Proc. Nadl. Acad. Sci. USA Vol. 81, pp.

5956-5960, October 1984

Biochemistry

Synthesis of a gene for human growth hormone and its expression in Escherichia coli
(trp promoter/hybrid plasmid/promoter efficiency/modified Shine-Dalgarno region)

Japan; lInstitute of Physical and Chemical Research, Wako, Saitama 351, Japan; and Institute for Molecular and Cellular Biology, Osaka University, Osaka 530, Japan

M. IKEHARA*, E. OHTSUKA*, T. TOKUNAGA*, Y. TANIYAMA*, S. IWAI*, K. KITANO*, S. MIYAMOTO*, T. OHGI*, Y. SAKURAGAWA*, K. FUJIYAMA*, T. IKARI*, M. KOBAYASHI*, T. MIYAKE*, S. SHIBAHARA*, A. ONOt, T. UEDAt, T. TANAKAt, H. BABAt, T. MIKIt, A. SAKURAIt, T. OISHI*, 0. CHISAKA, AND K. MATSUBARA *Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565, Japan; tFaculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060,
Communicated by Susumu Ohno, June 15, 1983
A gene coding for human growth hormone, ABSTRACT which consists of 192 amino acids, was chemically synthesized. The synthesis entailed ligating 78 deoxyribooligonucleotides, which had been synthesized on polymer supports by the phosphotriester method with frequently occurring amino acid codons of Escherichia coli. The chemically synthesized gene was inserted into an E. coli plasmid downstream from the E. coli thp promoter, with a modified ribosome-binding region carried on pBR322. E. coli cells transformed with this recombinant plasmid synthesized 2.9 x 106 molecules per cell of human growth hormone upon induction. The induced polypeptide was identical with natural human growth hormone in size and in immunological properties, as well as in biological activity as examined by the tibial test with hypophysectomized rats.

The improvement of techniques in chemical synthesis of deoxyribooligonucleotides has made possible the total synthesis of genes of 100-500 nucleotides (1-9). By this technique, genes with designed amino acid sequences can in principle be synthesized, and the products of their expression in bacteria can be examined. In the present work, a gene for human growth hormone (hGH) (10), which contains 191 amino acids and methionine, was totally synthesized by chemical means, using amino acid codons most frequently appearing in Escherichia coli (11-13) (Fig. 1). To establish a general procedure for efficient expression of genes for relatively large peptides, hGH was thought to be a suitable target. Use of the synthetic gene will provide sufficient sites for restriction enzymes, which should be useful in mutations of genes for replacement of protein domains. Rapid synthesis of 78 gene fragments of 7-26 bases was carried out, followed by sequential ligation to construct the gene of 584 base pairs (bp). This is the longest gene so far synthesized chemically. Separately, a modified trp promoter was constructed that allows expression of any synthetic genes with an initiation codon placed downstream of it. A recombinant plasmid carrying the promoter and the hGH gene produced the hormone in E. coli at a high level. The induced polypeptide was indistinguishable from the natural hGH in several features. The purified hormone was as active as the natural growth hormone in biological tests (14).

