Key words: Recombinant proteins, insulin, Escherichia coli, yeast, mammalian.
An ever growing population of diabetes patients in the world has encouraged the discoveries for recombinant insulin production as the traditional insulin products have large number of drawbacks as well as they are not cost effective. Recombinant insulin is mainly synthesized in E.coli as the host but there are various expression systems with efficient post-translational modifications and increase production of recombinant insulin along with being cost effective. E. coli being the most prevalent system most of the recombinant insulin is produced in E.coli however due lack of post-translational modification yeast and mammalian expression systems are being used. This review article put an insight on different methods used for insulin production.
1.0 Introduction and Background
Insulin is a well organized protein hormone secreted by the beta cells of pancreas. Insulin is a small molecule with a molecular weight of 5808 Da and made up of 51 amino acids. It is an important player in regulation of carbohydrates as well as metabolism of fats within the body 1, 2. Insulin is synthesized as single chain precursor molecule called as preproinsulin which has a 24 amino acid long signal peptide which facilitates the translocation of the polypeptide chain in the endoplasmic reticulum, once it transports this polypeptide to ER the signal peptide is removed which yields proinsulin. Different type of post translational modification takes place within the ER to form a folded proinsulin which is translocated to Golgi apparatus within which number of catalytically active enzymes act on the precursor polypeptide to form disulfide linked A and B chain of 21 and 30 amino acid long respectively to form active insulin1, 3. The dysfunction either in action of insulin or secretion of insulin or both results in hyperglycemic condition diabetes mellitus. It is a metabolic disorder caused due to metabolism disturbance in carbohydrates, fats or proteins. The effect of this disorder varies from improper functioning to loss of functions of different organs 4.
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Around 381.8 million adults in 219 different countries were estimated to suffer diabetes in the year 2013 and this number may rise to 591.9 million till the year 20355. This ever growing diabetes incidence has led to increase demand for insulin as a result to meet this demand various strategies need to be employed one of which is recombinant production of insulin.
1.1 Bacterial system.
Escherichia coli is an enterobacterium which is an workhorse used for cloning, genetic modification and most preferred for manufacturing recombinant proteins and biopharmaceuticals on large scale1, 6, 7. Due to availability of vast information regarding its physiology and genetics as well as use of molecular tools it is well studied and characterized host organism with large range of expression system1, 7. Other advantages include rapid expression of Proteins, High yields, Ease of culture and genome modiï¬cations less costly, mass production fast and cost effective9. The most important drawback of E.coli as a host for recombinant protein production is that it lacks most important post-translational modification (PTMs) such as formation of disulfide bonds, proteolytic processing, glycosylation and many others which important for the activity of eukaryotic proteins. As a result of these inefficient PTMs the recombinant protein produced in E.coli is inactive, insoluble and unstable1, 6, 7. In spite of this drawbacks variety of techniques have been develop to overcome this drawbacks and use E.coli as a host for recombinant protein production. These techniques include release of the recombinant protein in periplasmic space, denaturation and renaturation of inclusion bodies, discovery of variety of strains, use of strong promoters’ etc7.
In E.coli the recombinant insulin can be secreted out through the periplasmic space via the sec pathway. This pathway takes place I two steps wherein the signal sequence is necessary for transport of the premature protein to periplasmic space takes place in first step followed of formation of matured protein through processing in the second step. The signal sequence plays a very important role in secretion of recombinant protein. The characteristic feature of this signal sequence is that it contains10-20 amino acid long hydrophobic H-domain and 10 amino acid long N-domain which is positively charged, the hydrophobic nature allows secretion of protein in the periplasmic space. Mature protein is formed by cleavage of this signal sequence by signal peptidase7.
Table 1.1: signal sequences in E.coli 7.
In order to ensure proper disulfide bond formation in recombinant insulin in E.coli the proinsulin gene is combined along with disulfide oxidoreductase protein i.e. DsbA. This DsbA protein is normally present in the periplasmic space but in a low concentration thus increasing its amount in periplasm allows the production of proinsulin in its native soluble form with increased production8.
In prokaryotic bacteria such as E.coli recombinant protein are mostly produced as insoluble aggregates which render inactive protein called as inclusion bodies. These inclusion bodies contain highly pure form the protein and protect the protein from proteolytic degradation and can be isolated from the cells through simple centrifugation, homogenization using high pressure for industrial production etc. Followed by isolation these IB are solubilized with the help of guanidinium hydrochloride or by urea at a specific pH for protein of interest. The resulted protein after solubilization lacks its native conformation which can be achieved proper conditions for renaturing or use of chaperones like GroEL protein can be foled in its native conformation9.
1.2 Yeast system.
Yeast is well studied eukaryotic system and host for production of human recombinant proteins that require proper folding and post translational modifications10, 11, 12. There are numerous advantages for the use of yeast as recombinant system some of which are it can perform large number of post-translational modification ranging from N-linked and O-linked glycosylation to acylation, the proteins are properly folded in their active form and released in the extracellular medium which also facilitates its purification from the medium, the biopharmaceuticals produced using yeast as host system are relatively cheap as downstream processing is cost effective which facilitates its large scale production, increased amounts of recombinant proteins are made available through use of mutant strains 1, 6, 10 ,11, 12.
