A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli

Adiponectin is one of the most bioactive substances secreted by adipose tissue and is involved in the protection against metabolic syndrome, artherosclerosis and type II diabetes. Research into the use of adiponectin as a promising drug for metabolic syndromes requires production of this hormone in high quantities considering its molecular isoforms. The objective of this study is to produce recombinant human adiponectin by Pichia pastoris (P-ADP) as a cheap and convenient eukaryotic expression system for potential application in pharmaceutical therapy. For comparison, adiponectin was also expressed using the Escherichia coli (E-ADP) expression system. Adiponectin was constructed by overlap-extension PCR, and cloned in standard cloning vector and hosts. Recombinant expression vectors were cloned in the P. pastoris and E. coli host strains, respectively. SDS-PAGE and western blotting were used to detect and analyse expressed recombinant protein in both systems. Adiponectin was purified by affinity chromatography and quantified using the Bradford Assay. The results of this study indicated that P-ADP quantity (0.111 mg/mL) was higher than that of E-ADP (0.04 mg/mL) and both were produced in soluble form. However, P-ADP was able to form high molecular weights of adiponectin molecules, whilst E-ADP was not able to form isoforms higher than trimer. In addition, P-ADP was more active in lowering blood glucose compared with E-ADP. The two types of proteins were equally efficient and significantly decreased blood triglyceride and increased high density lipoprotein. We conclude that P. pastoris is able to produce high quantity of bioactive adiponectin for potential use in treatment of metabolic syndromes

RT-PCR amplification and cloning of partial DNA sequence coding for oil palm (Elaeis oleifera) phytoene synthase gene

The potential health benefits of carotenoids as anti cancer and antioxidant agents have recently been demonstrated. Oil palm, Elaeis oleifera in particular, is known to be the richest natural source for carotene. However, the species has not been commercially exploited due to its extremely low oil yield. The current work describes the isolation of a cDNA clone coding for phytoene synthase (psy) from E. oleifera by RT-PCR amplification. A pair of psy gene specific primers was successfully used to amplify a 899 bp fragment that codes for a partial length (300 amino acids) of oil palm psy. The DNA and amino acid sequences were shown to share a high level of identity to phytoene synthase from other plants at about 83%. Further analysis also showed the presence of conserved aspartate-rich catalytic domains within the clone. Work was also carried out to obtain the expression pattern of oil palm psy in developing fruits by real-time PCR analysis. Results indicated that the gene is highly regulated during the course of oil palm fruit development. The pattern of psy expression was shown to be well correlated to the accumulation of lutein in the young mesocarp and α- and β-carotenes in the older tissues. This observation demonstrated that oil palm psy was highly regulated for tissue development and accumulation of carotenes for storage

Food and Feeding Habits of Fishes in Brunei Bay, Malaysia

The study of the food and feeding habits of fishes is crucial in understanding their ecology. Food and feeding habits of the 30 fish species belonging to 22 families from Bukit Sari and Awat-awat of Lawas in the Bay of Brunei were studied on 11th February 2020 and 12th February 2020 respectively. Samples were collected using “Kabat” nets, casting nets, and seine nets. The dietary components of each species were studied and expressed as a percentage of numerical composition (N), percentage of weight composition (W), and percentage of frequency of occurrence (F). Diet compositions of the species were estimated using the Index of Relative Importance (%IRI) and trophic level (TROPHj). The major food and their Index of Relative Importance (%IRI) showed the highest was shrimps (64.25%) followed by crabs (11.78%), zooplankton (6.94%), fish (6.91%), algae (4.21%), plants (1.48%), mollusks (1.01%) and others below 1.0%. TROPHj value ranged from 2.0 to 4.2 and the trophic level value of 25 fish species was carnivorous, followed by 2 species (detritivorous and herbivorous) respectively, and 1 species (piscivorous). The findings of the study may offer important data for developing management plans for the region's fishing resources

Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo

Ductile fracture strain in uniaxial tensile test of plane specimen’s

This project was performed to determine the ductile fracture strain in uniaxial tensile test of plane specimen. In this project, uniaxial tensile test was performed for three difference material that is aluminum, brass and mild steel. The objective of the test is to identify the value of uniaxial fracture strain for these three difference materials. The specimens have been divided into three difference area: L, P-lateral and S-middle zones. The zones are measured using optical microscope before and after the tensile test to determine the value of ductile fracture strain at those zones. However, the result from tensile test gives an average value of ductile fracture strain. The second step was to determine the suitable point or area to get the accurate uniaxial ductile fracture strain. The element of the material with stress triaxiality,k = 0.33 is the location where the uniaxial fracture strain was occurred. Finite element analysis using MSC Patran/Marc 2008r1 software was used to determine the element with stress triaxiality,k = 0.33. In MSC Patran software, the specimen was divided into several nodes to represent the study location for lateral and middle zones. In this project, the model was divided into eleven points. Each point has differences values of stress triaxiality after ultimate tensile strength occurred. The finite element analysis data of engineering stress-strain curve was compared with experiment engineering stress-strain curve in order to determine the fracture point of the model. The state of stress for each material was determined in order to get the uniaxial ductile fracture strain nodes. The result shows that uniaxial ductile fracture strain occurred at nodes 2577 for aluminium and brass, while uniaxial ductile fracture strain for mild steel occurred at node 2598. It is also shown that the fracture strain at L and P-lateral zones was the nearest to the uniaxial ductile fracture strain