MATERIALS AND METHODS Deoxyoligonucleotides. Deoxyoligonucleotides with a chain length of 7-26 bases were synthesized by the phosphotriester solid-phase method, using 1% cross-linked polystyrene (15) as the support. Altogether 78 chains were prepared
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by addition of dinucleotide blocks to the nucleosides linked through a 3'-succinyl group to the resin. Tetranucleotide blocks prepared by condensation of dimers in the liquid phase were used for the synthesis of the 25-mer (A-UO; Fig. 1). Sixteen dinucleotide blocks were prepared with mesitylenesulfonyl-3-nitro-triazolide (16) by methods essentially as reported (17) and purified mostly by reversed-phase chromatography (18) under conditions described (19, 20). A typical synthesis of a 15-mer started with treatment of the 5'-dimethoxytritylated nucleoside resin (5 ,umol) with benzenesulfonic acid (17) (2%; 2 ml) for 1 min twice in a small vessel with a sintered glass plate and a stopcock at the bottom. In the case of deoxyadenosine resin, either zinc bromide (21) or 3% trichloroacetic acid (22) was used. A dimer (20 ,umol) was treated with MSNT (70 ,umol) in pyridine (200-300 Al) at 40'C for 20 min. The resin was treated with acetic anhydride (0.2 ml) in 4-dimethylaminopyridine-pyridine (0.1 M; 1.8 ml) for 3 min. The product was deblocked with 1,1,3,3-tetramethylguanidium 2-pyridinealdoximate in dioxane-H20 (0.5 M, 90% dioxane) (16) at 300C overnight and with concentrated ammonia at 550C for 5 hr. The dimethoxytritylated 15-mer was separated on a column (0.7 x 6 cm) of C-18 silica gel (Waters; 35-100 pum) with a gradient of acetonitrile (10%to35%; total, 200 ml) in 0.05 M ammonium acetate and treated with 80% acetic acid. The product was applied to a column (0.7 x 21 cm) of DEAE-Toyopearl 650S (Toyosoda) with a gradient of sodium chloride (0.1-0.3 M; total, 200 ml) in 7 M urea/20 mM Tris HCl, pH 7.5. The 15-mer was checked by high-pressure liquid chromatography (HPLC) on C-18 silica gel (Toyosoda, TSK-410 AK) and ion exchange (Toyosoda, TSK gel IEX540K). Impurities were removed by HPLC, and the purity and sequence were confirmed by mobility-shift analysis (23). Enzymes and Proteins. DNA ligase was obtained from Takara Shuzo and Boehringer Mannheim. Restriction endonucleases Cla I and Alu I were purchased from Boehringer Mannheim and Bethesda Research Laboratories. Other enzymes, including restriction endonucleases (Bgl II, BamHI, Sal I, Hinf I, and Hpa I) were obtained from Takara Shuzo. Methionyl hGH was a gift from H. Yamamoto (Sumitomo Chemical). Construction of the Promoters and the hGH Gene. A 0.5kilobase-pair DNA fragment carrying the intact trp promoter was extracted by Hinf I digestion from E. coli plasmid DNA, ptrp ED 5-1 (24). It was cloned into the unique EcoRI site of pBR322, using EcoRI linker. The upstream EcoRI site was cleaved by the enzyme and filled in with T4 DNA polymerase. Then, the remaining downstream EcoRI site was
Abbreviations: hGH, human growth hormone; SD sequence, ShineDalgarno sequence; bp, base pair(s).

5956

Biochemistry: Ikehara et aL

Proc. NatL Acad Sci. USA 81 (1984)

5957

1 10 20 Met Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Hinf IUl UO Cla I U2 U3 U4 V V Ica ATG TTC CCA ACT ATT CCA CTG AGTCGC CTG TTC GAT AAC GCG ATG CTG CGT GCG CAT CGT CTG CAC CAA CTG GCT TTC z TAC AAG GGT TGA TAA GGT GAC TCA fLCG GAC AAG CTA TTG C% TAC GAC GCA CGC GTA WA GAC GTG GTT GAC CGA A4G C L0Ot L1 L2 L3

A part
30 40 50 Asp Thr Tyr Gin Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr G C AT U6 TTGTU5 U0 Aiu I vU vC v GAC ACT TC CAG GAG TTC GAA GAA GYA TAC ATC CCG AAA GAA &G AM TAC A TTC Cl4 CAG MC CCA CAG ACC CTG IGA ATG GTC CTC AAG CTT CTT CGT ATG TAG GGC TTT CTT GTC TTT AG It.AAG GAA GTC TTG GGT GTC TGG A " L4 L5 L6 L0 B part 60 Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gin Gin U4 V U2 U3 US V TCG TTG TGT TTC TCT YAA AGT ATC CCG ACC CCT TCT AAC CGC GAA GAGVACC.CAG CAG AGC AAC ACA A~c AGA CTT TCA TAG GGC TGG GGA AGA TTGAGCG CTT CTC TGG GTC GTC " A Li L2 L3 L4
80 90 Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val
Lys Ser Asn Leu Glu Leu V U6 AAA TCG AAC CTT GAA CTG TTT AGC TTG GAA CTT GAC A L5

