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Constitutive expression of a native human interferon-alpha 1 gene in Escherichia coli
/fir. J. B;<>&m.Vol. 21, No. 9, pp. 983-985, 1989
0020-7 1I X/89 $3.00 + 0.00 Copyright i“ 1989 PergamonPress plc
Printedin Great Brit;tin.All rights reserved
~ONSTTTUTTVE EXPRESSION INTERFERON-alpha 1 GENE
OF A NATIVE HUMAN TN ESCHERICHIA COLI
DIMCHO BACHVAROV, KRASSIMIR ALEXCIEV, ADRIANA SARAFFOVA, VERA MAXIMOVA, IRINA TSANEVA and GEORGE MARKOV
IVAN IVANOV. NEDJALKA MARKOVA,
Institute of Molecular Biology, Bulgarian Academy of Sciences, 1I13 Sofia, Bulgaria
(Received 30 .Iunumy 1989) Abstract-l. A plasmid for constitutive expression of the human interferon-alpha I (hIFN-a I) gene in Escherichia coli is constructed on the basis of the cloning plasmid pBR322 using a strong synthetic promoter, synthetic ribosome binding site and a native hIFN-ccI gene excised from a chromosomal clone. 2. The yield of recombinant hIFN-a 1 from E. co/i LE392 cells transformed with the expression plasmid pJP, R,-hIFN-a 1 is evaluated to be 2-6 x IO’ U/l bacterial culture for metabolic shaker and 6-8 x 10’ U/t for fermentor.
INTRODUCTION Human interferon-alpha (hIFN-or) gene family, comprising more than 15 individual genes and pseudogenes, is one of the most extensively studied eukaryotic gene family (Hence et al., 1982; Langer and Pestka, 1985; Pestka, 1985). The hIFN-srl gene has been expressed first in Escherichia coli at a relatively low level (2 x IO4U/l culture) under the p-iactamase promoter (Pst I site of the plasmid pBR322) by Nagata et al. (1980a). Later on the yield of hIFN-a 1 has been increased IOOO-fold by fusing the gene to the luc promoter (Weissman et al., 1982). De Maeyer et al. (1982) used a chemically synthesized gene of hIFN-a 1 to compare the efficiency of expression of this gene under the control of both lac (wild type) and Zac UV.5 promoters. The yield of recombinant hIFN-a 1 obtained by these authors [as tested on human cells, known to be IO-50 times less sensitive to this interferon as compared with the MDBK (bovine) cells] were 0.6 x lO’U/l for the luc (wild type) and 6 x lO’U/l for the luc WV5 promoter respectively. The higher yield obtained with the luc LJV.5 promoter (which does not require catabolic activation) was stimulating foe us to study the efficiency of expression of the hIFN-a1 gene under the control of a totally insensitive to bacterial repressors promoter. For this purpose we used a strong synthetic promoter (the bacteriophage T5 early promoter) devoid of any operator sequences. This constitutive promoter (designated as P, ) was created by Jay and co-workers (Rommens er al., 1983) and its efficiency in controlling expression of heterologous genes in E. coli has been undoubtedly proven (Jay et al., 1984; Ivanov et al., 1987a,b). MATERIALS AND METHODS
Restriction endonucfeases and other enzymes as well as the reagents for oligonucleotide synthesis were purchased from Pharmacia, Sweden, All other chemicals were bought from Merck, F.R.G. and Sigma, U.S.A. The isotopes were product of Amersham, U.K. Oligonucleotides were synthesized by the phosphoramidite approach using automatic gene assembler (Pharmacia, 983
