Human proinsulin

We decided to investigate the self-association of human proinsulin, produced by recombinant DNA techniques, using large zone SEC. Due to the homology of human and porcine proinsulins, they would be expected to behave similarly under similar solution conditions. We repeated the experiment with human growth hormone described in the preceding section 

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FIGURE 15.3 Proinsulin is an 86-residue precursor to insulin (the sequence shown here is human proinsulin). Proteolytic removal of residues 31 to 65 yields insulin. Residues 1 through 30 (the B chain) remain linked to residues 66 through 87 (the A chain) by a pair of interchain disulfide bridges. 

J. S. Pati ick and A. L. Lagu, Determination of recombinant human proinsulin fusion protein produced in Escherichia coli using oxidative sulfitolysis and two-dimensional HPLC, Chem. 64 507-511 (1992). 

Figure 42-12. Structure of human proinsulin. Insulin and C-peptide molecules are connected at two sites by dipeptide links. An initial cleavage by a trypsin-like enzyme (open arrows) followed by several cleavages by a car-boxypeptidase-like enzyme (solid arrows) results in the production of the heterodimeric (AB) insulin molecule (light blue) and the C-peptide.

An alternative method (developed in the Eli Lilly research laboratories), entails inserting a nucleotide sequence coding for human proinsulin into recombinant E. coli. This is followed by purification of the expressed proinsulin and subsequent proteolytic excision of the C peptide in vitro. This approach has become more popular, largely due to the requirement for a single fermentation and subsequent purification scheme. Such preparations have been termed human insulin prb ... 

G Jager. Preparation of sequence 1-8 of human-proinsulin-peptide-C. (pGlu- from Z-Glu- hydrogenated in acetic acid). Chem Ber 106, 206, 1973. 

Human proinsulin has been synthesized in homogeneous solution from 11 protected fragments using azide coupling.1151 The difficulties with insoluble intermediates were sufficiently overcome to allow the 86-residue peptide to be synthesized. The product was de-protected, converted into the 5-sulfonate, and then reduced and reoxidized to form the three disulfide bonds. The product was extensively purified and analyzed, and shown to be pure proinsulin. This product could then be converted into insulin by the use of endopeptidases I and II from pancreatic (3-cell granules, together with carboxypeptidase H, which removed the four basic residues 31, 32, 64, and 65, and split out C-peptide.1 6 ... 

Insulin Preparations. Since diabetes mellitus is a defect of one or more of insulin production, secretion, or action, the administration of insulin replacement as a treatment for diabetes in the 1920s was a landmark discovery. Historically, most commercial insulin came from either bovine or porcine sources. Beef insulin differs from human insulin by three amino acid substitutions pork insulin differs by only one residue. For many years, standard insulin preparations were 70% beef and 30% porcine. However, the biosynthesis of human insulin has now displaced the animal insulins, especially bovine insulin which was more antigenic. Mass production of human insulin by recombinant DNA methods is achieved by inserting the human proinsulin gene into either E. coli or yeast and treating the resulting proinsulin to yield the human insulin molecule. Insulin preparations may be divided into four major types ... 

Mass production of human insulin and insulin analogs by recombinant DNA techniques is carried out by inserting the human or a modified human proinsulin gene into Escherichia coli or yeast and treating the extracted proinsulin to form the insulin or insulin analog molecules. 

Toma, A., Haddouk, S., Briand, J. P., Camoin, L., Gahery, H., et al. (2005) Recognition of a subregion of human proinsulin by class I restricted T cells in type 1 diabetic patients. Proc. Natl. Acad. Sci. USA 102, 10581-10586. 

Short, D. K, Okada, S., Yamauchi, K. and Pessin, J. E. (1998). Adenovirus-mediated transfer of a modified human proinsulin gene reverses hyperglycemia in diabetic mice. Am. J. Physiol. 275, E748-E756. 

A further example shows that one may add more amino acids to an amino acid ester -earbene complex by employing the customary methods of peptide chemistry (64). Starting from pentacarbonyl[GlyOMe (phenyl) -earbene]chromium(0) and using the. V-hydroxysuccinimide/dicyclohexyl-carbodiimide) (NHS/DCCD) method, we succeeded in synthesizing the sequence 14 to 17 of human proinsulin C-peptide (Scheme 3). 

Fig. 4. The amino acid sequences of human proinsulin, somatomedin C/IGF-I and IGF-II. From Ref 109...

Scheme 8.4 Stepwise synthesis of the tetrapeptide 18, containing the sequence 14-17 of the human proinsulin C-peptide, from the methoxy(phenyl)carbene complex 16 via the carbene chromium derivative 17 as the intermediate (Gly-OMe and Pro-OMe were the methyl esters of glycine and proline). The C—N coupling reactions in steps (1)—(4) were carried out using the DCCD/HOSU method, explained in [35]...

