(;zrw.

22 ( 19X3) 229- 235 229

tilsev~e~- Sc~rnce Publl.\hera

Secretion of interferon by Bacillus subtilis

(Recombinant DNA: a-amylase signal peptide; preprotein: antiviral activity)

llkka Palva, PGvi Lehtovaara, Leevi KGriiGnen, Mervi Sibakov *, Kari Cantell *, Catherine H. Schein **,
Kiyoko Kashiwagi ** and Charles Weissmann **

Recwdxr~ont DNA Luhortrro~v, Unrversi[l, of Helsinki, Huurmwninkufu 3, SF-00290 Helsrtlki 29, Tel. (3.5 80) 41X 51 I,
* Ntr~r~rrul Public He&h Institute, Mrrnnerheir~Gr~tie 166, SF-00280 Helsrnki 28 (Finlund) Tel. (35. X0)) 47441 md
** Imritur fiir Molekulrrhiologw I, Unwersrrdt Ziirich, X093 Ziirich (Sw~rt:erlund) Tel. (41. I) 37744 I/

(Received January ZSth, 1983)

(.4ccepted February I st. 1983)

SUMMARY

Bu~~il1u.ssuhtilis was transformed with a hybrid gene in which the sequence encoding the cu-amylase signal
peptide was joined by a linker to the sequence encoding mature human interferon a2 (IFN-(~2). The hybrid
preprotein was cleaved precisely following the last amino acid of the cu-amylase signal sequence and was
secreted at 0.5-l mg per liter. IFN-(~2, preceded by either one or six amino acids, has the same specific
antiviral activity as IFN-e2 itself.

INTRODUCTION al., 1981), the luc promoter or the P-lactamase

(h/a) promoter (M. Mishina, W. Boll and C.
A number of cloned human interferon IFN Weissmann, cited by Weissmann, 1981). The inter-
cDNAs have been expressed in E. coli to yield feron produced in E. coli is retained within the
proteins with IFN activity (Nagata et al., 1980; cell, albeit partly in the periplasmic space, even
Goeddel et al., 1980b; Gray et al., 1982; Tani- when the b/u signal sequence is fused to the inter-
guchi et al., 1980; Yelverton et al., 1981). High feron signal sequence (A. Hall and C. Weissmann,
yields of mature IFNs have been obtained from unpublished results).
IFN genes deprived of their signal sequences and For production purposes it might prove ad-
joined to the AUG codon of a prokaryotic promo- vantageous to generate microorganisms which
ter translation-initiation region, for example the secrete the desired protein into the medium. A
trp promoter (Goeddel et al., 1980a; Yelverton et microorganism which might provide a suitable
secretion system is B. subtilis. Hardy et al. (1981)
described the expression of hepatitis B core anti-
gen and the major antigen (VPI) of foot and
Abbreviations: bp, base pairs: IFN, interferon; SDS. sodium
dodecylsulfate: TCA, trichloroacetic acid; U. units: ::, novel
mouth disease virus in B. subtilis; however, secre-
joint; [ 1,indicates plasmid-carrier state. tion was not achieved.

