B

acterial Production of Human Insulin

ARTHUR D. RIGGS and the bacterial strains containing them produce large amounts of human insulin chains. We were not surprised that the insulin genes functioned, because we had earlier demonstrated the feasibility of this approach by producing somatostatin in E. coli.3
EARLY ASPECTS OF THE INSULIN PROJECT

uman insulin derived from bacteria is the first clinically proven product of recombinant DNA technology. I say proven because successful clinical trials of bacterially produced insulin were reported in the August 23, 1980, issue of Lancet.1 This represents an exciting milestone and, although not generally recognized, it is interesting to note that Dr. Levine played a key role in initiating the human insulin project. I will explain this statement later on, but first I will review some of the essential technical aspects of the project.2 As illustrated in Figure 1, two separate bacterial strains were constructed. One strain carried a man-made gene for the A-chain of human insulin and the other strain carried a gene for the B-chain of human insulin. Inside the bacterial cells, the insulin chains are made as tails on a rather large precursor protein, the enzyme beta-galactosidase. The insulin peptide chains are efficiently clipped from the precursor protein by treatment, in vitro, with cyanogen bromide. After purification of the separate insulin chains they are joined to give complete, active insulin. Some additional details of each of the various aspects of the project will be discussed more fully. A common misconception is that we cloned the human insulin gene and used it to direct the production of human insulin in Escherichia coli. We did not. As illustrated in Figure 2, there are two approaches to molecular cloning of genes for expression in bacteria. The most common approach starts with messenger RNA (mRNA) and uses reverse transcriptase to make a DNA copy (cDNA), which is then cloned. Our work used an entirely different approach. The structure and amino acid sequence of insulin has been known for many years. Therefore, we were able to use the genetic code to design, on paper, two small genes that contained the proper sequence of nucleotides to command a bacterial cell to produce human insulin. These genes are shown in Figure 3. Because the genetic code is degenerate (that is, there are usually several codons corresponding to each amino acid), the genes we designed and made have a sequence of nucleotides different from the natural coding sequences of human insulin. Yet these man-designed and man-made genes function very well

Since it is relevant to the Festschrift nature of this conference, I will discuss the history of the somatostatin and insulin projects, how they were started and why. The genes for somatostatin and human insulin were chemically synthesized by the triester method. Therefore, if there is a single key person in these projects, it is Dr. Keiichi Itakura, who directed the chemical synthesis aspects. In 1976 we at the City of Hope had a unique opportunity to add Dr. Itakura to our staff and to build upon our earlier collaborations on the synthesis and cloning of the lac operator, which is a small regulatory gene and the first biologically active synthetic gene.4 Because at first no money or space was available to Dr. Itakura, Dr. Ohno and I went to Dr. Levine, who was then the director of the City of Hope. We found that he quickly understood that the time was right to attempt to synthesize, clone, and express man-made genes. He actively supported our effort, and it is safe to say that the somatostatin and insulin projects would never have been done without his support and encouragement. He confessed later that he did not think that somatostatin would be of great benefit to diabetic patients, but he recognized the importance of developing the technology for the synthesis of genes and the bacterial production of hormones. Dr. Levine found space for Dr. Itakura and even came up with some seed money from City of Hope sources. However, it was necessary to find additional sources of support for most of the work. Dr. Itakura and I wrote a grant application for the somatostatin project and submitted it to NIH. A few months later, we received word that the grant had been rejected. In the written criticism that we requested, we were told that "This project seems to be just an intellectual exercise, because somatostatin is not likely to be of great thera-

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BACTERIAL PRODUCTION OF HUMAN INSULIN/ARTHUR D. RIGGS

E. COli

E. coli
CHEMICAL ONA SYNTHESIS 7* *

CLONED NATURAL GENE

GENETIC CODE

SYNTHETIC ONA

CLONED ARTIFICIAL GENE

{JOJ Beta ^ ^ VGaloctosidase

Insulin A Chain

^ Beta '^-y VGalactosidase

Insulin B Choin /

PROTEIN SEQUENCE

1. Partial Purification 2. Cleavage with CNBr 3. Purification of Insulin Chains

FIG. 2. Comparison of two approaches for molecular chning of genes to obtain the bacterial production of mammalian hormone and protein prod' ucts.