Saccharomyces cerevisiae is unicellular eukaryotic system and is a eukaryotic model system used to study and understand the metabolic processes under various physiological conditions. As wide range of information is made available through databases, genome sequence and different software for manipulating the genome has lead to discovery of engineered strains which are used to produce different biopharmaceuticals like insulin and its analog, vaccines, serum albumin 6, 12.
The main drawback of Saccharomyces cerevisiae as host system for production of recombinant protein is that it carries out large amount of high mannose N-linked glycosylation which is different from eukaryotic system as a result the half-life of the protein is greatly reduced and causes immune response in vivo10, 11, and 12. Hence large efforts are made to carry out the humanized pathway for N-glycosylation one of which is the use of different strain of yeast Pichia pastoris. Even Pichia performs high mannose N- glycosylation but the number of residues added is very small as compare to S. cerevisiae and increased amount of expression of proteins is observed in Pichia11.
Since 1980’s Saccharomyces Cerevisiae has been used for production of recombinant insulin used to treat diabetes mellitus. The production of this recombinant insulin is based on the expression system13. The secretory process and pathway of S. cerevisiae is multifunction and complex. The process involves transport of the protein across Golgi apparatus via endoplasmic reticulum and finally out of the cell through secretory granules. In the endoplasmic reticulum N-linked glycosylation and folding of protein takes place whereas post-translational modification takes place in Golgi apparatus by 160 different proteins 1013, . The gene responsible for secretion of pheromone α factor in the α type S. cerevisiae cells is engineered to produce proinsulin. The gene generally consists the following sequences:
- Pre sequence.
- Pro sequence
- 3 consensus sites where N-glycosylation takes place.
- KEX-2 endoprotease site at C-terminal.
The Pre sequence i.e. the signal peptide of this α factor is engineered with the cDNA of proinsulin gene which did not contain threonine amino acid in 30th position on the B-chain. The C- terminal is a synthetic peptide containing amino acid AAK. As S. cerevisiae cannot express human proinsulin it is secreted as pro-insulin in single chain form within the culture supernatant which is then purified. This purified proinsulin is then made active by trypsin mediated reaction of transpeptidation 10, 13.
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Pichia pastoris is also used for the production of insulin as it produces large amount of stable recombinant protein and adaptable to wide range of vector. The gene engineered in Pichia consists of insulin precursor (IP) whose amino acid sequences are chemically synthesized in such a way that can be expressed in Pichia. The IPgene contains its own stop codon. The gene construct consists following sequences
- Alcohol oxidase promoter.
- C-terminal sequence of α factor secretory signal from S. cerevisiae at the N-terminal
- Spacer sequence
- Linker sequence AAK
This entire gene construct is fused vector pPICZα and forms an expression vector pPICZα-IP. This expression vector is linearized with Sac1 and integrated into P. pastoris X-33 strain by electrophoresis. Transformed cells are selected on zeocin plates, this clones are grown in batch culture followed by methanol addition for formation insulin precursor. This insulin precursor is purified converted to pre-insulin and finally to insulin by transpeptidation , removal of spacer and linker and addition of threonine at 29th position in B-chain and removal of tert. Butyl14.
1.3 Mammalian system.
Diabetes is a hyperglycemic condition resulted either due insulin resistance (type II) or due autoimmune damage to the pancreatic beta cells (type I). In case of type I diabetes as large amount of insulin is required cell replacement therapy of either pancreatic cells or islets cell is widely applicable treatment. However immunosuppressive therapy, shortage of donors, low yields of insulin, large number of donor cells etc has lead to obstacle in the use of cell replacement therapy. In order to overcome this drawback different methodologies are incorporated like expression of insulin gene or insulin promoter factor in pluripotent cells of pancreas, differentiation of embryonic or adult stem cells into desired tissue15,16, 17,18.
The adult human liver cells have an astonishing capacity to differentiate into pancreas when transfected with pancreatic and duodenal homeobox gene-1 called as PDX-1. This developmental switching is an important factor in formation of pancreas and beta cells along with soluble factors i.e. SFs. In order to produce insulin secreting pancreatic cells the adult human liver cells are isolated and cultivated on complete DMEM medium and allowed to grow. These cells are then transfected with recombinant adenovirus which are replication deficient and encodes for cDNA of PDX-1, insulin-1 promoter from rat which is under the control of cytomegalovirus promoter. The human liver cells containing the recombinant adenovirus showed green fluorescence. Production of insulin was estimated by use of radioimmunoassay and increased seven folds after the treatment with SFs like EGF and nictotinamide17.
Human embryonic stem cells are capable of differentiating and can be grown under in vitro conditions resulting in specialized cells of adult tissue of interest under specific stimulation of substances that induces differentiation or feeder layer removal15,16 M12
The treatment of embryonic stem cell with activin A resulted initially in the formation of endoderm which was further differentiated into pancreatic cells on treatment with retinoic acid RA. These differentiated cells were later incubated in DMEM/F12 medium along with nicotinamide and after 5-8 days this cells were finally differentiated into beta cells which was determined by the presence of gene markers such as PDX-1, GLUT2 etc by RT-PCR19.
In order to meet large demand of insulin of increasing diabetic patients biopharmaceuticals have adopted a more reliable and promising technology for large scale insulin production using E.coli and yeast. Due to various obstacles in traditional methods and cost involved in insulin production novel method for recombinant insulin production are developed. Hence understanding various strategies using different host system plays a vital role in production of recombinant insulin.
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