A PCR-directed cell-free approach to optimize protein expression using diverse fusion tags

N-terminal fusion tags that enhance translation initiation or protein solubility are often used to facilitate protein overexpression. As the optimal tag for a given target protein cannot be predicted a priori, valuable time can be lost in cloning and manipulating the corresponding gene to generate different fusion constructs for expression analysis. We have developed a cell-free strategy that consolidates these steps, enabling the utility of a panel of nine fusion-tags to be determined within one to two days. This approach exploits the fact that PCR-amplified DNA can be used as a template for cell-free protein synthesis. Overlap/extension PCR using the TEV protease site as the overlap region allows the fusion of different T7 promoter (T7p)-tag-TEV DNA fragments with a TEV-gene-T7 terminator (T7ter) fragment. For tag sequences where the TEV site is not compatible, a short C?G? repeat (CGr) sequence can be used as the overlap region. The resulting T7p-tag-TEV-gene-T7ter constructs are then used as templates for PCR-directed cell-free protein synthesis to identify which tag-TEV-gene fusion protein produces the highest amount of soluble protein. We have successfully applied this approach to the overexpression of the Adiponectin hypervariable domain (AHD). Five of the nine N-terminal fusion tags tested enabled the synthesis of soluble recombinant protein. The best of these was the Peptidyl-prolylcis-trans isomerise B (PpiB) fusion tag which produces 1mg/ml amounts of soluble fusion protein. PpiB is an example of a new class of fusion tag known as the "stress-responsive proteins". Our results suggest that this cell-free fusion-tag expression screen facilitates the rapid identification of suitable fusion-tags that overcome issues such as poor expression and insolubility, often encountered using conventional approaches.     

<Emphasis Type="Italic">Lactococcus lactis</Emphasis> M4, a potential host for the expression of heterologous proteins

Background  

Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.     

Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb)

Background:Cytoglobin (Cygb) is a relatively newly identified globin protein that acts as an oxygen transporter in tissues like hemoglobin (Hb) in erythrocytes and myoglobin (Mb) in muscles. The natural oxidation of the Fe²⁺ ion in its heme group into metglobin (globin-Fe³⁺) made the loses of oxygen binding functions. It is known metHb and metMb can be reduced enzymatically using diaphorase or cyb5r3. However, metCygb reductase had not been previously identified. This study aims to analyze the reducing activity of bovine diaphorase on metCygb.Methods:Diaphorase was isolated from bovine erythrocyte and purified using gel filtration and cationic-exchanger chromatography. Its purity was verified by SDS-PAGE and western blot (WB). The metCygb was obtained from Cygb oxidation with potassium ferrocyanide and its reducing activity was determined by spectroscopy.Results:The diaphorase (MW=30.09 kDa) was purified 10.77-fold from crude enzyme with specific activity against metHb 8.479 U/mg. The purity was confirmed by WB using primary antibody anti-cyb5r3. The purified enzyme reduced metCygb at 0.785 µgmin^-1, which was 13.7 times less than the Vmax of metHb.DiscussionIn conclusion, the purified diaphorase from bovine erythrocytes did not significantly reduce metCygb rather than metHb, a natural substrate in cells.Key Words: Bovine Erythrocyte, Cytochrome B5 Reductase, Diaphorase, Metcytoglobin, Reduction     

Plasma Protein Profile of Lactating Women from Two Primary Health Centers in Jakarta,Indonesia

Background:Plasma protein profile test is a potential laboratory method to assess nutritional status especially albumin and globulins levels which reflects protein adequacy. The purpose of this study is to evaluate plasma protein profile of lactating women from two primary health centers in Jakarta.Methods:A cross-sectional study was conducted involving lactating women attending routine maternal examinations in two public primary health centers in Jakarta, Indonesia. The mother’s plasma total protein, albumin, globulin, and immunoglobulin levels were measured.Results:Sixty lactating women were recruited, mostly were 28 years old, slightly overweight, bearing two children, and their recent children were 2 months old. The mean total protein level was 8.13 g/dl, albumin 5.00 g/dl, globulin 3.18 g/dl, albumin: globulin ratio 1.558, mean total IgG level of 1255.98 and mean total IgM level of 135.819. All the measured plasma protein parameters were shown to be not correlated with maternal age, maternal BMI, or maternal parity.DiscussionThe plasma total protein, albumin, globulin, as well as total IgG and IgM level of lactating women in Jakarta were within normal range. These biochemical parameters were shown to be not correlated with anthropometrical data such as maternal age and BMI. The small and relatively homogenous samples were supposed to be the cause of such findings.Conclusion:The plasma protein profile of lactating women in Jakarta was adequate. Further studies are required to evaluate the eligibility of plasma protein profile as biochemical parameter of nutritional status in lactating women.Key Words: Blood protein, lactation, protein, albumin/globulin, immunoglobulin M/G     