037X-l 119,/83/$03.00 $2 1983 Elsewer Science Pubhshers

 PaI? ii ( 1982) has cloned the cu-amylase ge11c by W. Boll and M. Mishina as outlined in Weisa-
frcw R. trr?l~lo/iyuefuciens in pUB 1 10 and demon- mann (1981). In short, the IFN-a2 cDNA (Streuli
S[I atrd its expression in B. .vdui/i~. The rxoamylase et al., 1980) was cloned into the Hirldlll-PtiulI
ih preceded by a signal sequence of 31 amino acids fragment of pBK322. The signal sequence of IFN-
(l’alva et al., 198 1). which is accurately cleaved off cu2 and the first codon (TGT) of the mature se-
ili the 11ew host (Takkinen et al., 1983). Palva et al. quence (Streuli et al.. 1980) were cleaved off by
I 1982) have recently constructed a 5criza of becre- partial Scr113A digestion. After filling up the 5’
tron \-ectors consisting of the promoter, rihosome overhanging Stru3A end wlith DNA polymeraae.
binding site and signal sequence region of the the Hind111 linker ACAAGCTTGT (a gift from
pre-rr-amylasc gene in pUB1 10. When the E. colr Dr. Kay Wu) was added, restoring the Cya codon.
h/o grne devoid of the segment encoding its signal The f’stl site at the end of the cDNA (Streuli et
>eyuence was joined to the secretion vectors. active al., 1980) was replaced by a IfirzdIII site. The
enzyme was secreted into the growth medium of fIirzdII1 fragment containing the IFN-(r2 mature
the tldnsformed R. .suh/ilrs. provided that the full coding sequence was joined to MirldIII-cleaved
4igllal sequelIce of cu-amylase Has przsenl (Palva et pKTH53 or pKTH51. to yield the plasmida
‘il., 1982). pK’rH68 and pKTH93a, respectively.
In this report we show that biologically active pKTH93a was partially cleaved with !flirldllI,
human leukocyte interferon-a2 ib secreted by 8. digested with Sl nuclease and ligated with T4
.~&i//., transformed by a plasmid in which the DNA ligase. pKTH93 was identified as an IFN-
\eyue~~ce encoding mature IFN is appropriately producing clone. The relevant DNA and amino
jcjined to tu-amylase derived secretion vectors. ‘I’he acid sequences were determined as above. The
hybrid preprotein i, accurately cleaved after the nucleotide sequences at the junction regions were
last rebidue of the u-amylase signal sequence. re- determined from a 5’-“P-labeled HlrrfI site (posi-
gardless of the amino acid following it. tion 6X of the IFN-(Y~ sequence) by the method of
Maxam and Gilbert (1980).

(c) Preparation and purification of IFN-(r2 secreted

by transformed B. subtilis

(a) Bacteria B. .suhrilis transformed with pKTH68 was grown

in shake flasks, in 2 x L broth-starch medium [per
B. .s&ri/i.s lHh140 was derived from B. suhtilis liter: 10 g yeast extract (BBL), 20 g Bactotryptone
Marburg strain 6064 by multiple steps of muta- (Difco). IO g soluble starch (Merck), 10 g NaCl.
genesis with N-nitrosoguanidine and selection of 1 g (NH,),SO,. 7 g K,HPO,. 2 g KH,PO, and
colonies with low exoprotease and used in all 12.5 mg kanamycin sulfate] at 37°C’ to an AhS,) ot
experiments described. Similar levels of IFN activ- about 5. After centrifugation (5000 X g, 20 min).
ity were obtained with transformed B. .suhtrlis 6064. the protein from 20 titers of supernatant was pre-
cipitated with 3% TCA (Merck) at 4°C‘. The pre-
(b) Construction of IFN-a2 secretion plasmids (cf., cipitate was extracted with 95% ethanol and the
Fig. IA) extract was filtered through a Matrex blue column
(Amicon) ( I .5--2 mg protein/ml bed volume). After
Plasmid pKTH38 was cleaved with EcoKI and washing the column with 2 column volumes each
digested with BAL31; after addition of Hind111 of 20 mM Tris HCl (pH 8) and 0.2 M NaCl in 20
linkers and incubation with T4 DNA ligase. the mM Tris HCI (pH S), the IFN was eluted with
LINA was cloned in B. suhtih (Palva et al., 1982). 2 M NaCl in Tris HCI (pH 8). The proteins were
Several plasrnids were sequenced from the HilrdIII concentrated by precipitation with 6% TCA. dis-
site (Maxam and Gilbert, 19X0), and pKTH5 I and solved in 50 mM Tris . HCI (pH 8) at 5~- 10 mg/ml
pKTH53 (see Fig. I) were used for further work. and chromatographed on Sephadex G- 100 (bed
Plasmid pmatIFNa2::pBK322 was constructed volume, 75 times the sample volume) in 50 mM
 231

