellet and supernatant samples. The purification of recombinant protein was performed by Ni-NTA column.
In fed-batch cultivation mode, the early incubation time was desirable at 30 °C whereas the maximum amount of soluble α-Luffin was achieved from the extended protein synthesis period (12 and 24h post induction) at 25 °C.
Our founding showed that EnBase had a greater efficacy in producing higher soluble protein ratios compared to batch cultivation growth rate, however for cytotoxic proteins, incubation temperature and time need to be optimized. Owing to the advantages of natural toxins from RIP family for producing anticancer immune-conjugates, well optimization of this protein expression is of importance regarding industrial aspects. The optimized condition proposed here is promising in terms of large scale soluble production of α-Luffin without the need for refolding.

Xanthomonas campestris is a biopolymer producing gram negative bacterium. Production of xanthan biopolymer can be affected by different extrinsic factors as well as surfactants. Hitherto, effects of nonionic surfactants on xanthan production have been studied in a limited number of articles.
In the present study, nonionic surfactants were used to pursue their effects on improvement of xanthan production. Moreover, a number of cellular consequences upon the treatment were investigated with impacts on gum production.
Effects of different nonionic surfactants (Tween 20, Tween 80 and Triton X100) on xanthan production and Xanthomonas campestris cells were assessed by ultramicroscopy (SEM), changes in culture turbidity, leakage of sugars and ATP, and quality of xanthan (i.e. pyruvate content and determination of polymer molecular weight).
The nonionic surfactant Tween 20 increased ATP (3.2 folds) and sugar leakage (3.1 folds). Furthermore, they caused cell shape alteration. Tween 80 improved both xanthan production (11 g.L-1) and viscosity of the product (1368 cP), while the total biomass remained unchanged (2.2 g.L-1). Molecular weight of xanthan was enhanced (from 23 to 59 million Da). Toxic effect of 5% (v/v) Triton X 100 decreased the turbidity of culture to 120 NTU and total biomass was diminished to 1 g.L-1. Tween 20 caused the loss of ATP and sugar leakage and led to lower xanthan production. It had no effect on biomass content.
In general, amounts of surfactants that bacterial cells can tolerate seem to be helpful in substrate and metabolite transportation, and enzyme activities involved in xanthan biosynthesis and release. Surfactants induce harsh damages to cell barriers, preventing the growth and adversely affecting quantity and quality of xanthan gum.

Although advantages of immobilization of cells through entrapment in calcium alginate gel beads have already been demonstrated, nevertheless, instability of the beads and the mass transfer limitations remain as the major challenges.
The objective of the present study was to increase the stability, porosity (reduce mass transfer limitation), and cell immobilization capacity of calcium alginate gel beads.
Sodium alginate was mixed with various concentrations of the starch or sugar and gelled in 2% calcium chloride solution. During the gelling and curing, the starch or sugar leached out of the beads and created micro-pores.
Micro-porous beads prepared with starch were more stable and had higher immobilization capacity than those prepared with sugar. After 24 hours of incubation (curing) of the micro-porous beads prepared with starch in calcium alginate, the solubilization time in citrate buffer was 93 minutes compared to 41 minutes for the control beads (without starch). The compressive strength of the micro-porous beads was also higher (5.62 Mpa) than that of the control beads (5.54 Mpa). The optimal starch concentration for cell immobilization was 0.4%. With this starch concentration, the immobilized Bacillus subtilis and Saccharomyces cerevisiae cell densities were 5.6 × 109 and 1.2 × 108 cells/beads, respectively. These values were 36.5% and 74% higher than the value obtained for the control beads. This method of immobilization resulted in more uniform cell distribution.
Addition of starch to the sodium alginate solution before gelation in calcium chloride solution increased the stability of the beads, increased the immobilized cell density, and resulted in a more uniform cell distribution in the beads.

Coagulation Factor VII is a vitamin K-dependent serine protease which has a pivotal role in the initiation of the coagulation cascade. The congenital Factor VII deficiency is a recessive hemorrhagic disorder that occurs due to mutations of F7 gene. In the present study C91S (p.C91S) substitution was detected in a patient with FVII deficiency. This mutation has not been characterized by a functional study.
In this study, we aimed to evaluate the impact of C91S substitution on factor VII expression and function.
The F7 complete cDNA was isolated from HepG2 cell line and inserted into the pcDNA3.1 mammalian expression vector. The desired mutation was generated by the site-directed mutagenesis and the wild-type and mutated constructs were transfected into CHO-K1 cells. The protein activity and antigen level (antigen concentration) were validated in the culture medium and cell lysate of the transiently transformed cells. An immunocytochemistry procedure was also performed to evaluate the intracellular localization of the mutated and the wild-type FVII, as well.
The present in vitro study has demonstrated that C91S antigen expression was increased in the transfected CHO-K1 cells compared to the wild-type (WT) protein. Despite an increased protein secretion, the factor VII coagulant activity was diminished following C91S substitution when it was assessed by a standard one-stage analysis. In addition, the immunocytochemistry procedure revealed that there was no difference in the intracellular localization of the C91S mutated FVII compared to the WT protein.
Our results present that C91S mutation has an effect on the coagulation activity, secretion, biosynthesis, and probably folding of the FVII leading to the FVII deficiency.

Tumor necrosis factor- a (TNF-α) is a cytokine that was identified as a factor with a wide range of proinflammatory activities. The expression of bovine TNF-α in mammary tissue during pregnancy seems to have a role in development of the corresponding glands.
Single nucleotide polymorphisms (SNPs) were defined in 3′-flanking region of bovine TNF-α in cattle. Moreover, its association with performance traits in Holstein dairy cattle was evaluated.
The 3′-flanking region of TNF-α was screened by single strand conformation polymorphism (SSCP) and DNA sequencing in Holstein cattle breed. SAS statistical software was used to analyze the relationship between different genotypes of amplified fragment with milk production traits (daily milk yield, fat and protein percentage) and somatic cell score (SCS).
A total of 6 distinct SSCP patterns were observed. It was further revealed to be 3 novel SNPs. Statistical analysis revealed that different haplotypes of amplified fragment in the TNF-α 3′-region had a significant effect on average daily milk production (p The association identified in the 3′-flanking region of TNF-α may have potential to serve as candidate genetic marker for genomic selection in dairy cattle.