Editing Insulin (section)

=== Structural analysis and synthesis ===
{{multiple image
 | align = right
 | direction = vertical
 | width = 200
 | image1 = Insulin monomer 4INS.png
 | alt1 = Black-and-white ribbon diagram of a pig insulin monomer.
 | caption1 = [[Ribbon diagram|Richardson diagram]] of a [[pig|porcine]] insulin monomer, showing its characteristic [[secondary structure]]. This is the biologically active form of insulin.
 | image2 = Insulin hexamer 4INS.png
 | alt2 = Black-and-white ribbon diagram of a pig insulin hexamer, showing its characteristic quaternary structure. At the center is a pale blue-gray sphere representing a zinc atom.
 | caption2 = Richardson diagram of a porcine insulin hexamer. The sphere at the center is a stabilizing [[zinc]] atom, surrounded by coordinating [[histidine]] residues. This is the form in which insulin is stored in beta cells. {{PDB|4INS}}.
}}
Purified animal-sourced insulin was initially the only type of insulin available for experiments and diabetics. [[John Jacob Abel]] was the first to produce the crystallised form in 1926.<ref>{{cite journal | vauthors = Abel JJ | title = Crystalline Insulin | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 12 | issue = 2 | pages = 132–6 | date = February 1926 | pmid = 16587069 | pmc = 1084434 | doi = 10.1073/pnas.12.2.132 | bibcode = 1926PNAS...12..132A | doi-access = free }}</ref> Evidence of the protein nature was first given by [[Michael Somogyi]], [[Edward Adelbert Doisy|Edward A. Doisy]], and [[Philip Anderson Shaffer|Philip A. Shaffer]] in 1924.<ref>{{Cite journal| vauthors = Somogyi M, Doisy EA, Shaffer PA |date = May 1924 |title=On the Preparation of Insulin |url= https://www.jbc.org/content/60/1/31.full.pdf |journal=Journal of Biological Chemistry |volume=60 |issue=1 |pages=31–58 |doi = 10.1016/S0021-9258(18)85220-6 |doi-access=free }}</ref> It was fully proven when Hans Jensen and Earl A. Evans Jr. isolated the amino acids phenylalanine and proline in 1935.<ref>{{cite journal| vauthors = Jensen H, Evans EA |date=1 January 1935|title=Studies on Crystalline Insulin Xviii. the Nature of the Free Amino Groups in Insulin and the Isolation of Phenylalanine and Proline from Crystalline Insulin |url= https://www.jbc.org/content/108/1/1.full.pdf |journal=Journal of Biological Chemistry |volume=108 |issue=1 |pages=1–9 |doi=10.1016/S0021-9258(18)75301-5|doi-access=free }}</ref>

