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,Th~: Chemical World This Week kTEREL [ULEE1 UC1EGN EELtRS REIt!J Recombinant DNA research advo- ~,. cates have been~ promising insulin F-production as one of the practical achievements that the new field ,might bring. And several research t;;groups in the U.S. and Canada are .r!f _. r, speeding toward that end of making insulin in the test tube with a little help from some microbes. A team of scientists led by Dr. Walter Gilbert of Harvard University has just edged ± into the lead. That team has a rat in- sulin gene funcfioning in the bacte- rium Escherichia coli. ~.; A number of technical hurdles still .remain before microbial insulin pro- duction approaches pilot plant status. But the hardest parts of the run seem finished. That does not mean that the race is won, however. At least four groups are working feverishly, each with its own strategy for achieving insulin pro- duction. The short-term goal is _ making animal insulin, and Gilbert's group is very close to that. Eventual- ly, though, human insulin is desired, because its amino acid sequence dif- fers significantly from rat insulin. Presently, some of the most direct paths to making human insulin by recombinant DNA technology are not open. For example, in the U.S. certain kinds of experiments must be done in laboratories with maximum safety features, designated P4, according to National Institutes of Health guide- lines. But the one such certified lab, the Frederick (Md.) Cancer Center, simply is not available now for these = experiments. - Gilbert's group did some experi- ments in a P3 lab on the Massachu- setts Institute of Technology campus. Only a few certified P3 labs exist, scattered around the country, and their fairly stringent safety features are required for much of the research in which animal genes are moved into bacteria. Gilbert's coworkers include Dr. William Chick and Dr. Stephen Naber of the Joslin Diabetes Foun- dation of Boston in addition to a group at Harvard, Their present •uccess, to a large extent, overlaps a ~series of achievements announced' over the past 14 months. A look at Gilbert's work in that context pro- vides some clues as to how the re- maining problems might be solved. For example, Gilbert's group has F '.s3.. been working with the insulin gene from the rat. It is the rat insulin gene that first was moved into E. coli slightly more than a year ago by a team at the University of California, San Francisco, led by Dr. William J. Rutter and Dr. Howard A. Goodman (C&EN, May 30, 1977, page 4). Un- like the California group, Gilbert's group used pancreas tumor cells that overproduce insulin. The tumor cells. also produce more messenger RNA (mRNA) for insulin~ and its precursor protein, proinsulin. That makes the task of finding the message and con- verting it into DNA easier. The California group reported no insulin production. But by fall last year, news from another research group at the University of California, San Francisco, leaked out in Con- gressional hearings. That group had coaxed E. coli into making another, smaller peptide hormone (C&EN, Nov. 7, 1977, page 4). In this case, Dr. Herbert Boyer of UCSF and colleagues from there and from the City of Hope Medical Center in Los Angeles fooled E. coli into making the small hormone somato= statin with some elegant sleight of hand. The nucleotide sequence for that hormone was made chemically, and the sequence was made to serve the bacterium and the scientists' preferences. For instance, a sequence designating the amino acid methio- nine was put in. Then the entire hor- mone sequence was fused to a bacte- . 1 f? rial gene for the enzyme 0;galacto- sidase. That protein normally is excreted from the cell, and tKus this fusion provides a way to "wrap" the hormone for export. Once outside the cell, the hormone-still part of the sequence of the larger enzyme-was separated from its wrapper by a sim- ple chemical procedure, dependent on the extra methionine. I Insulin contains about 70 amino acids in two separate chains joinedby sulfhydryl bridges. In pancreas cells, it's made first as a single, larger chain from whi& the connecting piece is cut. Thus, it presents a more difficult problem than the much shorter so- matostatin. = Gilbert's group bypassed some of that problem and postponed another part. By using the rat proinsulin gene, it circumvented the difficulties of chemical synthesis of the large insulin gene. But the bacteria containing the gene does not make free insulin or proinsulin. Instead, the proinsulin is made with a "wrapper" (as in the so- matostatin case), the enzyme peni- cillinase. Though not the same en- zyme, like 0-galactosidase, penicil- linase is excreted from cells. However, because the Harvard group did not make the proinsulin gene chemically, there was no simple way to put in methionine and hence no simple way to remove the penicillinase. Proinsulin was detected by anti- bodies, and it is in fact bound cova- lently to penicillinase when excreted Events leading to microbial insulin production May 1977 Rat insulin gene incorporated in E. coli at University of California, San Francisco; no gene expression November 1977 E. coli'reported to take up DNA from higher cells in work at Stanford University November 1977 Clinically synthesized gene for somatostatin fused to E. coii enzyme gene at UCSF; gene expression obtained January 1978 Bacterial gene moved into yeast at Cornell UniversRy; gene stable but expression doubtful March 1978 Hormone-controlled gene for ovalbumin from chicken incorporated in E. coli at Baylor University;no expres- sion reported so far •

;Y1 Ix r': . 9 ro the bacterial cells. That mole fm cule is several steps removed from becoming free insulin. Though each 'Tof those steps can be done, the pro- I cedures are impractical. Also, Gil- bert's group says that the bacteria `make only about 100 copies of this l a One re earch group, whi hlincludes I x~w ~f _ : Dr. Saran A. Narang of the National Research Council of Canada and Dr. Ray Wu of Cornell University in Ith- Y~~ µ"'L aca, N.Y., has hopes of finessing the ~ `' roblem of making human insulin b f' ~ ~ p ,~~y ~ j making that gene chemically. That s„yf strategy also eliminates the bottle- ~ neck of waiting for a P4 facility. h Chemical synthesis of auman gene ' not restricted by regulations so the , , 'ear1y- stages of the research can pro- ' -•' ceed without restriction. Any re- '~ strictions in putting such material into bacteria may be dealt with later, and more P41abs might be readyby" then. In unconfirmed reports, C&EN ,pt` h ld tht B's i S asearneaoyer groupnan Francisco is working along similar ` lines as Narang,and' Wu, progressing :joward 'chemical synthesis of the human~ insulin gene. o l~t~/..'....... ' .. `l f•!a+' ~i . z3437:t1ta2 g J~. t;'?2 ~ ry ts`i~s I=e, ~ !/ .kL.n4r > f .' f ~ S t6~ r Y tS. s._ f ll..1J tl _';,.i ~n~' J~ <t.. r S' t :'Y 3 p!!a 1 i> ~ t3.'d tTaC '1.:} ~' .iJit~ 2 kf~= ti kLt.x}LC rz'r 2 r) +•it _a+}1.~~fff'~34~s~: j-l.`~ ~ ^~ iJ .L:~wn.~ :a , . n A. . .. ^~ .y. l I ..4 .x t -- .sr,•r q v--~ : . . . - . . .. . ... . f. . . .. ..,.