PREPARATION OF HUMAN INSULIN

PRESENTED BY:-

NAME:- RUSHAM DAS.

ROLL NO:-18901912041.

ACADEMIC SESSION:-3RD YEAR (6TH SEM).

PRESENTATION GUIDE:-MR. SAGAR SENGUPTA.

CONTENTS:-Introduction.What Is Diabetes Mellitus?What Is Insulin?History of Insulin Production.A New Era In Insulin Production.Basis Of Recombinant Insulin Preparation.How The Recombinant Human Insulin Is Made?Second Generation Recombinant Insulin.Chemically Altered Porcin Insulin.Conclusion.References.

INTRODUCTION:-

A few decades ago, it was realised that certain proteins could be used as pharmaceutical agents for the treatment of human diseases .How ever, the availability of such pharmaceutical products was very limited due to costly and complex process involved in their isolation and production.

The advent of recombinant DNA technology heralded a new chapter for the production of a wide range of therapeutic agents in sufficient quantities for human use.

WHAT IS DIABETES MELLITUS? -Diabetes mellitus is a genetically linked disease characterized by

increased blood glucose concentration (HYPERGLYCEMIA).

-The occurrence of diabetes is due to insufficient or inefficient insulin.

-When the blood glucose concentration exceeds about 180 mg/ml, glucose is excreted through urine.

-The more serious consequences of uncontrolled diabetes include (a)Kidney damage (NEPHROPATHY), (b)Eye damage (RETINOPATHY), (c)Nerve diseases (NEUROPATHY), (d)Circulatory diseases (ATHEROSCLEROSIS,STROKE).

*DIABETES IS THE LEADING CAUSE OF DEATH IN MANY COUNTRIES (AFTER CANCER AND HEART DISEASES)!!!

WHAT IS INSULIN?-It is a hormone produced from the β-cells of islets of Langerhans of

pancreas.

-Human Insulin has 51 amino acids, arranged in two polypeptide chains.

-The CHAIN A has 21 amino acids and CHAIN B has 30 amino acids.Both held together by disulfide bonds.

-Insulin facilitates the cellular uptake and utilization of glucose for the release of energy.

-In absence of Insulin, glucose accumulates in the blood stream at higher concentration which may lead to diabetes mellitus, a dangerous disease for the person.

HISTORY OF INSULIN PRODUCTION:-

-In early years insulin was isolated from pancreases of pigs and cows.

-Due to slight structural difference(by 1-3 amino acids) compared to human insulin, allergy was observed.

-Large number of animals had to be sacrificed for extracting insulin.*

*FOR INSTANCE 70 PIGS GIVING 5 KG OF PANCREATIC TISSUE HAVE TO BE KILLED TO GET INSULIN FOR TREATING A DIABETIC PATIENT FOR 1 YEAR!!!

A NEW ERA IN INSULIN PRODUCTION:--Concept of recombinant Insulin production was introduced for the

first time.

-Attempts were taken to produce human Insulin from the late 1970.

-It was July 1980,for the first time,17 human volunteers were administered recombinant Insulin for diabetes treatment at Guy’s Hospital, London and it worked well.

-Approval by concerned authorities, for using recombinant Insulin for the treatment of Diabetes Mellitus was given in 1982.

-In 1986,ELI LILLY company received approval to market human Insulin under the trade name HUMULIN.

*INSULIN IS THE FIRST EVER PHARMACEUTICAL PRODUCT OF rDNA TECHNOLOGY ADMINISTERED TO THE HUMANS.

BASIS OF RECOMBINENT INSULIN PREPARATION:--The basic technique consisted of inserting human insulin

gene and the promoter gene of Lac operon on to the plasmids of Escherichia coli (Strain no.812).

-The two chains separately synthesised are finally combined to form active insulin.

HOW THE RECOMBINANT HUMAN INSULIN IS MADE?

-The genes for Insulin Chain A and Chain B are separately inserted to the plasmids of two different Escherichia coli cultures.

-The Lac operon system consisting of inducer gene, promoter gene, operator gene and structural gene (β-Galactosidase) is used for expression of both the genes.

-The presence of Lactose in the culture medium induces the synthesis of INSULIN A and INSULIN B chains in separate cultures.

-The so formed Insulin chains can be isolated, purified and joined together to give exact human Insulin.

-The technique was improvised for improving yield and addition of signal peptide was done.

NEED OF SECOND GENERATION RECOMBINANT INSULIN:-

-After injecting Insulin, the plasma concentration of Insulin rises slowly so patient must be injected at least 15 minutes before meal.

-Decrease of Insulin levels is also slow , exposing the patients to a danger of Hyperinsulinemia.

-All this is due to the existence of therapeutic Insulin as a hexamer (six molecule associated),which dissociates slowly to the biologically active dimer and monomer.

*ATTEMPTS HAVE BEEN MADE IN RECENT YEARS TO PRODUCE SECOND GENERATION INSULINS BY SITE DIRECTED MUTAGENESIS AND PROTEIN ENGINEERING.

SECOND GENERATION RECOMBINANT INSULIN:--The second generation recombinant proteins are termed

as MUTEINS.

-A large number of Insulin Muteins have been constructed with an objective of faster dissociation of hexamers to biologically active forms.

-Insulin Lispro is among these with modified amino acid residues at position 29 and 30 of the chain b of Insulin. Other 2nd generation Insulins are Glargine and Lente.

*INSULIN LISPRO CAN BE INJECTED IMMEDIATELY BEFORE A MEAL AS IT ATTAINS THE PHARMACOLOGICALLY EFFICIENT LEVELS VERY FAST.

CHEMICALLY ALTERED PORCIN INSULIN:-

-Porcin Insulin differs from human Insulin just by one amino acid (Alanine in place of Threonine) at the C-terminal of the B Chain of Insulin.

-Biotechnologists have developed methods to alter the chemical structure of Porcin Insulin to make it identical to human Insulin.

-This chemically modified Porcin Insulin can also be employed for treating Diabetes Mellitus.

CONCLUSION:-Diabetes is a threat for the human race and so, strong steps

should be taken to eradicate it. Production of human Insulin got a giant leap after recombinant technology was used. Then to enhance its activity second generation Insulins were developed.

In fact, the future could see Insulin pills making diabetes management considerably more safe and convenient, not to mention eliminating prevalent needle phobia.

At this stage, Insulin remains too complex a protein to survive within the environment of the body. Leading pharmaceutical companies are thought to be using protein engineering to bring the insulin pill one step closer.

REFERENCES:-www.ncbi.nlm.nih.gov/pubmed/https://www.nlm.nih.gov/.../recombinant-dna-te

chnology-alternative.htmlhttps://books.google.co.in/bookswww.nature.com › Journal home › Archive ›

Table of Contents › Letteren.wikipedia.org/wiki/Insulinwww.diabetes.co.uk › InsulinBiotechnology BY U. Satyanarayana.Biochemistry BY U. Satyanarayana And U.

Chakrapani.