70

Uii U9 U8 U1 0 V v V v V U7 CTT CGT ATC TCG CTG CTT CTC ATT CAG TCG TGG CTG GAG CCA GTA CAG TTC CTG CGT TCG GTT TTC GCA AAC TCA GAA GCA TAG AGC GAC GAA GAG TAA GTC AGC ACC GAC CTC GGT CAT GTC AAG GAC GCA AGC CAA AAG CGTA TTG AGT A A A A L6 L7 L9 L8 L10
Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met

Gin

100
Phe Leu Arg Ser Val Phe Ala Asn Ser

110

120

U16 U14 U15 Rn'ILV U12 U13 v v v V CTG GTT TAC GGT GCG TCT GAC AGT AAC GTT TAC GAC CTG CTG AAA GAC CTT GAA GAA G rAG ACC CTG ATG GAC CAA ATG CCA CGC AGA CTG TCA TTG CM ATG CTG GAC GAC TTT CTG GAA CTT CTT CC TC TGG GAC TAC A A A A Lii L12 L13 L14 L15

CCA GCG L16

Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gin Thr Tyr Ser Lys Phe Asp Thr Asn Ser U3 U2 v U17 U18 v v V.Ui9Bql II Ui GGT CGC CTG GAA GAT GGTVTCA CCA CGC ACT GGT Ck ATC TC AAA CAG ACT TAC TCC AAA TTC GAT ACT AAC TCT

130

140

150

GACACTT CTA CCAL17AGT GGT GCGATGA CCA GCTA GAA~ TTT GTC LiTGA ATG AJG TTT AAGL2 CTA TGA TTG AGA A i A L18
C part
160
170

His Asn Asp

Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr U6 U8 U5 U7 U4 V V v v CAT AAC GAT GAT GCT CTG CTG AAA AAC TAC GGC CTG CTG TAC TGT TTC CGT AAA GAT ATG GAT AAA GTT GM ACT GTA TTG CTA CTA CGA GAC GAC TTT TTG ATG CCG GAC GAC ATG ACA AAG GCA TTT 9TA TAC CTA TTT CAA CTT TGA A Li A 16

Asp Ala

Leu Leu

L3

L4

15

190 180 Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe StopStop U9 U10 U12 Sal I V Uli v TCG TGT GGC TTC TAA TAG TTC CTG CGT ATC GTT CAG TGT CGT TCT GTT GAA GCA TAG CAA GTC ACA GCA AGA CAA CTT CCC AGC ACA CCG AAG ATT ATC aqct MG GAC " " " Li1 L10 L9 L8

&G

FIG. 1. Human growth hormone and its

synthetic gene.

cleaved by EcoRI, followed by BAL-31 digestion to eliminate a sequence between the trpE-coding region and its Shine-Dalgarno (SD) sequence. A chemically synthesized Cla I linker d(A-A-T-C-G-A-T-T) was inserted just downstream of the SD sequence. Sequence of the relevant region in the resulting plasmid pCT-1 is shown in Fig. 2. It has the trp promoter, the SD sequence for trpL (attenuator), along with another SD sequence of trpE in front of the Cla I linker,