A two-gene strategy increases iron and zinc concentrations in wheat flour,improving mineral bioaccessibility

Dietary deficiencies of iron and zinc cause human malnutrition that can be mitigated by biofortified staple crops. Conventional breeding approaches to increase grain mineral concentrations in wheat (Triticum aestivum L.) have had only limited success, and our understanding of the genetic and physiological barriers to altering this trait is incomplete. Here we demonstrate that a transgenic approach combining endosperm-specific expression of the wheat VACUOLAR IRON TRANSPORTER gene TaVIT2-D with constitutive expression of the rice (Oryza sativa) NICOTIANAMINE SYNTHASE gene OsNAS2 significantly increases the total concentration of zinc and relocates iron to white-flour fractions. In two distinct bread wheat cultivars, we show that the so called VIT-NAS construct led to a two-fold increase in zinc in wholemeal flour, to ∼50 µg g⁻¹. Total iron was not significantly increased, but redistribution within the grain resulted in a three-fold increase in iron in highly pure, roller-milled white flour, to ∼25 µg g⁻¹. Interestingly, expression of OsNAS2 partially restored iron translocation to the aleurone, which is iron depleted in grain overexpressing TaVIT2 alone. A greater than three-fold increase in the level of the natural plant metal chelator nicotianamine in the grain of VIT-NAS lines corresponded with improved iron and zinc bioaccessibility in white flour. The growth of VIT-NAS plants in the greenhouse was indistinguishable from untransformed controls. Our results provide insights into mineral translocation and distribution in wheat grain and demonstrate that the individual and combined effects of the two transgenes can enhance the nutritional quality of wheat beyond what is possible by conventional breeding.

Targeted expression of a vacuolar iron transporter and increased nicotianamine levels have combinatorial effects on iron and zinc levels and their distribution in wheat grain.     

A specific secretion system mediates PPE41 transport in pathogenic mycobacteria

Mycobacterial genomes contain two unique gene families, the so-called PE and PPE gene families, which are highly expanded in the pathogenic members of this genus. Here we report that one of the PPE proteins, i.e. PPE41, is secreted by pathogenic mycobacteria, both in culture and in infected macrophages. As PPE41 lacks a signal sequence a dedicated secretion system must be involved. A single gene was identified in Mycobacterium marinum that showed strongly reduced PPE41 secretion. This gene was located in a gene cluster whose predicted proteins encode components of an ESAT-6-like secretion system. This cluster, designated ESX-5, is conserved in various pathogenic mycobacteria, but not in the saprophytic species Mycobacterium smegmatis. Therefore, different regions of this cluster were introduced in M. smegmatis. Only introduction of the complete ESX-5 locus resulted in efficient secretion of heterologously expressed PPE41. This PPE secretion system is also involved in the virulence of pathogenic mycobacteria, as the ESX-5 mutant of M. marinum was affected in spreading to uninfected macrophages.     

Regulation of apoptotic effects by erythrocarpine E, a cytotoxic limonoid from Chisocheton erythrocarpus in HSC-4 human oral cancer cells

The aim of this study was to determine the cytotoxic and apoptotic effects of erythrocarpine E (CEB4), a limonoid extracted from Chisocheton erythrocarpus on human oral squamous cell carcinoma. Based on preliminary dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, CEB4 treated HSC-4 cells demonstrated a cytotoxic effect and inhibited cell proliferation in a time and dose dependent manner with an IC(50) value of 4.0±1.9 μM within 24 h of treatment. CEB4 was also found to have minimal cytotoxic effects on the normal cell line, NHBE with cell viability levels maintained above 80% upon treatment. Annexin V-fluorescein isothiocyanate (FITC), poly-ADP ribose polymerase (PARP) cleavage and DNA fragmentation assay results showed that CEB4 induces apoptosis mediated cell death. Western blotting results demonstrated that the induction of apoptosis by CEB4 appeared to be mediated through regulation of the p53 signalling pathway as there was an increase in p53 phosphorylation levels. CEB4 was also found to up-regulate the pro-apoptotic protein, Bax, while down-regulating the anti-apoptotic protein, Bcl-2, suggesting the involvement of the intrinsic mitochondrial pathway. Reduced levels of initiator procaspase-9 and executioner caspase-3 zymogen were also observed following CEB4 exposure, hence indicating the involvement of cytochrome c mediated apoptosis. These results demonstrate the cytotoxic and apoptotic ability of erythrocarpine E, and suggest its potential development as a cancer chemopreventive agent.