Tris . HCl (pH 8). Polyacrylamide gel electro- was replaced by a Hind111 linker, so that cleavage
phoresis of the combined peak fractions is shown with Hind111 and treatment with Sl nuclease would
in Fig. 2 (lane 5). IFN from B. subtilis transformed generate a blunt end, with the first triplet encoding
with pKTH93 was purified in a similar fashion, the first amino acid (Cys) of the mature IFN
starting with 20 liters of supernatant. except that molecule (M. Mishina. W. Boll and C. Weissmann,
the last step was chromatofocusing rather than as described in Weissmann, 1981. A Hind111 lin-
Sephadex chromatography. Specific activities and ker was also introduced at the end of the cDNA
yields are given in Table II. (replacing the PstI site following the 3’ end of the
non-coding region; see Fig. 1A; Streuli et al.,
(d) Amino acid sequence determination 1980). As shown in Fig. lA, the Hind111 fragment
containing the IFN-(u~ sequence was introduced
Amino terminal amino acid sequences were into the Hind111 site of pKTH53, and the plasmid
determined on 50 pg purified IFN by automated with the desired orientation. pKTH68, was identi-
Edman degradation using the Applied Biosystems fied and sequenced across the a-amylase-inter-
Gas Phase Sequenator Model 470a; the resulting feron gene junction. It encoded a fusion prepro-
phenylthiohydantoin derivatives of the amino acids tein, in which the signal sequence of a-amylase
were identified by reverse phase HPLC, all as was followed by the first four amino acids of
described by Hewick et al. (1981). On a separate mature a-amylase. two extra amino acids encoded
sample, the amino terminal amino acid was by the linker, and finally the complete IFN-(Y~
dansylated and the dansyl derivative identified by mature sequence in the same reading frame. This
two-dimensional thin-layer chromatography. All construction was carried out because the require-
analytical data are available on request. Analyses ments for the cleaving off of the n-amylase signal
were carried out by Dr. J. Smart, Biogen Inc. peptide in B. subtilis were not yet known and we
wished to retain the original sequence around the
cleavage point.
In a second construction the IFN-a2-containing
RESULTS AND DISCUSSION Hind111 fragment was inserted into the Hind111
site of the secretion vector pKTH51, in which the
(a) Expression-plasmid constructions Hind111 linker followed immediately after the last
amino acid of the signal sequence. Partial Hind111
pKTH38 is a derivative of pUBll0 (Palva et al., cleavage, treatment with nuclease Sl and religa-
1982) which contains a 560-bp B. amyloliquefaciens tion eliminated four nucleotides of the linker and
DNA fragment extending from a MboI site, about yielded a plasmid, pKTH93, which encoded the
400 bp upstream from the beginning of the pre-cy- cy-amylase signal sequence followed by an alanine
amylase coding region, to an EcoRI linker inserted residue (due to residual linker nucleotides) and the
following codon 30 of the cu-amylase sequence mature IFN-a2 sequence (Fig. 1C).
(Fig. 1; Palva et al., 1982). This plasmid was
cleaved with EcoRI, partially digested with BAL3 1 (b) Synthesis, purification and properties of IFN
and religated via Hind111 linkers. Plasmids con- from B. subtilis
taining the Hind111 linkers at various positions
relative to the signal and mature coding sequence B. subtilis IH6140 carrying plasmids pKTH68
were obtained (Palva et al.. 1982). pKTH53 and or pKTH93 was grown in shaker flasks, and cell-
pKTH51, in which the Hind111 linker followed the bound and secreted IFN were assayed at different
fourth a-amylase codon and the last signal peptide times. Maximum IFN activity was found in the
codon, respectively, were used for further con- beginning of stationary phase; more than 90% of
structions (Fig. 1). the activity ws found in the supernatant at this
The mature IFN-c~2 sequence was introduced time point (Table I) as well as at other stages of
into secretion plasmids as follows. The sequence growth (data not shown).
encoding the signal peptide of IFN+.2 sequence IFN was purified from the supernatant by acid
232

precipitation, extraction with ethanol, chromatog- Table II, IFNs produced by both plasmids had
raphy on Matrex blue. and Sephadex G-100 chro- specific activities, around 2 x IO’ U/mg, which is
matography or chromatofocusing. As shown in in the same range as found for purified mature

EcoR I

Hind III linkers

Pst I

Hind III Hind III

. I I AGTCGKGTTCGP#KG...