d(A-A-T-C-G-A-T-T). Oligonucleotides (0.2 A260 unit; 10 ug; -1.5 nmol) were phosphorylated by treatment with [y32P]ATP (500 pmol; 1-2 mCi/hmol; 1 Ci = 37 GBq) and polynucleotide kinase (3 units), pH 8.0 at 370C for 15 min, and then with unlabeled ATP (10 mmol) and polynucleotide kinase (1 unit) at 370C for 90 min. Phosphorylated fragments (5-8 pieces) and nonphosphorylated ends were mixed as shown in Fig. 2, then annealed at 70'C, in the presence of 66 mM Tris*HCI, pH 7.5/6 mM MgCl2/500 tkM ATP, in a volume of 270 ul. The mixture was cooled to 15C during 1 hr and treated with T4 DNA ligase (1.2 units; 4.4 units/mol) together with dithiothreitol (10 mM) at 15C for 13-15 hr. The reaction was checked by gel electrophoresis on 10% acrylamide. The products were precipitated or separated by the same electrophoresis when needed. For example, in the synthesis of the C part gene, the

(Fig. pCT-1 to yield plasmid phGH-1 its 3). To improve the efficiency of expression, the promoter follows. First, region region of phGH-1 was modified as the I site at the157 was + between the Hpa I site at -11 and Cla eliminated to remove the attenuator (trpL) along with the SD sequence of trpE. These were replaced by a synthetic oligonucleotide consisting of either 33, 35, or 37 nucleotide pairs, each carrying a SD sequence (A-A-G-G) and with appropriate termini. The resulting plasmids, pGH-L9, pGH-L11, and pGH-L13, carry 9, 11, or 13 bases upstream of the initiation codon (ATG) of the hGH gene (Fig. 3). Plasmid pGH-L8 has a similar structure to the others, except that the gene was inserted into the Taq I site just downstream of the trpL SD
Characterization of the Protein. Radioimmunoassay was carried out with a Phadebas hGH PRIST kit (26) and an EIKEN-hGH-I kit for the double-antibody method from Eiken
sequence.

larger three fragments (50 pmol) were phosphorylated at pH 9.6 and ligated at 20'C for 16 hr, and the mixture was treated with Cla I (30 units) at 370C for 6 hr then with Sal 1 (120 units) at 370C for 15 hr. The product (0.5 gg; -2.5 pmol) was isolated by 5% gel electrophoresis. A, B, and C parts of the gene were ligated as shown in Fig. 2, and the duplex (0.5 pmol) was joined to the Cla I/Sal I sites of the expression vector

5958

Biochemistry: Ikehara et aLP Proc. NatL Acad ScL USA 81 (1984)

1

15

B-I

2B
34
3

35

B-E

45 B-N
53

54

C-1
63

64 C U

nc-7
7

-4 -4 14 24
Clal

-4 44
Bg
8

1) T4ligase 2) Clal, Bgilx

A
v

1) T4Iigase
I S CSol ,. all CallI

A
v

A____

,2))ClaL Soal Sall A

v

-v-

AB

415b.p

)<

176b.p.

Bgif[

sail
C I 165bp

pCT 1/Cial, Sall

Clal

AB

Sall
HGH gene 584

bP

FIG. 2. Synthesis of the hGH gene.

Immunochemical. Biological activity of the induced product, which was fractionated by chromatography on DEAE-cellulose (Whatman DE-52), was tested on hypophysectomized rats (14).
RESULTS Synthesis of hGH Gene. The amino acid sequence of hGH and the synthetic gene fragments for methionyl hGH are shown in Fig. 1. The gene was divided into three parts-the amino terminal (A), the middle part (B), and the carboxyl terminal (C). Amino acid codons used in the present gene were chosen mainly from those found in a gene for elongation factor Tu (tufA) of E. coli (13). Homologous or complementary sequences were sought with a microcomputer, and codons were altered to avoid mismatches in duplex alignments. The amino acid codons used in the present synthesis are shown in Table 1, together with those found in a human gene (27). As indicated in Fig. 1, sequences for the restriction enzymes Cla I, Sal I, Hinf Bgl II were incorporated to clone the fragments into the vector and to facilitate modifications. A single cleavage sequence for each of these enzymes was designed. The methionine codon and the termination codons were also added at the appropriate positions. Dinucleotide blocks were mainly used, except for the synthesis of
pCT-1
Sal
pBR322 A r