Indirect Two-Site Immunoradiometric Assay of Human Proinsulin. The method used is that described by Rainbow et al. Plastic tubes coated with purified guinea pig anti-insulin antibodies are prepared as described above 200-jul samples containing human proinsulin are added to these coated tubes and incubated at 4° for 24 hr. After removal of the sample, tubes are washed twice with 400 /tl of NIGP buffer. Rabbit antibody to human C-peptide is diluted to 1/1000 in 50 mM sodium phosphate buffer, pH 7.4, containing 150 mM sodium chloride, 10 g of bovine serum albumin per liter, and 100 mg of guinea pig IgG per liter 200 /til are added to each tube. After a further 24 hr of incubation at 4 the tubes are washed twice as previously and 200 /u,l of I-labeled sheep anti-rabbit IgG (10,000 cpm) are added in the same buffer as that used for diluting the C-peptide antiserum. After a final 24 hr of incubation and two further washes as above, the tubes are counted. 

Kuglin, B., Cries, F. A., and Kolb, H. (1988). Evidence of IgG autoantibodies against human proinsulin in patients with IDDM before insulin treatment. Diabetes 37, 130-132. 

Lindbladh C, Persson M, Buiow L, et al. The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion. Biochem Biophys Res Gommun 1987 149 607-14. 

Principle. Accurate measurement of proinsuJin has been difficult for several reasons the blood concentrations are low antibody production is difficult most antisera cross-react with insulin and C-peptide, which are present in much higher concentrations the assays measure intermediate cleavage forms of proinsuhn and reference preparations of pure proinsulin were not readily available. Therefore few accurate data are availabie in the hterature on plasma proinsulin. These problems have, to a large extent, been overcome by the availability of biosynthetic proinsulin, which has allowed the production of monoclonal antibodies to proin-sulin and provided reliable proinsulin calibrators and reference preparations. An International Reference Preparation for human proinsulin (code 84/611) is available from the National Institute of Biological Standards and Controls (Potters Bar, United Kingdom). Earlier assays may have overestimated proinsulin concentrations. ... 

Dhahir FJ, Cook DB, Self CH. Amplified enzyme-linked immunoassay of human proinsulin in serum (detection limit O.lpmol/L). Clin Chem 1992 38 227-32. 

Houssa P, Dinesen B, Deberg M, Prank BH, Van Schravendijk C, Sodoyez-Goffaux F, et al. First direct assay for intact human proinsulin. Clin Chem 1998 44 1514-9. 

Birnbaum S, Billow L, Hardy K, Danielsson B, Mosbach K (1986) Automated thermometric enzyme linked immunoassay of human proinsulin produced by Escherichia coli. Anal Bio-chem 158 12-19... 

Structures of human proinsulin and insulin. Insulin is derived from proinsulin by cleavage at the dipeptides Arg-Arg and Lys-Arg to give A and B chains held together by disulfide bonds. In the pig, B30 is Ala. In the cow, A8 is Ala, AlO is Val, and B30 is Ala. Bovine and porcine insulins are used extensively in clinical practice. 

The flow-through TELISA has been automated and used to monitor the production of human proinsulin by genetically manipulated Escherichia coli cells. Good agreement with the results of an RIA was obtained. The short analysis time, reusability, and automatability of the flowthrough TELISA technique offer further prospects for application in fermentation control and clinical chemistry. 

Cross-reactivity with proinsulin and insulin was determined by measuring recombinant human proinsulin (Sigma-aldrich Co.) and WHO approved reference material (International Reference Preparation Code 66/304, NIBSC). 

Oyer PE, Cho S, Peterson JD, Steiner DF. Studies on human proinsulin. J Biol Chem 1971 246 1375-86. 

In addition, genetically engineered enzyme conjugates have been used in immunoassays. Thus a human proinsulin- . coli alkaline phosphatase conjugate was used for the determination of insulin or proinsulin. Concentrations lower than 1 fig ml could be determined in less than 15 min [30]. 

Moore, H.P.H., Walker, M.D., Ue, F. and Kelly, R.B. (1983) Expressing a human proinsulin DNA in a mouse ACTH-secreting cell. Intracellular storage, proteolytic processing and secretion on stimulation. Cell 35 531-538. 

FIGURE 60-1 Human proinsuUn and its conversion to insulin. The amino acid sequence of human proinsulin is shown. By proteolytic cleavage, four basic amino acids (residues 31, 32, 64, and 65) and the connecting peptide are removed, converting proinsulin to insulin. The sites of action of the endopeptidases PC2 and PC3 are shown. 

Figure 11. DNA sequence of synthetic human proinsulin by Gilbert-Maxam method (gel-pattern).

Table III. Alignment of the Amino Acid Sequence of Mouse NGF with Those of Human Proinsulin and Guinea Pig Insulin to Show Their Homology"... 

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