Hind III

Hind III

Hind Ill
 233

Fig. I. IFN-n2 secretion plasmids. (A) Construction of pKTH68 and pKTH93a plasmids (Palva et al., 1982). pKTH38 contains the
kanamycin resistance gene and replicon derived from pUBll0 (thin line), and the promoter, ribosome binding site (hatched box) and
the coding sequence (open box) of pre-a-amylase of B. amyloliquefacwns (Palva et al., 1982) up to codon 61. where an EcoRI linker is
inserted. pKTH38 was cleaved with EcoRI. the ends nibbled back to varying extents and joined via NindlIl linkers to yield pKTH51
and pKTH53. The DNA encoding the mature IFN-a2 sequence was excised from pmatIFN-a2::pBR322. inserted into the Hind111
site of pKTH51 and pKTH53 to yield pKTH68 and pKTH93a. respectively. Crosshatched box, IFN coding sequence; open box,
n-amylase or linker sequence; hatched box, a-amylase signal sequence; wavy line. IFN 3’ non-coding sequence. (a) and (b), nucleotide
sequence at the Hind111 sites of plasmids pKTH53 and pKTH51, respectively. (B) Structure of pKTH68 at the a-amylase-interferon
junction. (C) Construction of pKTH93. pKTH93a was partially cleaved with Hind111 and the ends were blunted and rejoined to give
pKTH93. For details, see MATERIALS AND METHODS, section b. The amino acids in capital letters were determined by
sequential Edman degradation of the purified proteins (Table II); those in lower case letters were inferred from the nucleotide
sequence

TABLE 1

Secretion of interferon synthesized in transformed B. suh/ihs

66 kD
E. suhr,lis lH6140 transformed with either pKTH68 or pKTH93
was grown at 37’C in shaker flasks in ZXL broth until 5.5 h
beyond midlogarithmic phase (early stationary phase). The cells
45 kD were collected by centrifugation, suspended in 100 mM K.
phosphate buffer (pH 6.5) and disrupted by sonication.

Transforming Interferon activity (IU/ml

plasmid original culture)’
24 kD Secreted Cell
associated

pKTH68 1-2x 10s 6x IO’

pKTH93 1 x IO” 6x IO’
18 kD
a IFN activity was assayed in the supernatant and in the
14 kD sonicate using the plaque reduction assay (Cantell et al., 1974).
de--- relative to a standard titrated against an international standard.

123456
IFN-CYZ from E. coli (M. Fountoulakis, unpub-
Fig. 2. Analysis of purified alpha interferons by SDS-poly-
acrylamide gel electrophoresis. SDS-polyacrylamide gel electro- lished results). Both preparations gave a single
phoresis was carried out essentially as described by Laemmli band upo; SDS-polyacrylamide gel electrophore-
(1970). Lane 1, marker proteins; from top to bottom, bovine sis (Fig. 2).
serum albumin (66 kDa). egg albumin (45 kDa). trypsinogen
(24 kDa), /3-lactoglobulin (18.4 kDa) and lysozyme (14.3 kDa)
(Sigma). Lanes 2, 4 and 6, purified mature IFN-a2 from E. co11 amino actds). lane 3. Ala-IFN-a2 from B. suhrilis (pKTH93;
(prepared by M. Fountoulakis: 165 amino acids). Lane 5. Val- 166 amino acids). IO- I2 pg of each IFN were applied to the
Asn-Gly-Thr-Gln-Ala-IFN-a2 from B. sub/i/is (pKTH68; I71 gel; staining was with Coomassie blue. (kD or kDa. kilodalton).
234

TABLE I1

Purification of IFN secreted by transformed 8. s&t&\

Each purification (see MATERIALS AND METHODS. aectmn c) was started with 20 liters of bacterial supernatanta. Protrln
concentrations were determined by the Coomassie blue method (Bradford. 1976) usmg bovine serum alhumln as standard. IFN was
determined by the cytopathic effect reduction assay as described by Nagata et ai. (1YXO) and expressed in mternational units (IC!: see
Table 1).
~- ~____ _____- ~~
IFN (1Ux lCH) IFN (IUx IO ‘) y weld
per mg protein
-__
pKTH68 pKTH93 $THhx pKTH93 pKTH64 pKTH43

Culture supernatant 16 7.x 0.003 N.L). 100 100

Ethanol extract 9 7.1 0.01) 0.0x S6 Yl
Matrex blue 4.5 2.1 1.5 I.6 7x 35
Sephadex G- 100 3 ._ 2 10
~hr~rn~~t~f~c~lsin~ _ 1.6 2.7 20

N.D. = not determined.