Insulin A Chain

O0QOO0

ooooooooo B Chain
Air Oxidation O0O0O0 s s

Insulin

oooaxooo Active Insulin

FIG. I. Schematic overview of the strains and procedures for the production of human insulin by bacteria. Two Escherichia coli strains were constructed haivng chemically synthesized insulin A- or B-chain genes inserted into the (2-galactosidase gene (/3-gaI) of a plasmid cloning vector. In vivo, a fused protein is made, mostly fi-galactosidase, but with an insulin tail joined by a methionine. In vitro, the insulin peptide chain is clipped off by treatment with cyanogen bromide. After separate purification, the insulin A- and B-chains are joined by air oxidation. (From Riggs and hakura.8) CHEMICAL DNA SYNTHESIS

emphasize the foresight and understanding of modern molecular biology that he displayed in his active encouragement of the somatostatin and insulin projects. I do not mean to criticize the NIH peer review process, because it is an excellent system. Given severely limited resources, some good projects will be missed. The rejection by NIH did not delay us because we were extremely fortunate to get a contract from Genentech, Inc., a newly established company consisting, at that time, of two people, Robert Swanson and Dr. Herbert Boyer. Our contract with Genentech meant that we were adequately funded and had the additional advantage of collaboration with Boyer and his colleagues. The rest of the story has already been published.2'3 In less than a year after funding, we had a bacterial strain producing somatostatin and, encouraged by these results, we quickly went on to make insulin.

peutic value. . . . The project is impractical and cannot possibly be completed in the time requested (three years)." I agree, of course, that the somatostatin project was an intellectual exercise. It was intended to be mainly a demonstration of feasibility. Dr. Levine understood this, and I want to

nce funding was obtained, Dr. Itakura set about making genes. For the insulin gene, it was necessary to make 29 oligonucleotides, which were then assembled and joined by ligation to make a total of 181 base pairs of duplex DNA (Figure 3). The DNA fragments were made by four people in about three months.

FIG. 3. Chemically synthesized human insulin genes. The genes for human insulin, B- and Achain, were designed from the amino acid sequences of the human polypeptides. The 5' ends of each gene have single-stranded cohesive termini for the EcoRl and Bam I restriction endonuckases for correct insertion of each gene into plasmid pBR322. A Hind/// endonuclease recognition site was incorporated into the middle of the B-chain gene for the amino acid sequence Glu-Ala to allow amplification and verification of each half of the gene separately before construction of the whole Bchain gene. The B- and the A-chain genes were designed to be built from 29 different oligodeoxyribonucleotides, varying from decamers to pentode camers. Each arrow indicates the fragment synthesized by the improved phosphotriester method, HI to H8 and BJ to B12 for the B-chain gene, and A/ to All for the A-chain gene. (From Crea et al.5)

B-Chain Gene
28
Mat Phi Vol Av Pro

29
L]f

30
Thr

A-Chain Gene

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BACTERIAL PRODUCTION OF HUMAN INSULIN/ARTHUR D. RIGGS

tion of the DNA fragments by high performance liquid chromatography.

\ .
MOLECULAR CLONING

I 1) TPST. I 2) Sd:co gel tj) B B El,N B

1 ) TPSTe 2) Silica gel

0"

0-

0"

0"

0"

0-

0"

0"

H r < 0 > Cl H :: Protected ond non protected bases 0 fln :: - C - ^ ^ OMe OMT -" dimeihoiylntyl

Figure 5 illustrates how the insulin A-chain was assembled, cloned, and positioned at the end of beta-galactosidase. Step 1 (shown in Figure 5) was joining the small (13 base average) oligonucleotides. Because they were designed to have complementary overlaps, they assembled themselves and were joined to give duplex DNA by the action of the T4 DNA ligase. The gene was designed to have restriction enzyme sites at each end (EcoRI on the left and BamHI on the right). Step 2 was preparation of the plasmid DNA cloning vector pBR322. Preparation included treatment with EcoRI and BamHI restriction enzymes, which cuts out a small piece of the plasmid and provides a site for insertion of the synthetic A gene. In step 3, the prepared plasmid and synthetic DNA were mixed and joined by T4 ligase, followed by transformation of E. coli and molecular cloning. A clone was obtained that contained a correct insulin A gene, as verified by direct DNA sequencing. Next, a DNA fragment containing most of the E. coli lac operon, including the lac promoter, operator, and the first 1006 amino acid codons of beta-galactosidase, was inserted (steps 4, 5, and 6). This led to a clone making insulin-beta-galactosidase fused protein.

