Transgenic plants

Transgenic plants

Introduction

Transgenic plants have offered an excellent platform for the recombinant production of protein. Gene encoding carrying the wanted or rather an expected protein is used to establish stable transgenic lines. Cloning of the latter into an expression construct that it must include a promoter and elements that take up the regulatory role to ensure proper RNA processing and efficient protein formation. This phenomenon of the construct is then integrated to make the nuclear genome of a plant, leading to a stable transfer of the transgenes and appearance of pharmaceutical Proinsulin in different generations. Two main hereditary strategies have been considered to place the appearance of gene constructs into the nuclear genome, these include: Agrobacterium-mediated development in dicots as in the case of transgenic peanut and blast of DNA- tungsten beads or coated gold in monocots plant species.

Transgene plant categories provide various strengths as an opportunity for pharming molecules: they are best for posterior recombinant pharmaceutical proteins’ production and are majorly accessible, as every occasion can be used to produce seeds, which multiplies the number of plants in each generation. Definitely, the capacity of production of recombinant pharmaceutical Proinsulin in transgenic plants is almost impossible because of the size of the land available for plant culture matters. The main disadvantage of transgenic plants is extended development period, low production yields, and likelihood spread of pharmaceutical crops on the environment and to the ecosystems by pollination and seed dispersal.

The rising cases of diabetes globally and the exploration of alternative insulin manufacturing approaches such as oral route or inhalation that depend majorly on higher doses is destined to increase the desire for recombinant insulin in the future. The laboratory processing of therapeutic recombinant proteins requires a host organism alongside the most efficient equipment for protein refolding and posttranslational modifications. Manufacturing of recombinant human insulin is achieved using Saccharomyces cerevisiae and E. coli for therapeutic use in humans. However, this paper reviews different approaches that can be explored to escalate the production of insulin that is biologically active and along with analogues in yeast and E. coli. The paper also discusses some of the transgenic plants with a significant expression system and which have the capability of being exploited to produce insulin for therapeutic use in humans.

In that regard, the paper draws facts from three primary sources with biotechnology literature. The focus of the discussion is to review the gene constructs used to obtain insulin and their respective promoters, enhancers, and silencers based on every author’s research and findings. The paper will primarily stick to three sources while generating the context of the paper. Each case is discussed basing on one article in order to bring out a clear review, as far as proinsulin production is concerned.

Case 1

According to the article “Enhanced recombinant insulin production in transgenic Escherichia coli that heterologously expresses carrot heat shock protein 70,” by Bomin Jang and Yeh-JinAhn, the process of acquiring Proinsulin will need a carrot plant (Daucus carota L.). From the carrot, a heat shock protein 70 (DcHsp70) is obtained. In that case, the protein will act as the promoter once inserted into the genome of E. coli. The process of adding the protein into the E. coli genome is referred to as homologous recombination. E. coli, in this case, is a transgenic organism. The expression of non-homologous DcHsp70 increases the chains of recombinant human Proinsulin and insulin A and B in the transgenic E. coli.

According to this article, the gene containing the promoter DcHsp70 with the constitutive bacterial lipoprotein promoter is inserted into the E. coli DNA strand. Lambda Red-mediated is used as a silencer while placing homologous recombination. Nuclear sequences belonging to the human proinsulin B and Proinsulin A chains are separately cultured in the pVFT 2S expression vector. The latter contains a 6xhistidine silencer as well as glutathione S-transferase (GST) tag. The isopropyl β-d-1-thiogalactopyranoside healing is a promoter that expresses levels of the insulin A and B and proinsulin chain fusion proteins is higher in the transgenic tissue lines that express DcHsp70 in a non-homologous form than as compared to control tissue line. The 6xHis-GST silencer has three distinct properties that express Proinsulin in tobacco’s protease etch virus. DcHsp70 is an active molecular content composition that increases the recombinant production of Proinsulin when expressed heterologously in E. coli.

Case 2

Drawing ideas from the article “Production of proinsulin in marker-free transgenic tobacco plants using CRE/loxP system,” by L. Zheng, Z. Y. Peng, Q. Q. Jiao, Y. Wang, F. Bian, S. J. Qu, S. B. Wan & Y. P. B, CRE/loxP system is used to develop a selective marker-free system to produce Proinsulin. The system is applied in transgenic plant. During the process, pRD29A is a promoter which is isolated from Arabidopsis. For the case of tobacco, CRE recombinase gene is controlled by the promoter. The latter controls the recombinase gene along with the selective NPTII gene between two loxP recombination sites. As for the case of tobacco, gene excision was applied in removal of the sequence between the two loxP sites. It is under the condition that 200 mM NaCl (enhancer) is present. PCR analysis shows that self-excision occurs in many transgenic plants. Transgenic peanut lacking a marker gene expressed Proinsulin significantly. The auto-excision removes the optional available marker gene from transgenic peanut. That, however, is the best method of obtaining an efficient method for producing bio-safe recombinant Proinsulin.