Effect of Codon Optimisation on the Production of Recombinant Fish Growth Hormone in Pichia pastoris

This study was established to test the hypothesis of whether the codon optimization of fish growth hormone gene (FGH) based on P. pastoris preferred codon will improve the quantity of secreted rFGH in culture supernatant that can directly be used as fish feed supplements. The optimized FGH coding sequence (oFGH) and native sequence (nFGH) of giant grouper fish (Epinephelus lanceolatus) were cloned into P. pastoris expression vector (pPICZαA) downstream of alcohol oxidase gene (AOX1) for efficient induction of extracellular rFGH by adding 1% of absolute methanol. The results showed that recombinant P. pastoris was able to produce 2.80±0.27 mg of oFGH compared to 1.75±0.25 of nFGH in one litre of culture supernatant. The total body weight of tiger grouper fingerlings fed with oFGH increased significantly at third (P<0.05) and fourth weeks (P<0.01) of four-week experiment period compared to those fed with nFGH. Both oFGH and nFGH significantly enhanced the final biomass and fish survival percentage. In conclusion, codon optimization of FGH fragment was useful to increase rFGH quantity in the culture supernatant of P. pastoris that can be directly used as fish feed supplements. Further studies are still required for large scale production of rFGH and practical application in aquaculture production

Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo

In vitro and in silico studies of chalcone synthase variant 2 in Boesenbergia rotunda and its substrate specificity

In this study, transformation of BrCHS var 2 into B. rotunda cell suspension culture, followed by chalcone synthase enzymatic assay and HPLC analysis was conducted to investigate whether the substrate specificity for BrCHS var 2 is either cinnamoyl-CoA or p-coumaroyl-CoA. The HPLC profile showed an increase in the amount of pinocembrin chalcone when cinnamoyl-CoA and malonyl-CoA were added but not p-coumaroyl-CoA. Molecular docking was performed to explore the binding of cinnamoyl-CoA and p-coumaroyl-CoA to BrCHS var 2 receptor and the docking results showed that cinnamoyl-CoA formed numerous hydrogen bonds and more negative docked energy than p-coumaroyl-CoA. Cinnamoyl-CoA showed good interactions with Cys 164 to initiate the subsequent formation of pinocembrin chalcone, whereas the hydroxyl group of p-coumaroyl-CoA formed an unfavorable interaction with Gln 161 that caused steric hindrance to subsequent formation of naringenin chalcone. Docked conformation analysis results also showed that malonyl-CoA formed hydrogen bonding with Cys 164, His 303, and Asn 336 residues in BrCHS var 2. The results show that cinnamoyl-CoA is the preferred substrate for BrCHS var 2

A PCR-based sex determination method for possible application in caprine gender selection by simultaneous amplification of the Sry and Aml-X genes.

Sex determination of livestock is performed to achieve the objectives of livestock breeding programmes. Techniques for sex determination have evolved from karyotyping to detecting Y-specific antigens and recently to the polymerase chain reaction (PCR), which appears to be the most sensitive, accurate, rapid and reliable method to date. In this study, a PCR-based sex determination method for potential application in goat breeding programmes was developed. Primers were designed to amplify a portion of the X amelogenin gene (Aml-X) on the X chromosome to give a 300 bp product and Sry gene on the Y chromosome to give a 116 bp product. PCR optimization was performed using DNA template extracted from a whole blood sample of Jermasia goats (German Fawn x Katjang) of both sexes. It was possible to identify the sex chromosomes by amplifying both male- and female-specific genes simultaneously in a duplex reaction with males yielding two bands and females yielding one band. The Aml-X primer set, which served as an internal control primer, did not interfere with amplification of the Y-specific sequence even when a low amount of DNA (1 ng) was used. The duplex reaction subjected to a blind test showed 100% (14/14) concordance, proving its accuracy and reliability. The primer sets used were found to be highly specific and were suitable for gender selection of goats

Molecular phylogenetics and sequence analysis of two cave-dwelling Dugesia species from Southeast Asia (Platyhelminthes: Tricladida: Dugesiidae)

Khang, Tsung Fei, Tan, Siew Hwa, Panha, Somsak, Mohamed, Zulqarnain (2017): Molecular phylogenetics and sequence analysis of two cave-dwelling Dugesia species from Southeast Asia (Platyhelminthes: Tricladida: Dugesiidae). Raffles Bulletin of Zoology 65: 515-524, DOI: http://doi.org/10.5281/zenodo.535784