AUO (25-mer), where tetramer blocks were used. As expected, the use of larger oligonucleotides as condensing units was found to be advantageous in obtaining the pure product, although synthesis of protected tetranucleotides was time consuming. All fragments were finally examined by HPLC on reversed-phase and mobility shift analyses (23). Construction of Plasmids Carrying the hGH Gene Under Control of the trp Promoter. Enzymatic ligation of the 78 chemically synthesized oligonucleotides was carried out by joining 5-10 pieces in the first step. The subfragments thus obtained were further ligated to yield 415-bp (A+B segment: amino-terminus portion) and 176-bp (C segment: carboxylterminus portion) polynucleotides. The C-segment was integrated into a plasmid, replacing the region between the Cla I and Sal I sites of pCT1 (Fig. 2) to generate phGHC-1. After amplification, the 165-bp C segment was extracted by digestion with Bgl II and Sal I, and ligated to the 415-bp A+B segment to yield the whole hGH gene, which was inserted into the plasmid pCT1 as shown in Fig. 2. The resulting plasmid phGH-1 carries the 584-bp synthetic gene under the control of the trp promoter and the SD sequence for trpE. The attenuator was next removed from phGH-1, and the synthetic gene was placed at different distances from the trpL SD sequence A-A-G-G (Fig. 3). Plasmids pGH-L8,
trpL

Ptrp
-300
-1 0

trpL
T

S.D. TaqI attenuator S.D. ClaI HpaI It -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ----0 --GTTAAC--1---0 TA +2C7TG--+--TGA ------+-1+-1--GAGAAICGA +1 4
+27 +71

trpE

pBR322

Sall

+1 40 +1 54

pHGH-1
p H -1 G

Ptrp
HpaI
-10

replace right ClaI-Sall fragment with synthetic HGH gene (584b.p.)

trpL

trpL
TaqI
+27
+71

trpE

Sail

S.D. +14

attenuator S.D. Clal

+140 +154

HGH gene

Sail

-CJ-------"---tI--300

C--1---AAGGGTATCGACAATG---TAG-------GAGAATCGA
remove TaqI(+22)-ClaI(+157) fragmentyielding pGH-L8 or replace HpaI(-11)-ClaI(+157) fragment with synthetic DNA fragments ,yielding pGH-L9,pGH-Ll l,pGH-Ll3 trpL S.D. Clal HGH gene
----------------+1

HpaI
pGH-L8 pGH-L9
pGH-L1
-10

AACTAGTACGCAAGTTCACGTAAAAAGGGTATCGA1'g
AAC-------------------------GTATCATCGAg
MC-------------------------GTATC
G

AC-------------------------GAAGAM-----------------------------

pGH-L13

1-----------

FIG. 3. Construction of plasmids containing the synthetic hGH gene. pCT-1 and phGH-1 were obtained as described in Materials and Methods. Dotted lines are identical to the sequence of the trp operon (25). To replace Hpa I/Cla I fragments, DNA duplexes of 33, 35, and 37 bp were prepared by joining four synthetic fragments in each duplex.