Sweden) and following the instruction manual of the producer. Piasmid DNA for analytical purposes was isolated by the mini-prep method of Birnboim and Doty (1979) and for preparative use by the hydroxylapatite method of Bachvarov and Ivanov (1983). DNA colony hybridization with synthetic ohgonucleotides was carried out as recommended by Meinkoth and Wahl (1984) and the RNA colony hybridization with the same probes was performed as already described (Ivanov and Gigova, 1986). -DNA was sequenced bv the chemical method of Maxam and Gilbert (1977). Competent bacteria were prepared and transformed with plasmids as recommended by Hanahan (1983). Clear lysates for hIFN-a 1 testing were prepared from SA,,,-U recombinant cells cultivated in L-broth supplemented with I % glucose and IO pg/ml tetracycline. The cells were harvested by centrifugation and sonicated in I ml of TE (10 mM Tris, pH 7.4, 1mM EDTA) containing 0. I % SDS. The biological testing (determination of the protective effect against the cytopathic action of the VSV) was performed
on Wish cells. RESULTS AND DISCUSSION
The expression vector pJP, RghIFN-a 1 was constructed as shown in Fig. 1. As seen from the figure, this plasmid is a derivative of the cloning vector pBR322 in which a DNA fragment consisting of consecutively linked (5’+3’) synthetic constitutive promoter (P,), synthetic ribosome binding site (RBS, R,) and a chromosomal hIFN-a 1 gene is substituted for the 29 bp DNA fragment excised by EcoR I and Hind III. The structure of the P, promoter as linked to the artificial RBS Rg was published elsewhere (Ivanov et al., 1987a). The hIFN-a 1 gene was excised from a chromosoma1 DNA clone previously sequenced in our laboratory (unpublished data). The nucleotide sequence of the region in this clone taking up the nucieotides from -45 (nucleotide position, counting from A of the start-codon ATG of the hIFN-cr 1 leader sequence) to + 629 (located downstream of the stop-codon TAA) was exactly the same as published by Nagata et al. (1980b). The hIFN-sc 1 coding sequence was excised from this genomic clone by double digestion with Hinf I
984
IVAN
Eco
Hind
RI
IVAN~V
III
‘1, Hind
III
Ligase
Hind 111
[Hind
Ill
ECORI Hind
Hind
Fig. I. Construction
Ill
Ill
of the expression plasmid pJP, R,hIFN-a I.
(cleavage site at -45) and Eco RI (recognition site at f625). The physical map of this fragment is presented in Fig. 2. As seen from the figure, there are two Pvu II sites in this fragment: one located at nucleotide position +44 in the leader sequence and the other one in the middle part of the hIFN-a 1 gene (nt position +346). In order to remove the nucleotides preceding the first Pvu II site the Hinf I/EcoR I fragment was partially digested with Pvu II and the longer DNA fragment was isolated. Then a synthetic linker composed of two ohgonucleotides (9 nt and 13 nt long) was ligated to the 5’-blunt ended Pvu II/EcoR I DNA fragment with the purpose of introducing a Hind III site, one start-codon ATG and to restore the codons of the signal sequence destroyed by the enzyme Pvu II. The 3’-end of the fragment was modified by ligating a EcoR I/Hind III adapter. The hIFN-cr 1 gene thus modified coded for 177 aminoacids, 166 of which belonged to the mature hIFN-cr 1, 10 to the leader sequence and one additional methionine. This gene was inserted in the Hind III site of the expression vector between the synthetic RBS R, and the Tc’ gene. In this construction the
HI
P
I
I
B
et
al.
