Insulin chain

The initial approach to recombinant insulin production taken entailed inserting the nucleotide sequence coding for the insulin A- and B-chains into two different E. coli cells (both strain K12). These cells were then cultured separately in large-scale fermentation vessels, with subsequent chromatographic purification of the insulin chains produced. The A- and B-chains are then incubated together under appropriate oxidizing conditions in order to promote interchain disulfide bond formation, forming human insulin crb ... 

Jimenez et al. (2002) proposed a molecular model for the insulin protofilament based on these data and on electron cryomicroscopy (cryo-EM) reconstructions of insulin fibrils. The fibrils show a number of twisted morphologies that seem to be alternative packings of similar protofilaments. The protofilaments have cross sections of 30x40 A. The authors suggest a complete conversion to / -structure and model the amyloid monomer as having four jS-strands (Fig. 3B). Each insulin chain contributes two of these jS-strands, and the chains align in a parallel stack, constrained by the interchain disulfide bonds. One pair of stacked /i-stran ds is curved... 

DNA coding for the 21-amino-acid chain and the other carrying the DNA sequence coding for the 30-amino-acid chain. In both plasmids, the DNA sequence was linked to instructions for another protein, an enzyme protein. The bacteria produced two fused proteins of enzyme and one of the two insulin chains. 

Other proteins show much bigger changes [e.g. the tyrosine of carb-oxypeptidase1 and the ends of the insulin chains (Hodgkin2)]. Still others seem to show gross changes [e.g., phospholipases (our own unpublished work)]. Finally, there are proteins that are almost random—chromogranin A and phosphovitin.3... 

Isocratic RPC was also successful in separating the position isomers of mono-iodinated insulin. The 125-iodination by the lactoperoxidase method was carried out in phosphate buffer containing 6 M urea and led to the iodination of tyrosyl units, i.e., to substitution in the positions A14, A19, B16, or B26. The retention of the mono-iodinated insulin chains increased in the order A19 < B26 < B16 < A14 on a C 18 column (150 x 4.0 mm dP = 3 pm). The eluent contained 1 % triethylammonium tri-fluoroacetate in water (pH 3) and acetonitrile78). 

One of the requirements in MALDI-MS analysis is the use of a liquid matrix. The electrowetting-on-dielectric (EWOD) method has been used to move and mix droplets containing proteins and peptides with the liquid matrix, all of which were situated at specific locations on an array of electrodes. With this method, insulin (1.75 pM), insulin chain B (2 pM), cytochrome c (1.85 pM), and myoglobin (1.45 pM) have been analyzed [518]. 

In a later paper (1949), Sanger described how he used these techniques to determine the first few amino acids (amino-terminal end) of each insulin chain. To analyze the B chain, for example, he carried... 

Immediately decapitated and injected with insulin chain A (1.45 nmol)... 

Fig. 12. Evolution of insulin chains. Plots of the sequence divergence of replacement (A) and silent (B) substitutions against divergence time, x, A-chains +, B-chains O, signal peptides. Reprinted, with permission, from Hahn et al. (1983).

Separation of the 131/-labelled S-sulphonated A and B insulin chains and of125l-labelled aromatic sulphonic acids... 

INOC = [(4-pyridinylmethoxy)carbonyl)], 241 myo-Inositol selective glycosylation, 270 Inserted DNA sequences, 243-245 Insulin synth. by genetic engineering, 244-245 Insulin -chain chemical synthesis, 239-240 Introns, 242... 

Di Abietes. Di Abietes treatment was first changed to daily injections j of Humulin instead of beef insulin. Humulin is now mass-produced by recombinant DNA techniques that insert the human DNA sequences for the insulin A and B chains into the Escherichia coli or yeast genome (see Chapter 17). The insulin chains that are produced are then extracted from the media and treated to form the appropriate disulfide bonds between the chains. As costs have fallen for production of the synthetic human insulins, they have replaced pork insulin and the highly antigenic beef insulin. 

B) Arginine is a basic amino acid that binds to the a-carboxylic acid groups at the N-terminals of insulin chains. 

E) Arginine has a side chain that forms peptide bonds with the carboxyl terminals of the insulin chains. 

Recombinant DNA techniques are used to produce proteins that have therapeutic properties. One of the first such proteins to be produced was human insulin. Recombinant DNA corresponding to the A chain of human insulin was prepared and inserted into plasmids that were used to transform E. coli cells. The bacteria then synthesized the insulin chain, which was purified. A similar process was used to obtain B chains. The A and B chains were then mixed and allowed to fold and form disulfide bonds, producing active insulin molecules (Fig. 17.11). Insulin is not glycosylated, so there was no problem with differences in glycosyltransferase activity between E. coli and human cell types. 

Ans. The amino acid residue in the pig insulin chain must be in a portion of the chain whose conformation is not greatly affected by whether the residue is Thr or Ala, or that portion of the protein is not involved in the physiological functioning of insulin. 

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