Biochemistry: Ikehara et aL
Table 1. Codon usage in synthetic (and natural) hGH gene First Second position position A U C (5' end) Phe 0 (3) U Ser 5(3) Tyr 0 (3) Phe 13 (10) Ser 1 (5) Tyr 8 (5) Term. 1 (0) Leu 0 (1) Ser 2 (3) Ser 6 (1) Leu 1 (0) Term. 1 (1) 2 (1) Pro 1 (0) His C Leu 5 (1) 1 (2) Pro 0 (5) His Leu 1 (8) 1 (2) Pro 5 (2) Gln Leu 0 (4) Gln 12 (11) Leu 19 (12) Pro 2 (1) A Thr 6(1) Asn 0 (0) Ilie 2 (2) Asn 9 (9) Thr 4 (4) lie 6 (6) Thr 0 (4) le 0 (0) Lys 9 (1) Thr 0 (1) Met 4 (3) Lys 0 (8) Val 5 (0) G Ala 2(1) Asp 7 (2) Val 0 (3) AlaO0(5) Asp 4 (9) Glu 11 (6) Ala 2 (1) Val 2 (0) 3 (8) Glu Ala 3 (0) Val 0 (4)
pGH-L9, pGH-L11, and pGH-L13, respectiveiy,
nine codon.
carry the

~~~Proc.

NatL Acad. Sci. USA 81

(1984)

5959

Cys 4 (2) Cys 0 (2)

Term. 0 (0) Trp 1 (1) Arg 7 (1) Arg 4 (4) Arg 0 (0) Arg 0 (1) Ser 3 (1) Ser 1 (5) Arg 0 (0) Arg 0 (5) Gly 4 (0) Gly 2 (5) Gly 0 (0) Gly 2 (3)

Third position (3' end) U C A G U C A G U C A G U C A G

SD sequence 8, 9, 11, and 13 bases upstream of the methio-

in the coagulated form. Thin-section electron micrographs (Fig. 5), in fact, show a large mass of uniformly stained material.

Expression of the Gene in E. coli. The synthetic gene inserted in the plasmid, and the modified regions around the SD sequences were identified by sequence analyses (28). The plasmids were used to transform E. coli HB1O1 (29), and the transformants were examined for expression of the hGH gene upon induction by 3-indolylacrylic acid at eariy logarithmic phase (24). The cells were collected after 23 hr of induction, lysed, and the proteins were analyzed by electrophoresis in reducing NaDodSO4/ polyacrylamide gels (30). The results are shown in Fig. 4. E. coli cells carrying pGH-L9 synthesized the product most efficiently. From radioimmunoassay, the yield of hGH was esTransformation and

Analysis of the Induced Protein. The molecular weight of product was estimated from the mobility in reducing gel electrophoresis as 21,000 (Fig. 4). The amino termi nus of the product was analyzed by Edman degradation and found to
the be methionine.

timated to be 169

ji~g

per ml of culture

medium,

or

2.9

106

molecules per cell. As shown in Table 2, pGH-L8, pGH-L11, and pGH-L13 also yielded hGH at a higher level upon induction

Immunological properties of the induced peptide were exby two procedures. Table 2 summarizes the results of radioimmunoassay of hGH in extracts from bacteria containing expression plasmids by the solid method (26). Biological activity of the induced peptide was examined in hypophysectomized rats. In tests of weight increase and increase in the width of the proximal epiphyseal cartilage of the tibia, the synthesized hGH showed equal activity to that of the natural growth hormone.
amined

compared

to

phGH-1.

E.

coli

cells

mids had to be treated with 6 M

carrying these plasguanidine hydrochloride

DISCUSSION
the growth of bones and currently used for treatment of hypopituitary. dwarfism (10). Other biological activities can be studied if hGH is available in quantity. The present study has. shown that a totally synthesized hGH gene was efficiently expressed in E. ccli under the control of the trp promoter. Enzymatically synthesized cDNA corresponding to the major part of the hGH gene has also been cloned, together with a synthetic hGH is known to be

responsible for

(31). The

amount of hGH in cells without the treatment was

is

almost one-tenth of that observed after denaturation. This

suggests that
Mr

some

of the induced

proteins

in cells

might be

1-- AA--

HGH
L13 GH L8

10O3

GH

L8

L9

L9Lii1L13

5gg1lOptg

92.5 68~
43I

Table 2. Radioimmunoassay of hGH in E. ccli extracts

Plasmid*

Induction
+

milliunits/
mit
67.7

pag/mit
33.9 3.6 107.1 10.7 168.7 13.6 108.5 13.0
117.2 8.7

phGH-1
25.7