Tc’ gene is devoid of its own promoter (which is destroyed by eliminating the EcoR I/Hind III DNA fragment from pBR322) and its transcription is dependant on the activity of the synthetic promoter P, . This makes it possible to use tetracycline as selective medium against segregation of the plasmid and to maintain the structure of the P, promoter. The expression plasmid pJP, R,-hIFN-a 1 thus constructed was used for transformation of E. coli LE392 cells and recombinant clones containing the hIFN-cc I gene in right orientation were selected by DNA and RNA colony hybridization using a 15 nt long 3ZP-labelled oligonucleotide complementary to the hIFN-cc I mRNA. The efficiency of the synthetic regulatory elements (promoter and RBS) in the expression plasmid pJP, R,-hIFN-cc 1 to initiate expression of the native hIFN-cr 1 gene was studied by measuring the accumulation of recombinant hIFN-cc 1 in the producing cells. Figure 3 illustrates the production of recombinant product as a function of the bacterial growth. As seen from the figure, the hIFN-cc 1 was synthesized continuously and the maximum of accumulation was achieved at the late logarithmic phase. In order to compare productivity of the recombinant strain constructed in our laboratory with that of the others we tested the interferon activity on human (Wish) cells. Our results showed that the yield of hIFN-cr 1 was 2-6 x 10’ U/l for metabolic shaker and 6-8 x lO’U/l for 16 I. fermentor. These values are close to those reported by De Maeyer et al. (1982) for the expression of the hIFN-cc 1 gene under the lac Ii V5 promoter. To measure the specific activity of the recombinant hIFN-cc I some amount of this product was isolated by using affinity chromatography column containing monoclonal antibodies specific for hIFN-a 1 covalently bound to BrCN-activated Sepharose 4B. The specific activity as measured on Wish cells was evaluated to be about lO’U/mg protein which corresponded to the value (2 x IO’ Ujmg) reported by Weissman et al. (1982). Based on this value we estimated that the content of recombinant hIFN-a 1 in the producing cells was l-2% of the total cellular protein. This yield was much lower as compared with the yields of other proteins like hIFN-y (Jay et aI., 1984), human tetrameric calcitonin (Ivanov and Jay, 1987) CAT (E. Jay et al., unpublished data) etc., obtained by using the same regulatory elements, where the yield of recombinant product exceeded l&15% of the total bacterial protein. We failed to improve the yield of hIFN-crl by modifying the structure of the synthetic RBS and the distance between the RBS and the start-codon ATG. Taking into account the literature data and the results obtained in our laboratory we could conclude:
P
HIU,E
100 bp
I
HI
I
Fig. 2. Restriction map of the chromosomal Hinf I/EcoR I DNA fragment containing the hIFN-a I gene.
Interferon-alpha
25
20
15
g 2
10
S-
I gene expression
985
synthesized human alpha l-interferon gene. Proc. natn. Acad. Sri. U.S.A. 79, 4256-4259. Hanahan D. (1983) Studies on transformation of Escherichia coli with plasmids. J. mol. Biol. 166, 557-580. Hence K., Brosius J., Fujisawa A., Fujisawa J., Haymra J., Hochstadt J., Kovacic T., Pasek M., Schambock A., Schmid J., Todokoro K., Walchli M.. Nagata S. and Weissman C. (1982) J. mol. Biol. 185, 227-260. Ivanov I. and Gigova L. (1986) RNA colony hybridization method. Gene 46, 287-290. Ivanov I. and Jay E. (1987) Gen de la calcitonina humana: Synthesis quimica y expresion in E. co/i. Inter/bon y Biothchnologia 4, 48-82. _ Ivanov I., Tam J., Wishart P. and Jay E. (1987a) Chemical synthesis and expression of the human calcitonin gene. Gene 59, 2233230. Ivanov I.. Gieova L. and Jav_.E. (1987b) Chemical synthesis and expresiion in E. coli of a human ValR-calcitonin gene by fusion to a synthetic human interferon-gamma gene. FEBS Lerr. 210, 5660. Jay E., Rommens J., Pomeroy-Cloney L., MacKnight D., Lutze C., Wishart P., Harrison D., Liu W.-Y., Asundi V., Dawood M. and Jay F. (1984) High-level expression of a chemically synthesized gene for human interferon-gamma using a prokaryotic expression vector. Proc. nafn. Acad. Sri. U.S.A. 81, 229&2294. Langer J. A. and Pestka S. (1985) Structure of interferons. Piarmac. Ther. 27, 371401. Maxam A. and Gilbert W. (1977) A new method for sequencing DNA. Proc. n&. kcad. Sci. U.S.A. 74, 560-564. Meinkoth J. and Wahl G. (1984) Hybridization of nucleic acids immobilized on solid supports. Analyf. Biochem. 183, 267-284. Nagata S., Taira H., Hall A., Johnsrud L., Streuli M., Ecsddi J., Ball W., Cantell K. and Weissman C. (1980a) Synthesis in E. coli of a polypeptide with human leukocyte interferon activity. Nature 284, 316320. Nagata S., Mantei N. and Weissman C. (1980b) The structure of one of the eight or more distinct chromosomal genes for human interferon alpha. Nafure 287, 401408. Pestka S. (1985) Interferon from 1981 to 1985. Merh. I
5 / /
/
I
I
I
2
4
6
[h’J
Fig. 3. Production
of recombinant hIFN-a growth phase.