The amino acid structure of insulin was first characterized in 1951 by [[Frederick Sanger#Sequencing insulin|Frederick Sanger]],<ref name="Stretton_2002" /><ref name="sanger">{{cite journal | vauthors = Sanger F, Tuppy H | title = The amino-acid sequence in the phenylalanyl chain of insulin. I. The identification of lower peptides from partial hydrolysates | journal = The Biochemical Journal | volume = 49 | issue = 4 | pages = 463–81 | date = September 1951 | pmid = 14886310 | pmc = 1197535 | doi = 10.1042/bj0490463 }}; {{cite journal | vauthors = Sanger F, Tuppy H | title = The amino-acid sequence in the phenylalanyl chain of insulin. 2. The investigation of peptides from enzymic hydrolysates | journal = The Biochemical Journal | volume = 49 | issue = 4 | pages = 481–90 | date = September 1951 | pmid = 14886311 | pmc = 1197536 | doi = 10.1042/bj0490481 }}; {{cite journal | vauthors = Sanger F, Thompson EO | title = The amino-acid sequence in the glycyl chain of insulin. I. The identification of lower peptides from partial hydrolysates | journal = The Biochemical Journal | volume = 53 | issue = 3 | pages = 353–66 | date = February 1953 | pmid = 13032078 | pmc = 1198157 | doi = 10.1042/bj0530353 }}; {{cite journal | vauthors = Sanger F, Thompson EO | title = The amino-acid sequence in the glycyl chain of insulin. II. The investigation of peptides from enzymic hydrolysates | journal = The Biochemical Journal | volume = 53 | issue = 3 | pages = 366–74 | date = February 1953 | pmid = 13032079 | pmc = 1198158 | doi = 10.1042/bj0530366 }}</ref>  and the first synthetic insulin was produced simultaneously in the labs of [[Panayotis Katsoyannis]] at the [[University of Pittsburgh]] and [[Helmut Zahn]] at [[RWTH Aachen University]] in the mid-1960s.<ref name="Katsoyannis_1964">{{cite journal|vauthors=Katsoyannis PG, Fukuda K, Tometsko A, Suzuki K, Tilak M|year=1964|title=Insulin Peptides. X. The Synthesis of the B-Chain of Insulin and Its Combination with Natural or Synthetis A-Chin to Generate Insulin Activity|journal=Journal of the American Chemical Society|volume=86|issue=5|pages=930–32|doi=10.1021/ja01059a043}}</ref><ref name="pmid5881570">{{cite journal | vauthors = Kung YT, Du YC, Huang WT, Chen CC, Ke LT | title = Total synthesis of crystalline bovine insulin | journal = Scientia Sinica | volume = 14 | issue = 11 | pages = 1710–6 | date = November 1965 | pmid = 5881570 }} {{free access}}</ref><ref name="Marglin_1966">{{cite journal | vauthors = Marglin A, Merrifield RB | title = The synthesis of bovine insulin by the solid phase method | journal = Journal of the American Chemical Society | volume = 88 | issue = 21 | pages = 5051–2 | date = November 1966 | pmid = 5978833 | doi = 10.1021/ja00973a068 }}</ref><ref>{{cite journal | vauthors = Costin GE | title = What is the advantage of having melanin in parts of the central nervous system (e.g. substantia nigra)? | journal = IUBMB Life | volume = 56 | issue = 1 | pages = 47–9 | date = January 2004 | pmid = 14992380 | doi = 10.1080/15216540310001659029 | doi-access =free  | publisher = Time Inc. | s2cid = 85423381 }}</ref><ref name="isbn1-4020-0655-1">{{cite book |vauthors = Wollmer A, Dieken ML, Federwisch M, De Meyts P | title = Insulin & related proteins structure to function and pharmacology | publisher = Kluwer Academic Publishers | location = Boston | year = 2002 | isbn = 978-1-4020-0655-5 | url = https://books.google.com/books?id=Ula72_FSwy8C&q=Panayotis%20Katsoyannis&pg=PP11 }}</ref> [[Synthetic crystalline bovine insulin]] was achieved by Chinese researchers in 1965.<ref name="Zou2015">{{cite journal | vauthors = Tsou CL |author-link1=Chen-Lu Tsou |script-title=zh:对人工合成结晶牛胰岛素的回忆 |trans-title=Memory on the research of synthesizing bovine insulin|journal= 生命科学 [Chinese Bulletin of Life Science] |year=2015 |volume=27 |issue=6 |language=zh-hans |pages=777–79}}</ref> The complete 3-dimensional structure of insulin was determined by [[X-ray crystallography]] in [[Dorothy Hodgkin]]'s laboratory in 1969.<ref name=":2">{{cite journal | vauthors = Blundell TL, Cutfield JF, Cutfield SM, Dodson EJ, Dodson GG, Hodgkin DC, Mercola DA, Vijayan M | title = Atomic positions in rhombohedral 2-zinc insulin crystals | journal = Nature | volume = 231 | issue = 5304 | pages = 506–11 | date = June 1971 | pmid = 4932997 | doi = 10.1038/231506a0 | bibcode = 1971Natur.231..506B | s2cid = 4158731 }}</ref>