~~~7.3
214.2

pGH-L8

18.4 411%t-l-

~~~21.4
337.5 ~~~27.2 217.1

pGH-L9
1,W.
am-

Molecules per cell 4.9 x i0 4.9 x 104 1.7 x 106 1.6 x i05 2.9 x 106
2.0 x i05 1.7 x 106

...
X

12.3

ME

_:;.

.-

pGH-L11

~~~26.0
234.4

19 2)7 16 16 (%)
Analysis of the induced protein by NaDodSO4/POIYacrylamide gel electrophoresis. 3-Indolylacrylic acid (IAA) was used as the inducer. A mixture of proteins containing cytochrome C,

pGH-L13

FIG.
4.

~~~17.4
ND

1.8 x 105 1.9 X 106 1.3 x i05

pCT-1

NDND

i3-lactoglobulin, a-chymotrypsinogen, ovalbumin, bovine albumin, phosphorylase b, and myosin (H chain) was used markers. hGH, methionyl hGH.

serum
as

size

ND, not determined. *In E. coli HB1O1. tOne milliliter per culture.

~~ND

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Proc. NatL Acad Sci. USA 81 (1984) Biochemistry: Ikehara et aLP

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4. 5. 6. 7.

8.

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9.

FIG. 5. Electron micrograph of E. coli. (a) Cells containing pHG-L9. Cells were stained with glutaraldehyde and osmium tetraoxide. (b) Cells containing pCT1.

10.
11.
12. 13. 14.

amino-terminal gene, and expressed in E. coli under the control of lac (26) or tac (32, 33) promoters. It was shown that pGH-L9, which carried the SD sequence of trpL 9 bases upstream from the initiation codon, produced methionyl hGH most efficiently. The production of methionyl hGH was about 5 times that of phGH-1, which contained the attenuator and the SD sequence of trpE. This efficiency is also 10 times higher than that reported previously (32, 33). The distance of the synthetic gene from the trpL SD sequence A-AG-G seemed to affect the efficiency of the expression. As shown in Table 2, the efficiency for pGH-L9 is significantly higher than that for pGH-L8, pGH-L11, or pGH-L13. The usage of amino acid codons most frequently appearing in E. coli should favor the expression, because it was shown that there was a good correlation between the frequency of codon usage and the amount of corresponding tRNAs. Expression vectors used in this study can be applied to any genes having proper sequences for restriction enzymes and the initiation and termination codons. Any proteins of -200 amino acids may be prepared by the recombinant DNA technique in bacteria by use of chemically synthesized genes. Site-directed mutations of amino acids or domains can be designed by replacing nucleotides in the corresponding region of the gene fragments. The present hGH gene was also designed to facilitate modification of hGH by replacement of deoxyoligonucleotides.
The authors thank Drs. J. Kawamura and T. Terao of the National Institute of Hygiene of Japan for the biological testing of the product. We thank Dr. A. Matsushiro and T. Wada of the Microbiology Disease Institute of Osaka University and Dr. S. Aida of the JEOL Co. for electron microscopy. We are indebted to Dr. H. Yamamoto of the Sumitomo Chemical Co., Ltd., for his generous gift of methionyl hGH. 1. Khorana, H. G., Agarwal, K. L., Buchi, H., Caruthers, M. H., Gupta, N. K., Kleppe, K., Kumar, A., Ohtsuka, E., RajBhandary, U. L., van de Sande, J. H., Sgaramella, V., Terao, T., Weber, H. & Yamada, T. (1972) J. Mol. Biol. 72, 209-217. 2. Khorana, H. G. (1979) Science 203, 614-625. 3. Itakura, K., Hirose, T., Crea, R., Riggs, A. D., Heynecker,

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22.

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24. 25.

26.
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30. 31. 32.

33.