The IFN from B. s~~~~fi.~(pKTH6~~ had a mo- tem, where the usual approach (Goeddel et al..
bility corresponding to an M, of 18000, distinctly 1979) yields as primary product the methionyl
slower than that of lFN-cu2 (apparent M,, 16800). derivative of the eukaryotic protein. This
while the product of B. .suhri/i,v[pKTH93] (ap- methionine residue is cleaved off by bacterial
parent M,, 17 000) has migrated only slightly enzymes to a variable extent, depending perhaps
slower than IFN-(~2. The amino terminal amino on the nature of the protein, the bacterial strain
acid sequences of the products. as determined by and the fermentation conditions. Talmadge et al.
automated Edman degradation. were Val-Asn- (l!%Oa,b) have found that fusion proteins in which
Gly-Thr-Gln-Ala-X-Asp-Leu-Pro for pKTH6X and part of the ampicillinase signal sequence is fused
Ala-Cys-X-Leu-Pro for pKTH93 (X: amino acid to part of the preproinsulin signal sequence are
was not identified). transported into the periplasmic space of E. co/i
This means, as can be seen in Fig. I, B and C, and cleaved accurately to yield proinsulin. how-
that hybrid pre-IFN molecules synthesized in B. ever, no secretion into the medium was observed.
subf~~is. comprising the cu-amylasr signal peptide Comparable constructions with preillterfer~~n did
and the mature IFN-cu2 polypeptide, were cleaved not lead to accurate cleavage (A. Hall. M.
after the Ala residue in position 31, exactly as the Hunkapiller. C. Weissmann, unpublished results).
natural pre-cu-amylase (Takkinen et al.. 1983). We It is of interest to note that the addition of up to 6
have not ascertained whether the cells contain amino acid residues to the amino terrn~ilus oi
biologically inactive, uncleaved IFN precursor. The IFN-a2 does not diminish the specific antiviral
fact that accurate cleavage occurred both when the activity of the protein.
signal peptide was followed by the Val-Asn-Gly- Another potential advantage of the B. .ruhtllis
Thr sequence derived from the mature cu-amylase, system is that the eukaryotic protein is secreted
or by an Ala residue (encoded by a linker residue) into the medium and therefore has a higher initial
followed by the IFN-cu2 sequence suggests that purity than when it is recovered from cell extracts
accurate processing by B. suhrilis of at least some or cell lysates. The fact that the foreign protein is
hybrid preproteins is determined solely by the not accumulated within the cell. where it can
signal peptide. It is therefore likely that construc- impair the cell metabolism and growth, should in
tions in which the signal peptide is fused precisely principle allow much higher yields of protein per
to the IFN-(w2 polypeptide will be cleaved to re- liter of culture; some strains of B. suhtilis contain-
lease mature IFN-a2 without any supernumerary ing the cloned a-amyiase gene produce several
amino acid residues. In this regard the approach grams of n-amylase per liter. and there is no
may be superior to expression in the E. co/i sys- reason to assume that this could not be achieved
 235

for other proteins. Such yields would be greater by Hewick. R.M., Hunkapiller, M.W.. Hood, L.E. and Dreyer.
a factor of ten than those hitherto achieved with E. W.J.: A gas-liquid phase peptide and protein sequenator. J.
Biol. Chem. 256 (1981) 7990-7997.
co/i in the most favourable cases.
Laemmli, U.K.: Cleavage of structural proteins during the
assembly of the head of hacteriophnge T4. Nature 227
(1970) 680-685.
Maxam. A.M. and Gilbert. W.: Sequencing end-labeled DNA
ACKNOWLEDGEMENTS with base-specific chemical cleavages. in Grossman. L.. and
Moldave, K. (Eds.), Methods in Enzymology. Vol. 65.
Academic Press. New York. 19X0. pp. 499-560.
The project was supported by the Finnish Na-
Nagata. S.. Taira, H.. Hall. A.. Johnsrud. L.. Streuli. M.,
tional Fund for Research and Development Ecaodi, J.. Boll. W., Cantell, K. and Weissmann, C.:
(SITRA) and Biogen S.A. We thank John Smart, Synthesis in E. co/r of a polypeptide wth human leukocyte
Biogen Inc., for carrying out the amino acid se- interferon activity. Nature 284 (1980) 316-320.
quence determination and Eila Kujamaki, Monika Palva. I.: Molecular cloning of n-amylase gene from Ruullus
un~,vloliquefacrens and its expression in B. whtllu. Gene I9
Walchli and Barbara Alberti for technical assis-
(1982) X1-87.
tance.
Palva. I., Pettersson. R.F.. Kalkkinen. N.. Lehtovaara, P.,
Sarvas, M., Siiderlund, H., Takkinen, K. and KBBrl%inen.
L.: Nucleotide sequence of the promoter and NH,-terminal
signal peptide region of the a-amylase gene from Bocrllus

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