bacterial insulin production nears reality

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The Chemical World This Week BACTERIAL INSULIN PRODUCTION NEARS REALITY Recombinant DNA research advo- cates have been promising insulin production as one of the practical achievements that the new field might bring. And several research groups in the U.S. and Canada are 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 functioning 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 oil 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 success, 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 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- rial gene for the enzyme 0-galacto- sidase. That protein normally is excreted from the cell, and thus 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. Insulin contains about 70 amino acids in two separate chains joined by sulfhydryl bridges. In pancreas cells, it's made first as a single, larger chain from which 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. coli enzyme gene at UCSF; gene expression obtained January 1978 Bacterial gene moved into yeast at Cornell University; 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 4 C&EN June 19, 1978

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The Chemical World This Week

BACTERIAL INSULIN PRODUCTION NEARS REALITY Recombinant DNA research advo­cates have been promising insulin production as one of the practical achievements that the new field might bring. And several research groups in the U.S. and Canada are 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 in­sulin gene functioning in the bacte­rium Escherichia coli.

A number of technical hurdles still remain before microbial insulin pro­duction 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 pro­duction. The short-term goal is making animal insulin, and Gilbert's group is very close to that. Eventual­ly, though, human insulin is desired, because its amino acid sequence dif­fers 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 guide­lines. But the one such certified lab, the Frederick (Md.) Cancer Center, simply is not available now for these experiments.

Gilbert's group did some experi­ments in a P3 lab oil the Massachu­setts 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 Foun­dation of Boston in addition to a group at Harvard, Their present success, to a large extent, overlaps a series of achievements announced over the past 14 months. A look at Gilbert's work in that context pro­vides some clues as to how the re­maining problems might be solved.

For example, Gilbert's group has

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). Un­like 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 con­verting 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 Con­gressional 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 somato­statin 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 methio­nine was put in. Then the entire hor­mone sequence was fused to a bacte­

rial gene for the enzyme 0-galacto-sidase. That protein normally is excreted from the cell, and thus 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 sim­ple chemical procedure, dependent on the extra methionine.

Insulin contains about 70 amino acids in two separate chains joined by sulfhydryl bridges. In pancreas cells, it's made first as a single, larger chain from which the connecting piece is cut. Thus, it presents a more difficult problem than the much shorter so­matostatin.

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 so­matostatin case), the enzyme peni­cillinase. Though not the same en­zyme, like 0-galactosidase, penicil­linase 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 anti­bodies, 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. coli enzyme gene at UCSF; gene expression obtained

January 1978 Bacterial gene moved into yeast at Cornell University; gene stable but expression doubtful

March 1978 Hormone-controlled gene for ovalbumin from chicken incorporated in E. coli at Baylor University; no expres­sion reported so far

4 C&EN June 19, 1978

from the bacterial cells. That mole­cule is several steps removed from becoming free insulin. Though each of those steps can be done, the pro­cedures are impractical. Also, Gil­bert's group says that the bacteria make only about 100 copies of this amalgamated protein per cell.

One research group, which includes Dr. Saran A. Narang of the National Research Council of Canada and Dr. Ray Wu of Cornell University in Ith­aca, N.Y., has hopes of finessing the problem of making human insulin by making that gene chemically. That strategy also eliminates the bottle­neck of waiting for a P4 facility. Chemical synthesis of a human gene is not restricted by regulations, so the early stages of the research can pro­ceed without restriction. Any re­strictions in putting such material into bacteria may be dealt with later, and more P4 labs might be ready by then. In unconfirmed reports, C&EN has learned that Boyer's group in San Francisco is working along similar lines as Narang and Wu, progressing toward chemical synthesis of the human insulin gene. •

Chemical stocks match spring market pickup Common stocks of leading U.S. basic chemical producers continue very much in the middle of the pack in 1978 after several years on either end. This spring, the middle was not a bad place to be, since the entire stock market went through a sharp and completely unexpected upsurge.

C&EN's index of leading chemical stocks climbed 14% to 193 (1954 = 100) by the end of the second week of June from 169 just before the dra­matic upswing began in April. This index is a weighted average for Allied Chemical, American Cyanamid, Cel-anese, Dow Chemical, Du Pont, Monsanto, and Union Carbide.