F/G. 4. The chemical synthesis of oligodeoxyribonuckotides by the improved triester method.5

It is apparent that techniques have developed to the point where the genes for altering bacteria can be made with relatively modest effort. Actually, the techniques have been improved considerably since the insulin project and much less time would be needed now. Figure 4 illustrates the triester chemical synthesis method that was used to make the insulin genes.5 Starting with nucleosides, a library of fully protected triester trimers, such as 1 and 2, are made. The type 2 trimer, with the anisole 3'protecting group, will become the 3' end of the oligonucleotide. The type 1 trimer is bifunctional, and depending on treatment (either mild acid or base), will be either the 5' end component or an internal sequence component. Because of the chlorophenyl protecting groups attached by ester linkage to the phosphate groups (forming phosphotriesters), the trimers and intermediate oligonucleotides (e.g., 3, 4, 5, 6, 7) are not water soluble. Therefore, all condensations and purifications are done in nonaqueous solvents such as chloroform. Trimers can be condensed to yield hexamers (e.g., 3 + 4 yields 6) and nexamers can be condensed to yield dodecamers (e.g., 6 + 7 yields 8, still in fully protected triester form). The next-to-last step in a typical synthesis is the removal of all protecting groups by treatment with acetic acid and NH 4 OH, generating the desired water-soluble singlestranded DNA fragment. The last step is a careful purifica-

Lombdo ploc

FIG. 5. The construction of a plasmid DNA containing a synthetic insulin A-chain gene inserted at the end of a (3-galactosidase gene. The procedures are described in the text, and details are given in Goeddel et al.2 Only an explanation of the symbols is given here. The symbol A represents the synthetic A-chain gene. pBR322 is a well-characterized plasmid cloning vector containing two antibiotic resistance genes, ampicillin (Amp) and tetracycline (Tet), and several convenient restriction endonuclease sites including Eco Rl (Rl) and Bam HI (Bam). Lambda plac is a lambda transducing phage carrying the entire E. coli operon, which includes the lac promoter (?), the lac operator (O), and the entire (i-galactosidase structural gene (Z). There is an Eco Rl endonuclease site to the left of the operon and abo one near the end of the (i-galactosidase gene; thus, the lac operon DNA fragment can be readily obtained. The phenotype of the bacterial strains successfully infected with the desired plasmid is shown. For example, the A-chain-producing strains would be ampicillin resistant (AmpR), tetracycline sensitive (Tets), and the colonies would be Blue on a special indicator agar called Xg.

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EXPRESSION

Approximately 20% of the bacterial protein produced by the insulin strains was found to be beta-galactosidase-insulin fused protein. The fused protein is insoluble and was enriched to more than 50% purity by low speed centrifugation. The insulin chains then were cleaved from the fused protein by treatment with cyanogen bromide in 70% formic acid at room temperature overnight. The free insulin chains were converted to S-sulfonated derivatives and purified by ion exchange chromatography, gel filtration, and reverse phase high performance chromatography. In our published work,2 the B-chain was not purified to homogeneity. However, this now has been done. No major problems were encountered. S-sulfonated derivatives of the insulin chains were made before purification to ensure stability by preventing the premature formation of disulfide bonds. Fortunately, the best method for joining chains6 starts with the S-sulfonated derivatives (Figure 6). With a fivefold excess of A-chain, up to 80% correct joining of the B-chain to the A-chain can be obtained. To detect insulin chain product by the bacteria, we adapted the Katsoyannis reconstitution procedure6 to the microgram scale. Using the procedure outlined in Figure 6, we had no difficulty producing radioimmune active insulin in control experiments and with partially purified bacterial insulin chains.2 The microreconstitution assay was used to follow the individual insulin chains during purification.