Hans E. Weber discovered preproinsulin while working as a research fellow at the University of California Los Angeles in 1974. In 1973–1974, Weber learned the techniques of how to isolate, purify, and translate messenger RNA. To further investigate insulin, he obtained pancreatic tissues from a slaughterhouse in Los Angeles and then later from animal stock at UCLA. He isolated and purified total messenger RNA from pancreatic islet cells which was then translated in oocytes from ''[[Xenopus laevis]]'' and precipitated using anti-insulin antibodies. When total translated protein was run on an SDS-polyacrylamide gel electrophoresis and sucrose gradient, peaks corresponding to insulin and proinsulin were isolated. However, to the surprise of Weber a third peak was isolated corresponding to a molecule larger than proinsulin. After reproducing the experiment several times, he consistently noted this large peak prior to proinsulin that he determined must be a larger precursor molecule upstream of proinsulin. In May 1975, at the American Diabetes Association meeting in New York, Weber gave an oral presentation of his work<ref>Weber, H.E. (1975) Diabetes 24, 405. (see figure)</ref> where he was the first to name this precursor molecule "preproinsulin". Following this oral presentation, Weber was invited to dinner to discuss his paper and findings by [[Donald Steiner]], a researcher who contributed to the characterization of proinsulin.  A year later in April 1976, this molecule was further characterized and sequenced by Steiner, referencing the work and discovery of Hans Weber.<ref>Chan SJ, Keim P, Steiner DF.  Cell-free synthesis of rat preproinsulins: Characterization and partial amino acid sequence determination.  Proc Natl Acad Sci. USA 1976;73:1964-1968.</ref> Preproinsulin became an important molecule to study the process of transcription and translation.

The first genetically engineered ([[Recombinant DNA|recombinant]]), synthetic human{{efn|Molecularly identical to human insulin: same sequence, same set of [[post-translational modifications]].}} insulin was produced using [[Escherichia coli|''E. coli'']] in 1978 by [[Arthur Riggs (geneticist)|Arthur Riggs]] and [[Keiichi Itakura]] at the [[Beckman Research Institute]] of the [[City of Hope National Medical Center|City of Hope]] in collaboration with [[Herbert Boyer]] at [[Genentech]].<ref name="urlGenentech" /><ref name="urlRecombinant DNA technology in the synthesis of human insulin" /> Genentech, founded by Swanson, Boyer and [[Eli Lilly and Company]], went on in 1982 to sell the first commercially available biosynthetic human insulin under the brand name [[Humulin]].<ref name="urlRecombinant DNA technology in the synthesis of human insulin"/> The vast majority of insulin used worldwide is biosynthetic recombinant human insulin or [[Insulin analog|its analogues]].<ref name="pmid23222785" /> Recently, another recombinant approach has been used by a pioneering group of Canadian researchers, using an easily grown [[safflower]] plant, for the production of much cheaper insulin.<ref>{{Cite web|url=https://www.ctvnews.ca/new-source-of-insulin-blossoming-on-the-prairies-1.479043|title=Safflowers may provide new insulin source {{!}} CTV News|website=www.ctvnews.ca|access-date=12 November 2019|date=February 2010}}</ref>

Recombinant insulin is produced either in yeast (usually ''[[baker's yeast|Saccharomyces cerevisiae]]'') or ''E. coli''. In yeast, insulin may be engineered as a single-chain protein with a [[Kexin|KexII endoprotease]] (a yeast homolog of PCI/PCII) site that separates the insulin A chain from a C-terminally truncated insulin B chain.  A chemically synthesized C-terminal tail containing the missing threonine is then grafted onto insulin by reverse proteolysis using the inexpensive protease trypsin;<ref name="pmid11030562">{{cite journal | vauthors = Kjeldsen T | title = Yeast secretory expression of insulin precursors | journal = Applied Microbiology and Biotechnology | volume = 54 | issue = 3 | pages = 277–86 | date = September 2000 | pmid = 11030562 | doi = 10.1007/s002530000402 | s2cid = 9246671 | url = http://w3.ualg.pt/~jvarela/biotecnol/pdf/humaninsulin.pdf | archive-url = https://web.archive.org/web/20170927000623/http://w3.ualg.pt/~jvarela/biotecnol/pdf/humaninsulin.pdf | archive-date = 27 September 2017 }}</ref> typically the lysine on the C-terminal tail is protected with a chemical protecting group to prevent proteolysis.  The ease of modular synthesis and the relative safety of modifications in that region accounts for common insulin analogs with C-terminal modifications (e.g. lispro, aspart, glulisine).  The Genentech synthesis and completely chemical synthesis such as that by [[Bruce Merrifield]] are not preferred because the efficiency of recombining the two insulin chains is low, primarily due to competition with the precipitation of insulin B chain.