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Recombinant DNA technology for insulin synthesis induced by E.coli

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Chapter I

Sajeda bader

waed aljazwy

Abdulrahman elsaber

ajbah alhaj

Sanad shaker

sendiana almzeny

Supervised by

  • Mohammed AlGhazali,B,Sc.M,Sc.Med

 

Introduction

Chapter I

I.1. Introduction

An insulin deficit in an organism or a lack of cell response to the produced hormone leads to diabetes mellitus. This usually incurable illness manifests as high blood sugar (hyperglycemia).

Human insulin is a hormone produced in the β-cells of the pancreatic islets, responsible for glucose metabolism regulation [4]. Human insulin is a polypeptide with a molecular mass of 5808 Da. The hormone consists of two chains: chain A (21 amino acids) and chain B (30 amino acids) connected by two disulphide bridges. Additionally, chain A contains an intrachain disulphide bond [5]. A new bacterial host strain (E. coli 20) was obtained at the Institute of Biotechnology and Antibiotics. Also, a new pIBAINS expression vector was constructed.

Discovery

Chapter II

I.2. Discovery

- In 1869, while studying the structure of the pancreas under a microscope, Paul Langerhans.

-In 1889, the physician Oskar Minkowski, in collaboration with Joseph von Mering

-In 1901, by the American physician and scientist Eugene Lindsay Opie, when he isolated the role of the pancreas to the islets of Langerhans

- in 1916-1921, Nicolae Paulescu developedan aqueous pancreatic extract.

CHAPTER II

II.1. Evolution and species distribution

Insulin may have originated more than a billion years ago.[21] The molecular origins of insulin go at least as far back as the simplest unicellular eukaryotes.[21] Apart from animals, insulin-like proteins are also known to exist in the Fungi and Protista kingdoms.[22]

Insulin is produced by beta cells of the pancreatic islets in most vertebrates.[17]

Chapter II

II.1.1. Gene

The preproinsulin precursor of insulin is encoded by the INS gene.[20][21]

II.1.2. Alleles

A variety of mutant alleles with changes in the coding region have been identified. A read-through gene, INS-IGF2, overlaps with this gene at the 5' region and with the IGF2 gene at the 3' region.[22]

Chapter III

CHAPTER III

III.1. Biotechnology in the pharmacology

 Biotechnology is being defined as organisms or biological molecules, which are used in the industrial production. This term includes processes used for centuries, such as the discovery in more recent years of genetic engineering [27].

-Biotechnology in the pharmacology is being called biotechnology pharmaceutical, which is producing biopharmaceuticals. These are proteins with therapeutic meaning and recently nucleic acid used in the gene therapy, which are being produced due to genetic engineering or traditional biotechnology.

III.2. Recombinant DNA technology

The idea for recombinant DNA was first proposed by Peter Lobban, a graduate student of Prof. Dale Kaiser in the Biochemistry Department at Stanford University Medical School. Recombinant DNA technology is the technique, which allows DNA to be produced via artificial means. The procedure has been used to change DNA in living organisms and may have even more practical uses in the future. It is an area of medicine, which is at present in its initial phase of the overall concerted effort [12].

Recombinant DNA technology

Currently, recombinant DNA technology has attracted headlines when it has been used on animals, either to create identical copies of the same animal or to create entirely new species. One of these new species is the GloFish™, a type of fish that seems to glow with a bright fluorescent coloring. While they have become a popular aquarium fish, they have other uses as well. Scientists hope to use them to help detect polluted waterways, for example.

CHAPTER IV

IV.1. Aim of study

Benefits of using E. coli 20 strain is that ownership of a patented strain allows the invention to be commercialized on the pharmaceutical industry market.

Production of insulin by recombinant DNA of E.coli 20.

To see whether any updates and recent studies related to this study.

Chapter IV

Material and methods

IV.2. Material and methods

Escherichia coli 20 IBA 1 strain is a laboratory derivative of E.coli CSH50R [ATCC: 39111] [6–8]. Antifoam 204, L-proline, thiamine hydrochloride, tetracycline hydrochloride, trizma base, Triton X-100, lysozyme, EDTA, sodium bicarbonate, citraconic anhydride, ethanolamine and other catalyting media agents. The expression plasmid pIBAINS is a derivative of plasmid pBR322 [ATCC: 31344]. Briefly, pIBA was prepared through amplification of PCR using plasmid pBR322.

-CytR repressor gene is a derivative of the E. coli CSH50R strain. It is a laboratory strain derived from E.coli K-12, Gram (-) negative bacterium belonging to the Enterobacteriaceae family. It is a strain used for transformation.

So, by lacking ( isolation ) the CytR repressor gene, the uncontrolled induction of insulin production excessively will be occur.

-PCR will be used for the gene transcription leading to efficient insulin synthesis.

Isolation of CytR repressor gene

After isolation performed according to the protocol described in Material & Methods.

Purification

Summary At the end of the whole process, the human insulin from the culture media by a three-step purification procedure .

Results and discussion

IV.3. Results and discussion

Genetic construction of pIBAINS plasmid

The human insulin gene was composed according to the following formula: L-B-X-A. In the formula, L is a leader peptide represented by the modified SOD gene, B is human insulin chain B, X is a short peptide Lys-Arg connecting chain B with chain A

Conclusions

IV.4. Conclusions

Diabetes belongs to so called “lifestyle” or “civilization diseases” – it is a major threat to the human health and well-being globally. Up to now, the most effective method to treat this disease in the case of hyperglycemia is still insulin delivery. Thus, it is meaningful to establish an effective and economical protocol for insulin production. To conclude, we reported here the construction of a new pIBAINS expression vector and the establishment of a new bacterial host strain E. coli 20, which is able to produce human insulin with high efficiency. The plasmid does not integrate into the genome and remains stable for at least 80 generations without antibiotic selection. we have mentioned the whole process from cell culture through inclusion body isolation and protein purification.

we hope this study may take the biotechnologists interest against industrial and economical protocols for insulin production…

End

CHAPTER V

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End

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