I in bacterial
first, that the constitutive gene expression is advantageous for the hIFN-crl gene and second, that the yield of 6-7 x lO’U/l culture (or l-2% of the total cellular protein) seems to be maximal one for the recombinant hIFN-a 1 from E. coli. Acknowledgement-The authors are thankful to Professor E. Jay (University of New Brunswick, Canada) for his permission to use the P, promoter in their constructions.
Enzym. 119, 3-14. REFERENCES
Bachvarov D. and Ivanov 1. (1983) Large scale purification of plasmid DNA. Prep. Biochem. 13, 161-166. Birnboim H. C. and Doly J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523. De Maeyer E., Skup D., Prasad K., De Maeyer J., Williams B., Meacock P., Sharpe G., Pioli D., Hennam J., Schuch W. and Atherton K. (1982) Expression of a chemically
Rommens J., MacKnight D., Pomeroy L. and Jay E. (1983) Gene expression: chemical synthesis and molecular cloning of a bacteriophage T5 (T5P25) early promoter. Nucleic Acids Res. 11, 5921-5940. Weissman C., Nagata S., Boll W., Fountoulakis M., Fujisawa A., Fujisawa J., Hence K., Mantei N., Ragg H., Schein C., Schmid J., Shaw G., Streuli M., Taira H., Todokoro K. and Weidle U. (1982) Structure and expression of human alpha interferon genes. UCLA Symp. Mol. Cell. Biol. 25, 295-326.
0020-7 1I X/89 $3.00 + 0.00 Copyright i“ 1989 PergamonPress plc
Printedin Great Brit;tin.All rights reserved
~ONSTTTUTTVE EXPRESSION INTERFERON-alpha 1 GENE
OF A NATIVE HUMAN TN ESCHERICHIA COLI
DIMCHO BACHVAROV, KRASSIMIR ALEXCIEV, ADRIANA SARAFFOVA, VERA MAXIMOVA, IRINA TSANEVA and GEORGE MARKOV
IVAN IVANOV. NEDJALKA MARKOVA,
Institute of Molecular Biology, Bulgarian Academy of Sciences, 1I13 Sofia, Bulgaria
(Received 30 .Iunumy 1989) Abstract-l. A plasmid for constitutive expression of the human interferon-alpha I (hIFN-a I) gene in Escherichia coli is constructed on the basis of the cloning plasmid pBR322 using a strong synthetic promoter, synthetic ribosome binding site and a native hIFN-ccI gene excised from a chromosomal clone. 2. The yield of recombinant hIFN-a 1 from E. co/i LE392 cells transformed with the expression plasmid pJP, R,-hIFN-a 1 is evaluated to be 2-6 x IO’ U/l bacterial culture for metabolic shaker and 6-8 x 10’ U/t for fermentor.