The rise in C&EN's index this spring compares quite well with the 12% increase in the widely followed index of Dow Jones 30 Industrials (DJI) during the same stretch. The DJI, which includes Allied, Du Pont, and Union Carbide, climbed nearly 90 points in this period to 859.

Despite keeping slightly ahead of the general stock market pace this spring, chemical stocks have not re­gained much of their luster from the glamour days of 1975. Wall Street security analysts are beginning to recommend attention to chemical equities again, but with restraint. The basic reason is that chemical pro­ducers are still caught in a greater profit-pinching cost-price squeeze

than are most other manufacturers. Behind this squeeze is a greater than average unused production capacity in important basic chemicals.

The result is that chemical stocks are still much further down from their peak in recent years than are indus­trial stocks. C&EN's chemical stock index is now 37% below its record of 307 in February 1976.

Among individual chemical stocks, performance has varied in the spring market offensive. The top performer has been American Cyanamid, a stock noted for stable movement in a nar­row range. Cyanamid's 26% gain this spring puts the stock actually 3% ahead of its former peak in 1975.

The next-best stock performance among the seven largest chemical firms is Dow's. Long-suffering Dow common stock rose 17% from April to June but is still 53% under its early 1976 peak.

After Dow, close to average stock price increases have come this spring for Celanese, Du Pont, and Monsan­to. Du Pont stock rose 15%, and the other two, 12%.

Laggards this spring among large chemical producers have been Allied Chemical and Union Carbide. Allied stock rose 9% from before the market surge to early June and now is 24% less than the recent peak in 1974. Carbide stock rose just 4% and re­mains 47% off from early 1976.

What will happen now? Explana­tions for the spring market push are somewhat vague, having to do largely with technical factors such as "excess bearishness" and large uncommitted cash reserves at mutual funds and others. Actually, the big market jump is hard to rationalize in view of steeply rising interest rates, which create competition from bonds and possibly presage an economic down­turn. Optimists say that the market already has discounted a downturn, that rising interest rates will quell inflation, and that caution is some­how too high for the market to drop. Pessimists aren't so sure. •

U.S. group impressed by China's technology "China is developing one hell of a petrochemical industry," says Dr. Alan Schriesheim, director of Exxon Research & Engineering Co.'s corpo­rate research labs. "China is making a really first class effort in pharma­ceutical chemistry, particularly ex­ploring development of pharmaceu­ticals from traditional Chinese herbs and medicinal materials," notes Dr. Ronald C. Breslow, chairman of the department of chemistry at Columbia

Peking petrochemical complex visited by U.S. group has sign saying "Follow Chairman Mao's revolutionery lines and march forward victoriously"

University. "I was really amazed by the great progress made in all areas. The state of their technology is a lot more advanced than I had antici­pated," comments Dr. Jacob Bigele-isen of the University of Rochester.

These impressions come from members of the first U.S. delegation in pure and applied chemistry to visit China, who returned last week from an intensive three-and-a-half-week tour of some 45 research institutes, universities, and chemical plants. Their visit is part of an ongoing ex­change program between the Com­mittee on Scholarly Communication with the People's Republic of China (CSCPRC) and China's Scientific & Technical Association.

The trip had two aims: to see what Chinese scientists are doing (each group member will assess a chemical subfield in a joint published report); and to explore the possibilities for closer future relations between U.S. and Chinese chemists (the group re­ceived a bit of encouragement).

The top-flight delegation included 10 chemists, an expert on Chinese science policy, and a CSCPRC staff member. It was chaired by Nobel Laureate Glenn T. Seaborg, with Dr. John D. Baldeschwieler of California Institute of Technology as deputy chairman. Other members included Dr. James A. Ibers of Northwestern University, Dr. Thurston E. Larson of the University of Illinois, Dr. Richard S. Stein of the University of Massa­chusetts, Dr. Yuan T. Lee of the University of California, Berkeley, and Dr. James Wei of Massachusetts Institute of Technology.

The first CSCPRC delegation to China this year, the group found much evidence of the new high na­tional priority that China is giving to development of science and technol-

June 19, 1978 C&EN 5