Hassan A. I. Ramadan

Cell factories for insulin production

The rapid increase in the number of diabetic patients globally and exploration of alternate insulin delivery methods such as inhalation or oral route that rely on higher doses, is bound to escalate the demand for recombinant insulin in near future. Current manufacturing technologies would be unable to meet the growing demand of affordable insulin due to limitation in production capacity and high production cost. Manufacturing of therapeutic recombinant proteins require an appropriate host organism with efficient machinery for posttranslational modifications and protein refolding. Recombinant human insulin has been produced predominantly using E. coli and Saccharomyces cerevisiae for therapeutic use in human. We would focus in this review, on various approaches that can be exploited to increase the production of a biologically active insulin and its analogues in E. coli and yeast. Transgenic plants are also very attractive expression system, which can be exploited to produce insulin in large quantities for therapeutic use in human. Plant-based expression system hold tremendous potential for high-capacity production of insulin in very cost-effective manner. Very high level of expression of biologically active proinsulin in seeds or leaves with long-term stability, offers a low-cost technology for both injectable as well as oral delivery of proinsulin.

Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives

Escherichia coli is the most preferred microorganism to express heterologous proteins for therapeutic use, as around 30% of the approved therapeutic proteins are currently being produced using it as a host. Owing to its rapid growth, high yield of the product, cost-effectiveness, and easy scale-up process, E. coli is an expression host of choice in the biotechnology industry for large-scale production of proteins, particularly non-glycosylated proteins, for therapeutic use. The availability of various E. coli expression vectors and strains, relatively easy protein folding mechanisms, and bioprocess technologies, makes it very attractive for industrial applications. However, the codon usage in E. coli and the absence of post-translational modifications, such as glycosylation, phosphorylation, and proteolytic processing, limit its use for the production of slightly complex recombinant biopharmaceuticals. Several new technological advancements in the E. coli expression system to meet the biotechnology industry requirements have been made, such as novel engineered strains, genetically modifying E. coli to possess capability to glycosylate heterologous proteins and express complex proteins, including full-length glycosylated antibodies. This review summarizes the recent advancements that may further expand the use of the E. coli expression system to produce more complex and also glycosylated proteins for therapeutic use in the future.

Molecular detection of adulteration in chicken products based on mitochondrial 12S rRNA gene

Abstract The aim of this study is to detect the fraudulent in chicken products constitutes in order to protect consumers in Saudi Arabia from illegal substitutions. Two different approaches were used in this study, direct sequencing of specific fragments of amplified mitochondrial 12S rRNA gene in addition to species-specific PCR primers for confirmation of the obtained Blast search results. The results showed that all processed chicken products were identified as chicken (Gallus gallus) by 90–98% homology depending on obtained sequence quality. Samples labeled with chicken luncheon (samples tested in this study) were identified as turkey meat (Meleagris gallopavo) by 98% homology, suggesting adulteration with inedible parts of turkey in chicken luncheon ingredients. The results showed also that not only chicken luncheon was mixed with inedible parts of turkey but also all chicken products tested in this study (chicken balls, chicken burger, chicken sausage and chicken mined meat) contained this turkey meat. Applying methods used in this study could be useful for accurate and rapid identification of commercial processed meat.

Molecular characterization of Saudi local chicken strains using mitochondrial DNA markers

Abstract The current study was carried out to investigate and estimate the genetic diversity of native breeds based on cytochrome b (cyt-b) gene of mitochondrial DNA information. The obtained sequences of cyt-b gene segment have TAA as a stop codon at 488 position with no insertions or deletion in all individuals of both native chicken strains. The blast results showed that no variation was found among individuals within both native chicken strains, but when a comparison was established among them and other species of genus Gallus the variation is exploring, additionally many mutant sites were detected as single nucleotide polymorphisms (SNPs) in different sites. The phylogenetic trees exhibited three different groups. The results revealed that the native chicken strains were closely related to the cluster of Gallus gallus and subspecies of Gallus, suggesting that they may be separated from the same origin. According to this result and previously studies, the native chicken strains are genetically closer to Gallus gallus and it could be successfully distinguished from the other wild types of Gallus chicken based on cyt-b gene information. We recommended that the governmental concerns for native chicken strain should be enhanced to screen its genetic structure for large scale in the Kingdom of Saudi Arabia.

Biological Identifications Through DNA Barcodes

Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon ‘barcodes’. It was established previously that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. A new tools were developed recently to be complementary markers for (COI) DNA barcoding.

Allele mining in the caprine alpha-lactalbumin (LALBA) gene of native Saudi origin

ABSTRACT Alpha-lactalbumin (α-LA) is a distinctive whey protein found in bovine milk and the milk of other mammalian species. Its main function in the lactating mammary glands is involved in lactose biosynthesis, which makes it a potential quantitative trait locus. The aim of this study is to determine the incidence of single nucleotide polymorphisms (SNPs) within the exonic and intronic portions of the caprine α-LA gene (LALBA) in native Saudi breeds (Ardi, Habsi and Harri) in relation to utility traits. Blood samples were collected from 300 animals (100 for each breed). Genomic DNA was extracted and a 268 bp fragment of the LALBA gene comprising exon 3 and its flanking area was amplified. Subsequent digestion with MvaI restriction endonuclease resulted in two different banding patterns: αLA A1/αLA A1 and αLA A1/αLA A2, and two allelic forms, i.e. αLA A1 and αLA A2. Nucleotide sequencing of the designated LALBA amplicons was done and, following successful BLAST analysis, the sequences were submitted to GenBank (NCBI Accession No. KM267632, KM267633, KM267634 and KP940442). SNPs were searched within breeds of Saudi origin and homology was sought among caprine, ovine and bovine species. A C > T transition was identified within the fifth nucleotide of the silent α-LA A2 allele. Phylogenetic analysis on the basis of LALBA nucleotide sequence of Saudi goats indicated similarity with evolutionarily related sheep, while the origin of bovine species and deer was located some distance away.

Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System

Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system.