INTRODUCTION Human interferon-alpha (hIFN-or) gene family, comprising more than 15 individual genes and pseudogenes, is one of the most extensively studied eukaryotic gene family (Hence et al., 1982; Langer and Pestka, 1985; Pestka, 1985). The hIFN-srl gene has been expressed first in Escherichia coli at a relatively low level (2 x IO4U/l culture) under the p-iactamase promoter (Pst I site of the plasmid pBR322) by Nagata et al. (1980a). Later on the yield of hIFN-a 1 has been increased IOOO-fold by fusing the gene to the luc promoter (Weissman et al., 1982). De Maeyer et al. (1982) used a chemically synthesized gene of hIFN-a 1 to compare the efficiency of expression of this gene under the control of both lac (wild type) and Zac UV.5 promoters. The yield of recombinant hIFN-a 1 obtained by these authors [as tested on human cells, known to be IO-50 times less sensitive to this interferon as compared with the MDBK (bovine) cells] were 0.6 x lO’U/l for the luc (wild type) and 6 x lO’U/l for the luc WV5 promoter respectively. The higher yield obtained with the luc LJV.5 promoter (which does not require catabolic activation) was stimulating foe us to study the efficiency of expression of the hIFN-a1 gene under the control of a totally insensitive to bacterial repressors promoter. For this purpose we used a strong synthetic promoter (the bacteriophage T5 early promoter) devoid of any operator sequences. This constitutive promoter (designated as P, ) was created by Jay and co-workers (Rommens er al., 1983) and its efficiency in controlling expression of heterologous genes in E. coli has been undoubtedly proven (Jay et al., 1984; Ivanov et al., 1987a,b). MATERIALS AND METHODS
Restriction endonucfeases and other enzymes as well as the reagents for oligonucleotide synthesis were purchased from Pharmacia, Sweden, All other chemicals were bought from Merck, F.R.G. and Sigma, U.S.A. The isotopes were product of Amersham, U.K. Oligonucleotides were synthesized by the phosphoramidite approach using automatic gene assembler (Pharmacia, 983
Sweden) and following the instruction manual of the producer. Piasmid DNA for analytical purposes was isolated by the mini-prep method of Birnboim and Doty (1979) and for preparative use by the hydroxylapatite method of Bachvarov and Ivanov (1983). DNA colony hybridization with synthetic ohgonucleotides was carried out as recommended by Meinkoth and Wahl (1984) and the RNA colony hybridization with the same probes was performed as already described (Ivanov and Gigova, 1986). -DNA was sequenced bv the chemical method of Maxam and Gilbert (1977). Competent bacteria were prepared and transformed with plasmids as recommended by Hanahan (1983). Clear lysates for hIFN-a 1 testing were prepared from SA,,,-U recombinant cells cultivated in L-broth supplemented with I % glucose and IO pg/ml tetracycline. The cells were harvested by centrifugation and sonicated in I ml of TE (10 mM Tris, pH 7.4, 1mM EDTA) containing 0. I % SDS. The biological testing (determination of the protective effect against the cytopathic action of the VSV) was performed
on Wish cells. RESULTS AND DISCUSSION
The expression vector pJP, RghIFN-a 1 was constructed as shown in Fig. 1. As seen from the figure, this plasmid is a derivative of the cloning vector pBR322 in which a DNA fragment consisting of consecutively linked (5’+3’) synthetic constitutive promoter (P,), synthetic ribosome binding site (RBS, R,) and a chromosomal hIFN-a 1 gene is substituted for the 29 bp DNA fragment excised by EcoR I and Hind III. The structure of the P, promoter as linked to the artificial RBS Rg was published elsewhere (Ivanov et al., 1987a). The hIFN-a 1 gene was excised from a chromosoma1 DNA clone previously sequenced in our laboratory (unpublished data). The nucleotide sequence of the region in this clone taking up the nucieotides from -45 (nucleotide position, counting from A of the start-codon ATG of the hIFN-cr 1 leader sequence) to + 629 (located downstream of the stop-codon TAA) was exactly the same as published by Nagata et al. (1980b). The hIFN-sc 1 coding sequence was excised from this genomic clone by double digestion with Hinf I
984
IVAN
Eco
Hind
RI
IVAN~V
III
‘1, Hind
III
Ligase
Hind 111
[Hind
Ill
ECORI Hind
Hind
Fig. I. Construction
Ill
Ill
of the expression plasmid pJP, R,hIFN-a I.
