Insulin cloning

Human insulin was the first animal protein to be made in bacteria in a sequence identical to the human pancreatic peptide. Expression of separate insulin A and B chains were achieved in Echerichia coli K-12 using genes for the insulin A and B chains synthesized and cloned in frame with the... 

A solution to the problem of introns is to isolate mRNA extracted from the human pancreas cells that make insulin. These cells are rich in insulin mRNA from which introns have already been spliced out. Using the enzyme reverse transcriptase it is possible to convert this spliced mRNA into a DNA copy. This copy DNA (cDNA), which carries the uninterrupted genetic information for insulin can be cloned. Although yeast cells (Saccharomyces) can splice out introns it is normal practice to eliminate them anyway by cDNA cloning. 

Once a gene is cloned it is necessary to convert the information contained in it into a functional protein. There are a number of steps in gene expression (i) transcription of DNA into mRNA (ii) translation of the mRNA into a protein sequence and (iii) in some instances, post-translational modification of the protein. In discussing these steps in more detail, expression of a cloned insulin gene will be used as an example. 

Fig. 24.5 Insertion of a cloned insulin gene into a vector carrying a bacterial promoter. The arrow indicates the direction of transcription. If we suppose the bacterial promoter is derived from the lactose operon then transcription will be initiated only in the presence of lactose.

Fukumoto, H., et al. Cloning and characterization of the major insulin-responsive glucose transporter expressed in human skeletal muscle and other insulin-responsive tissues. J. Biol. Chem. 1989, 264, 7776-7779. 

The first step in biopharmaceutical development is the selection of a clone in a specific cell line. Whole-mass analysis, if possible, is a fairly simple and powerful tool at this stage to verify the successful expression and translation of the desired protein. VanAdrichem et al.65 described the use of MALDI MS to monitor protein expression in several mammalian cell lines like CHO DXB11, CHO SSF3, and hybridomas. Quantitative MALDI-TOF MS measurements of an IgG antibody and insulin during large-scale production in hybridoma cells were comparable to affinity chromatography results. 

Ishihara, H., Sasaoka, T, Hori, H., Wada, T., Hirai, H., Harata, T, Langjois, W.J., and Kohayashi, M., 1999, Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin siganhng. Biochim. Biophys. Res. 

Along with the production of insulin, many other medical uses have been achieved for recombinant DNA. This includes the production of erythropoetin, a hormone used to stimulate production of red blood cells in anemic people tissue plasminogen activator, an enzyme that dissolves blood clots in heart attack victims and antihemophilic human factor VIII, used to prevent and control bleeding for hemophiliacs. These three important genetically engineered proteins were all cloned in hamster cell cultures. 

Figure 4.2. cloning of insulin from cDNA isolated from pancreas, the main tissue responsible for synthesis of insulin, and inserted into plasmid vectors that permit expression in host cells. 

Roep, B. O., Arden, S. D., de Vries, R. R. P., and Hutton, J. C. (1990). T-cell clones from type 1 diabetes patient respond to insulin secretory granule proteins. Nature (London) 345, 632-634. 

Rattus norvegicus (Sprague-Dawley [14,21] female Sprague-Dawley [26] male Wistar [28] clone 9 cells express higher amounts of the enzyme after insulin treatment [25]) [1, 3, 7, 10-14, 16, 18, 21-28]... 

Nellis, M.M. Doering, C.B. Kasinski, A. Danner, D.J. Insulin increases branched-chain cr-ketoacid dehydrogenase kinase expression in Clone 9 rat cells. Am. J. Physiol., 283, E853-E860 (2002)... 

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