Insulin precursors

Kjeldsen, T. 2002. Yeast secretory expression of insulin precursors. Applied Microbiology and Biotechnology 54, 277-286. 

There are several other examples of enzyme peptide synthesis. The conversion of porcine insulin into a human insulin precursor, by replacing the B chain C-terminal with threonine, is still an important alternative to microbially produced human insulin. Peptides that are produced from ethyl esters of L-amino acids by BioEurope are used as ingredients of cosmetics. 

Treatment of patients with diabetes mellitus for the control of hyperglycemia often used to produce a more rapid onset and shorter duration of action than human regular insulin GI 124617 insulin precursor PID gl24617 Lys(B28)Pro(B29)-human insulin PDB ID ILPH... 

Recombinant insulin precursor MI3 Study of the target protein per se and selection of appropriate binding sites Solid-phase combinatorial chemistry (64 ligands) solution-phase synthesis of a sub-library Affinity chromatography SPR 15,18,19... 

Markussen J, Fiil N, Hansen MT et al (1990) DNA-sequence encoding biosynthetic insulin precursors and process for preparing the insulin precursors and human insulin. US Patent 4,916,212... 

Peptides from the insulin precursor are good examples for peptide biomarkers present in blood. Insulin itself is used as a therapeutic compound but, at the same time, it is also used as a biomarker to diagnose diabetes (Table 6.2). In addition, byproducts released during hormone processing are used as diagnostics. 

The insulin molecule consists of 51 amino acids arranged in two chains, an A chain (21 amino acids) and B chain (30 amino acids), that are linked by two disulfide bonds. Proinsulin is the insulin precursor that is first processed in the Golgi apparatus of the beta cell where it is processed and packaged into granules. Proinsulin, a single-chain 86-amino acid peptide, is cleaved into insulin and C-peptide, a connecting peptide. These are secreted in equimolar portions... 

Swinn RA, Wareham NJ, Gregory R, Curling V, Clark PM, Dalton KJ, et al. Excessive secretion of insulin precursors characterizes and predicts gestational diabetes. Diabetes 1995 44 911-5. 

Fig. 13.1 S. cerevisiae expression vector used for production of recombinant insulin. The S. cerevi-siae/E. coli shuttle vector is composed of the following genetic units 1) The transcription promoter and terminator of the S. cerevisiae TPIl (triose phosphate isomerase) gene flanking the DNA encoding the leader-insulin precursor. 2) The TPIl gene from Schizosaccharomyces pombe (TPIlp)...

The three N-linked glycosylation sites of the a-leader have been shown to be important - but not essential - for the ability of the a-factor leader to secrete a-factor [32, 33], In addition, mutation of all three-consensus sites for N-linked glycosylation decreased the quantity of secreted insulin precursor to 10% [34], In contrast to what was found with the a-leader, elimination of the two consensus sites for N-linked glycosylation in one of the newly developed leaders, actually improved the ability to facilitate secretion of the insulin precursor [31],... 

Fig. 13.4 Conversion of insulin precursor to recombinant human insulin using transpeptidation. The C-peptide is removed from the partially purified insulin precursor using a serine protease (e.g., trypsin). By inclusion of a threonine ester (T ) and using appropriate reaction conditions, it is possible to couple threonine to Lys using...

Polymerization of the Insulin Precursor Influences Secretion Yield and Retention in the Vacuole... 

The polymerization of insuHn and proinsulin to dimers and hexamers is a very important process which takes place in the pancreas and impacts upon the pharmaceutical application of insuHn, because the dissociation of the polymers is the rate-limiting processes in the absorption and action in the tissues of the biological active monomeric insulin [45]. The polymerization diminishes osmotic pressure and hy-drophobicity and improves solubility. A positive correlation between expression yield and the degree of polymerization was found [46] that supports yeast in vivo polymerization of insulin precursors and accentuates its importance. However, polymerization can also account for a drawback by retention of product in the vacuole [47]. Intracellular retention of a substantial quantity of the synthesized insulin precursor indicated that the insulin precursor followed two different intracellular routes in the late secretory pathway [12, 47]. Constitutive secretion to the culture supernatant may reflect saturation of a sorting mechanism in the late Golgi due to overexpression. The kexin cleaves the leader-insulin precursor peptide in a late Golgi compartment to yield free insulin precursor, and... 

Insulin precursor expression system 1039 Insulin-like growth factor type 2 receptor (IGF2R) 218 Insuman 13, 470 Insuman basal 469 Interaction discovery mapping 1325 Interannular heterobivalent inhibitor 403 Interannular homobivalent inhibitor 400 interferon (IFN) 4, 466, 1253... 

Protnsuttn. Single chain insulin precursor consisting of the insulin A and B chains and a connecting polypeptide (C-peptide). which contains 30-35 amino acids the number and sequence of these amino adds are species da -pendent. Its presence was discovered in a human islet cell adenoma D. F. Steiner, P. E. Oyer. Proc. Nat, Acad. Set USA 57, 473 (1967). Conversion of proinsulin to insulin has a half-time of about 1 hour in rat islets in vitro it is postulated that proteolytic enzymes cleave proinsulin at the sites... 

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