(cleavage site at -45) and Eco RI (recognition site at f625). The physical map of this fragment is presented in Fig. 2. As seen from the figure, there are two Pvu II sites in this fragment: one located at nucleotide position +44 in the leader sequence and the other one in the middle part of the hIFN-a 1 gene (nt position +346). In order to remove the nucleotides preceding the first Pvu II site the Hinf I/EcoR I fragment was partially digested with Pvu II and the longer DNA fragment was isolated. Then a synthetic linker composed of two ohgonucleotides (9 nt and 13 nt long) was ligated to the 5’-blunt ended Pvu II/EcoR I DNA fragment with the purpose of introducing a Hind III site, one start-codon ATG and to restore the codons of the signal sequence destroyed by the enzyme Pvu II. The 3’-end of the fragment was modified by ligating a EcoR I/Hind III adapter. The hIFN-cr 1 gene thus modified coded for 177 aminoacids, 166 of which belonged to the mature hIFN-cr 1, 10 to the leader sequence and one additional methionine. This gene was inserted in the Hind III site of the expression vector between the synthetic RBS R, and the Tc’ gene. In this construction the
HI
P
I
I
B
et
al.
Tc’ gene is devoid of its own promoter (which is destroyed by eliminating the EcoR I/Hind III DNA fragment from pBR322) and its transcription is dependant on the activity of the synthetic promoter P, . This makes it possible to use tetracycline as selective medium against segregation of the plasmid and to maintain the structure of the P, promoter. The expression plasmid pJP, R,-hIFN-a 1 thus constructed was used for transformation of E. coli LE392 cells and recombinant clones containing the hIFN-cc I gene in right orientation were selected by DNA and RNA colony hybridization using a 15 nt long 3ZP-labelled oligonucleotide complementary to the hIFN-cc I mRNA. The efficiency of the synthetic regulatory elements (promoter and RBS) in the expression plasmid pJP, R,-hIFN-cc 1 to initiate expression of the native hIFN-cr 1 gene was studied by measuring the accumulation of recombinant hIFN-cc 1 in the producing cells. Figure 3 illustrates the production of recombinant product as a function of the bacterial growth. As seen from the figure, the hIFN-cc 1 was synthesized continuously and the maximum of accumulation was achieved at the late logarithmic phase. In order to compare productivity of the recombinant strain constructed in our laboratory with that of the others we tested the interferon activity on human (Wish) cells. Our results showed that the yield of hIFN-cr 1 was 2-6 x 10’ U/l for metabolic shaker and 6-8 x lO’U/l for 16 I. fermentor. These values are close to those reported by De Maeyer et al. (1982) for the expression of the hIFN-cc 1 gene under the lac Ii V5 promoter. To measure the specific activity of the recombinant hIFN-cc I some amount of this product was isolated by using affinity chromatography column containing monoclonal antibodies specific for hIFN-a 1 covalently bound to BrCN-activated Sepharose 4B. The specific activity as measured on Wish cells was evaluated to be about lO’U/mg protein which corresponded to the value (2 x IO’ Ujmg) reported by Weissman et al. (1982). Based on this value we estimated that the content of recombinant hIFN-a 1 in the producing cells was l-2% of the total cellular protein. This yield was much lower as compared with the yields of other proteins like hIFN-y (Jay et aI., 1984), human tetrameric calcitonin (Ivanov and Jay, 1987) CAT (E. Jay et al., unpublished data) etc., obtained by using the same regulatory elements, where the yield of recombinant product exceeded l&15% of the total bacterial protein. We failed to improve the yield of hIFN-crl by modifying the structure of the synthetic RBS and the distance between the RBS and the start-codon ATG. Taking into account the literature data and the results obtained in our laboratory we could conclude:
P
HIU,E
100 bp
I
HI
I
Fig. 2. Restriction map of the chromosomal Hinf I/EcoR I DNA fragment containing the hIFN-a I gene.
