Showing 4 results for Genetic Engineering
S. Sheykhi , M. Amininasab, B. Saffari, S. Abdi,
Volume 9, Issue 1 (1-2018)
Abstract
Aims: Identifying the structure and function of alpha-Synuclein protein can lead to the development of appropriate treatments for Parkinson disease. The aim of the current study was to investigate DNA cloning and the expression of alpha-Synuclein protein in E. coli.
Materials and Methods: In this experimental study, the sequence of encoding alpha-Synuclein in pRK172 recombinant plasmid was amplified by Polymerase Chain Reaction (PCR), using best primers. The synthesized DNA was, then, digested by restriction enzymes and cloned into pET28a and recombinant plasmid was transferred into the expression strain of E. coli (BL21) by Calcium Chloride method. The expression of alpha-Synuclein gene was induced by Isopropyl-Beta-D-Thiogalactoside (IPTG) and the expression of alpha-Synuclein was investigated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) method. Sequencing was done, using the ClustalW algorithm by the BioEdit 5.0.9 program.
Findings: In products of DNA enzymatic digestive reactions and pET28a plasmid with restriction enzymes, the size of the fragments indicated the correctness of the enzymatic reactions. The synthesized DNA and pET28a plasmid were 407 and 5369 nucleotides, respectively. The translation of the sequence of the cloned fragment revealed a 100% similarity to the human alpha-Synuclein protein. In expressing the recombinant protein in comparison with negative control samples, adding IPTG increased the expression of alpha-Synuclein protein in all samples, especially 2 hours after induction. Most of alpha-Synuclein expressed from the pET28a-alpha-Synuclein plasmid accumulated in the bacteria as incorporated objects.
Conclusion: The alpha-Synuclein protein is cloned into the pET28a plasmid and formation of the objects incorporated by alpha-Synuclein is confirmed by the expression of the pET28a-alpha-Synuclein system and paves the way for producing this protein in high scale.
R. Ghasemi , H. Hashemzadeh , H. Razavi , B. Yakhchali ,
Volume 9, Issue 1 (1-2018)
Abstract
Introduction: Growth hormone is a non-glycosylated polypeptide strand of the pituitary glands of all vertebrates that has a wide range of biological activities and considering the importance of this hormone and its importance and diverse therapeutic applications in medicine, its recombinant production can be of great importance. In recent decades, protein engineering and genetic engineering have resulted in a high level of expression and production of this protein in a variety of hosts, including Escherichia coli bacteria using new techniques and methodes, hormone purification and assay are carried out easily. Therefore, the aim of this review was to investigate the production of recombinant human growth hormone (rhGH) and future challenges.
Conclusion: One of the problems of the expression and purification of the human growth hormone may involve that maybe noted the production of inclusion bodies in the expression of recombinant proteins in the cell cytoplasm, the contamination caused by host proteins, low protein recovery from these inclusion bodies, low protein secretion into the Periplasmic space, high cost of production, especially in Purification stage and so on. Due to the lack of need for glycosylated hormone and high efficiency and simplicity of work, bacterial systems, especially Escherichia coli, are the most economical and effective systems for the expression of heterologous proteins. The hormone purification stage is usually the most costly process. Therefore, an optimal design for achieving the highest target protein recovery with the elimination of all contamination from the final product and reducing the purification step is required.
S. Shamriz, H. Ofoghi,
Volume 10, Issue 2 (7-2019)
Abstract
Microalgae are microscopic algae found in a wide range of habitats including freshwater and marine systems. Over the last decades, biotechnological processes based on microalgae have been receiving increasing interest due to their potential to produce large quantities of valuable products used as human food supplements, pharmaceuticals and animal feed. Microalgae have also been proved as an efficient and cost-effective platform for recombinant protein production. Most progress in this field has been achieved using Chlamydomonas reinhardtii, a photosynthetic unicellular alga which has been mostly considered as a model organism in different studies. High growth rate, ease of cultivation, well-established genetics and the ability to perform post-translational modifications are the most beneficial attributes that have made C. reinhardtii an attractive system for the expression of recombinant proteins. In this review, we focus on C. reinhardtii as a novel platform for the development of advanced recombinant products and compare them with other commonly used expression systems. We also present a comprehensive overview of its structure, life cycle, culture systems, and media in detail and then discuss the strategies for engineering its three different genomes to produce recombinant cells. Finally, algal culture collections in the world are introduced.
Leila Pourhang, Zahra Ghorbanzadeh, Mehrbanoo Kazemi Alamuti, Seyyed Mohammad Mousavi Pakzad, Elahe Moatamed, Mona Mapar, Aliakbar Ebadi, Mohammad Reza Ghaffari*, Katayoun Zamani, Ghasem Hosseini Salekdeh, Behzad Ghareyazie, Motahhareh Mohsenpour,
Volume 14, Issue 1 (3-2023)
Abstract
Production of drought tolerant crop is an important strategy for avoiding water scarce crisis. Improvement of the root structure leading to the higher yield and seed quality. In this study, three genes affecting root structure, drought tolerance and phosphorous absorbance are used in producing hybrid constructs used for the rice transformation. Three genes: a serine/threonine protein kinase (PSTOL1), a gene from the cytokinin oxidase/dehydrogenase family (OsCKX4) and a transcription factor induced under stress from the NAM-ATAF-CUC family (OsNAC5) isolated from the rice wild cultivars are cloned under separate regulatory elements in the T-DNA region of the Agrobacterium binary vector. OsNAC5 gene was cloned under RCc3 root specific promoter and PSTOL1 gene under ubiquitin promoter. Also, OsCKX4 gene was cloned once under ubiquitin promoter and once under RCc3 promoter. Two hybrid multi-gene constructs named pUhrN5CkPstol and pUhrCkPstol harboring multiple genes are synthetized and used for the gene transformation into the Hashemi cultivar. Gene transfer was done to callus obtained from mature rice seeds. Transgenic plants were confirmed using PCR analysis. From the number of 107 regenerated plants in which the presence of transgenes was proved, 14 transgenic events were finally obtained. Root structure of the T0 plants showed drastic phenotypic difference in comparison to the non-transgenic ones. By now, one transgenic event harboring CKX4 and PSTOL1 is confirmed to have a homozygous line in T2 generation. It is hoped that genetic engineering of rice for enhanced root structure lead to drought tolerance, reduce water consumption and improve yield under stress conditions.