In our laboratory scale experiments2 we obtained approximately 1 mg of insulin chain per liter of culture. These yields were high enough to stimulate large-scale commercial development work, but clearly they will need to be improved if human insulin is to become as cheap as bovine insulin.
COMMERCIALIZATION

A(SS0 3 ) 4 + B(SSO3 ) 3'2 40 fiq

10 fMQ

hen we had demonstrated the scientific feasibility and had published the initial findings, my part was essentially over. However, there was still a tremendous amount of work to be done before human insulin could become a useful clinical agent. The insulin project was done under contract with Genentech, Inc., and therefore they had responsibility for the scale-up and development work. Genentech quickly reached an agreement with Eli Lilly, Inc., and these two companies have worked extremely diligently and competently to bring the human insulin to the public. In the scale-up of a procedure from liter quantities to hundreds of thousands of liters, one can expect some difficulties, and many a potential product falters at this stage. Fortunately, no major difficulties have been encountered, and although there is still need for improvement of the yield, the present results are so encouraging that Lilly has committed 40 million dollars to bring human insulin to large-scale production by late 1982. Amounts of pure human insulin sufficient for clinical trials already have been made. Although much work remains to be done, and neither the time nor the costs of large-scale production can be predicted with any certainty at this time, we do know that the present results are so encouraging that a tremendous effort is being made.
CLINICAL TRIALS

20/i.l pH 4.5 2-mercaptoethanol 95, 10 min.

A(SH), + B(SH)2
Air 0 2 pH 10.6, ON. s s

B Insulin \
Standard Radioimmunoassay
FIG. 6. Insulin chain joining according to the Katsoyannis procedure. Ssulfonated B-chain is mixed with a fourfold excess of S-sulfonated Achain, and then free sulfhydryb are produced by reduction with mercaptoethanol. After removal of the excess mercaptoethanol by extraction with ethyl acetate, the pH is adjusted to 10.6 and the chains are joined by air oxidation at room temperature overnight (O.N.). A standard radioimmunoassay (Pharmacia) was used to measure radioimmune insulin.

From earlier work2'6 we knew that all chemical tests and animal studies indicated that the insulin produced from bacteria was identical to natural insulin. Nevertheless, it is gratifying to see the results of the first clinical trials reported by Keen and co-workers.1 The insulin from bacteria was virtually indistinguishable from porcine insulin in lowering plasma glucose levels in normal human volunteers. Most important, there were no adverse side reactions. One can argue that human insulin made by joining A- and B-chains should be purer and less likely to cause adverse reactions. However, this must be proved, and, of course, additional longer-term studies are underway.1 From the City of Hope National Medical Center, 1500 East Duarte Road, Durate, California 91010.

REFERENCES

Keen, H., Pickup, J. C , Bilous, R. W., Glynne, A., Viherti, G. D., Jarrett, R. J., and Marsden, R.: Human insulin produced by recombinant DNA technology: safety and hypoglycaemic potency in healthy men. Lancet 2: 398-401, 1980. 2 Goeddel, D. V., Kleid, D. G., Bolivar, F., Heyneker, H. L ,

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Yansura, D. G., Crea, R., Hirose, T., Kraszewski, A., Itakura, K., cal synthesis of genes for human insulin. Proc. Natl. Acad. Sci. and Riggs, A. D.: Expression in Escherichia coli of chemically syn- USA 75: 5765-69, 1978. 6 Katsoyannis, P. G., Trakatellis, A. C., Johnson, S., Zalut, C., thesized genes for human insulin. Proc. Natl. Acad. Sci. USA 76: and Schwartz, G.: Studies on the synthesis of insulin from natural 106-10, 1979. 3 Itakura, K., Hirose, T., Crea, R., and Riggs, A. D.: Expression and synthetic A and B chains. II. Isolation of insulin from recombiin Escherichia coli of a chemically synthesized gene for the hormone nation mixtures of natural A and B chains. Biochemistry 6: 2642 -54, 1967. somatostatin. Science 198: 1056-63, 1977. 7 4 Chance, R. E., Kroefif, E. P., and Hoffman, J. A.: In National Heyneker, H. L , Shine, J., Goodman, H. M., Boyer, H. W., Institutes of Health Conference on Insulin and Growth Hormone. Rosenberg, J., Dickerson, R. E., Narang, S. A., Itakura, K., Lin, S-y., and Riggs, A. D.: Synthetic lac operator DNA is functional in In press. 8 Riggs, A. D., and Itakura, K.: Synthetic DNA and medicine. vivo. Nature 263: 748-52, 1976. 5 Crea, R., Kraszewski, A., Hirose, T., and Itakura, K.: Chemi- Am. J. Hum. Genet. 31: 531-38, 1979.
r

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