On the other hand, the CTB-PFx3 gene construct has a high proinsulin expression, especially when subjected to a psbA enhancer. Within the transgene peanut’s structure, there is aadA silencer responsible for conferring the resistance to spectinomycin. However, the upstream promoter carries the resistance report to the chloroplast vector. Chloroplast vectors include lettuce DNA flanking regions (tRNA/tRNA) or native tobacco. The latter facilitates homologous recombination. In that regard Glycine–Proline–Glycine– Proline (GPGP) is applied to avoid steric hindrance.

On the other hand, the furin cleavage site (RRKR) is a unique enhancer stated by this article and which is not mentioned in the other pieces. The enhancer removes of the CTB construct following delivery. The C-peptide/A-chain junctions and the B-chain are two additional furin cleavage sites that replace the native PC3 and PC2 cleavage sites. They are the silencers in the proinsulin construct. Their location, the cleavage disulphide bonds, and sites are compared with initial Proinsulin.

Fig 1: Location of furin cleavage sites.

Case 3

The rising cases of diabetes have forced scientists to come up with insulin production to meet the demand. The insulin is rather alternative and, as such, is called Proinsulin. Based on the literature “Transgenic Expression and Identification of Recombinant Human Proinsulin in Peanut,” by Zheng Ling, Jiao Qi-Qing uses peanut (Arachis hypogaea L.) to represent bio-reactors which in turn expresses stable and bio-safe Proinsulin. Proinsulin analogues (FAIA and LAIA) are applicable as promoters according to this literature. Unlike the other two articles, this article under discussion is different from other pieces in a manner that amino acids play a crucial role in the desired gene construct. (FAIA) is the fast-acting proinsulin analogue comprising of a Gly. The latter is inserted between Cys19 and Gly20. The two are promoters in the peanut cells that are responsible for the expression of the gene proinsulin fragments. Also, a Pro28Asp does the same function in the B chain. There is also an Asn21Gly substitution found in the A chain, and all work together as silencers. For the case of the peanut is a transgene plant, four plasmids constructs pCAMBIA2301-Oleosin-LAIA, pROKII-Flag-FAIA, pCAMBIA2301-Oleosin-FAIA and pROKII-Flag-LAIA can express the desired gene properties in a transgene peanut. So then, recombinant proinsulin forms once the plasmids are transferred into a peanut.

Transgenic peanut expression systems have illustrated a high-capacity production of Proinsulin. Transgenic peanut’s leaves and seeds portray a long-term stability. As a result, its constructs are used to stockpile Proinsulin until it is needed. Expressed as insulin-oleosin fusion in Arabidopsis thaliana seeds, and the insulin that accumulates in the transgenic seeds could significantly lower the glucose levels to a similar point as commercially available Proinsulin.

The most common recombinant human Proinsulin for non-homologous expression is Proinsulin, which contains a BC-A construct and comprises of a longer in vivo half-life than mature Proinsulin. Long-acting insulin analogues (LAIA ), medium-acting insulin analogues (MAIA), and Proinsulin’s analogues (FAIA). These insulin analogues were obtained by changing several amino acids in the A-Chain. For example, regarding the latter, Glargine and Lispro are some of the amino acids located in the construct of a peanut. Peanuts are known for their oilseeds crop globally. The property of peanuts’ seeds to store fats and protein serves as the best reservoir for heterologously expressed Proinsulin; thus, we sought to express the human insulin gene in peanut seeds. Since the Proinsulin is prone to N-terminal degradation and highly unstable, fusing a flag tag with Proinsulin helps in protecting the N-terminus from degradation.

Conclusions

Molecular pharming or rather gene constructions among the transgenic organisms has undergone several reviews recently, and many more are expected in the coming future of this technology. Optimistic expectations have been gathered from the same. A number of studies have proven the ability in some plant species, including tobacco, peanuts, and the ability of several plant species and classes to produce recombinant pharmaceutical proteins and peptides. This method has already been put into practice in instances of emerging trends, where the short lifespan has been proved to be ideal for mass production of recombinant proteins as the solutions to epidemic solutions. On the contrary, their importance for the development of food functionals still fails to meet the threshold, as well as the achievement of its maximum ability in the production of bioactive peptide production. Improvement in plant’s avenues, improvements of genetic engineering techniques, and their continuous research, this, therefore, leads to evolution in the production of heterologous bioactive peptides, which results in the formation of the ACEI pharming project.

The beginning of genome deletion techniques (with the merit of site-particular gene addition), as in the case of CRISPR/Cas9 methodology, will definitely increase and accelerates event transformation of plants and therefore leads to an improvement

in genetically modified species for the purpose of molecular pharming.