Interferon-alpha
25
20
15
g 2
10
S-
I gene expression
985
synthesized human alpha l-interferon gene. Proc. natn. Acad. Sri. U.S.A. 79, 4256-4259. Hanahan D. (1983) Studies on transformation of Escherichia coli with plasmids. J. mol. Biol. 166, 557-580. Hence K., Brosius J., Fujisawa A., Fujisawa J., Haymra J., Hochstadt J., Kovacic T., Pasek M., Schambock A., Schmid J., Todokoro K., Walchli M.. Nagata S. and Weissman C. (1982) J. mol. Biol. 185, 227-260. Ivanov I. and Gigova L. (1986) RNA colony hybridization method. Gene 46, 287-290. Ivanov I. and Jay E. (1987) Gen de la calcitonina humana: Synthesis quimica y expresion in E. co/i. Inter/bon y Biothchnologia 4, 48-82. _ Ivanov I., Tam J., Wishart P. and Jay E. (1987a) Chemical synthesis and expression of the human calcitonin gene. Gene 59, 2233230. Ivanov I.. Gieova L. and Jav_.E. (1987b) Chemical synthesis and expresiion in E. coli of a human ValR-calcitonin gene by fusion to a synthetic human interferon-gamma gene. FEBS Lerr. 210, 5660. Jay E., Rommens J., Pomeroy-Cloney L., MacKnight D., Lutze C., Wishart P., Harrison D., Liu W.-Y., Asundi V., Dawood M. and Jay F. (1984) High-level expression of a chemically synthesized gene for human interferon-gamma using a prokaryotic expression vector. Proc. nafn. Acad. Sri. U.S.A. 81, 229&2294. Langer J. A. and Pestka S. (1985) Structure of interferons. Piarmac. Ther. 27, 371401. Maxam A. and Gilbert W. (1977) A new method for sequencing DNA. Proc. n&. kcad. Sci. U.S.A. 74, 560-564. Meinkoth J. and Wahl G. (1984) Hybridization of nucleic acids immobilized on solid supports. Analyf. Biochem. 183, 267-284. Nagata S., Taira H., Hall A., Johnsrud L., Streuli M., Ecsddi J., Ball W., Cantell K. and Weissman C. (1980a) Synthesis in E. coli of a polypeptide with human leukocyte interferon activity. Nature 284, 316320. Nagata S., Mantei N. and Weissman C. (1980b) The structure of one of the eight or more distinct chromosomal genes for human interferon alpha. Nafure 287, 401408. Pestka S. (1985) Interferon from 1981 to 1985. Merh. I
5 / /
/
I
I
I
2
4
6
[h’J
Fig. 3. Production
of recombinant hIFN-a growth phase.
I in bacterial
first, that the constitutive gene expression is advantageous for the hIFN-crl gene and second, that the yield of 6-7 x lO’U/l culture (or l-2% of the total cellular protein) seems to be maximal one for the recombinant hIFN-a 1 from E. coli. Acknowledgement-The authors are thankful to Professor E. Jay (University of New Brunswick, Canada) for his permission to use the P, promoter in their constructions.
Enzym. 119, 3-14. REFERENCES
Bachvarov D. and Ivanov 1. (1983) Large scale purification of plasmid DNA. Prep. Biochem. 13, 161-166. Birnboim H. C. and Doly J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523. De Maeyer E., Skup D., Prasad K., De Maeyer J., Williams B., Meacock P., Sharpe G., Pioli D., Hennam J., Schuch W. and Atherton K. (1982) Expression of a chemically
Rommens J., MacKnight D., Pomeroy L. and Jay E. (1983) Gene expression: chemical synthesis and molecular cloning of a bacteriophage T5 (T5P25) early promoter. Nucleic Acids Res. 11, 5921-5940. Weissman C., Nagata S., Boll W., Fountoulakis M., Fujisawa A., Fujisawa J., Hence K., Mantei N., Ragg H., Schein C., Schmid J., Shaw G., Streuli M., Taira H., Todokoro K. and Weidle U. (1982) Structure and expression of human alpha interferon genes. UCLA Symp. Mol. Cell. Biol. 25, 295-326.