- Email: [email protected]
[62] Cloning, expression, and purification of rat IFN-α1
[62]
RAT IFN-al
441
Concluding Comments Hybridization of the murine genomic library to the 32P-labeled human alpha interferon gene (642 bp AvaII-pLeIFLrA25 fragment) resulted in strong positive signals with virtually no background. The probe chosen represented over 90% of the coding region and contained no poly(A) sequences and no introns. Since we were cross-hybridizing between species and since the extent of the homology between human and mouse interferon genes was unknown, the stringency of the hybridization solution was lowered from a standard concentration of 50% formamide to a lower stringency of 40% formamide. This method proved successful in the isolation of a genomic murine alpha interferon gene. After the nucleotide sequence of the gene was determined, it was possible to construct an effective expression vector. It should be noted that since the gene isolated was a genomic clone rather than a cDNA clone, it could possibly contain introns. An open reading frame of 190 amino acids was determined from the sequence. By comparison of the nucleotide sequence with that of the nucleotide sequence of the human alpha interferon genes, TM it was concluded that the gene contained no introns, but contained a signal peptide of 23 amino acids followed by a cysteine which corresponded to Cys 1 of the human gene. Therefore, cysteine was concluded to be the first amino acid of the mature protein and the expression vector was appropriately designed and constructed as described above. 7 14 S. Pestka, Arch. Biochem. Biophys. 221, l (1983).
[62] C l o n i n g , E x p r e s s i o n , a n d P u r i f i c a t i o n o f R a t I F N - a l
By
PETER H. VAN DER M E I D E , REIN DIJKEMA, MARTIN CASPERS, KITTY VIJVERBERG, and H U U B SCHELLEKENS
This chapter describes the introduction of a structural gene for a rat alpha interferon subtype (rat IFN-a) into Escherichia coli to obtain an efficient expression system for the production of large quantities of rat interferon. This would provide a more economic way of production than does the system employing a rat continuous cell linO and also make it possible to study the properties of a single gene product. DNA comple1 p. H. van der Meide, J. Wubben, K. Vijverberg, and H. Schellekens, this volume [31].
M E T H O D S IN E N Z Y M O L O G Y , V O L . 119
Copyright It) 1986 by Academic Press, Inc. All rights of reproduction in any hwm reserved.
442
GENE ISOLATION AND EXPRESSION
[62]
mentary to rat interferon mRNA isolated from Ratec cells induced by Sendai virus was cloned and amplified in E. coli. A purified cDNA fragment identified by hybridization with the mouse a2 gene was then used as a probe for the isolation of the chromosomal genes from a rat gene library. In this way, a 2.5 kb EcoRI fragment encoding a complete rat IFN-a gene (rat IFN-al) was isolated and placed under the control of a hybrid tetracycline-tryptophan promoter into the EcoRI site of pMBL604 (a derivative plasmid of PBR322). E. coli strains carrying this recombinant plasmid synthesized biologically active interferon. The procedure for the production and purification of this type of interferon will be described. Also attempts to increase production with other host/vector systems are described and some characteristics of rat IFN-al will be discussed. Cloning of the Chromosomal Rat IFN-a Gene
Preparation, Purification, and Identification of l F N m R N A from Induced Ratec Cells For the construction of cDNA clones, total cytoplasmic RNA was isolated from Ratec cells actively synthesizing interferon. To establish at what time after virus infection a maximum amount of mRNA coding for rat interferon was present in the cells, poly(A) ÷ RNA was isolated at different times after Sendai infection and translated in Xenopus laevis oocytes (see below). The pattern of IFN activity in this assay revealed an optimum at about l0 hr after infection (see Fig. 1). At that time, total RNA (from 101° cells) was isolated from the cells by extraction with a buffer containing l0 mM Tris.HCl (pH 7.9), 1 mM EDTA, 150 mM NaCl, and 0.65% Nonidet P40 for 5 min at 4°. After centrifugation, the supernatant was mixed with an equal volume of a buffer containing 20 mM Tris.HC1 (pH 7.9), 20 mM EDTA, 300 mM NaCl, 7 M urea, and 1% SDS. The mixture was then extracted twice with phenol. The poly(A) ÷ RNA fraction (about 250/xg) was isolated by repeated batchwise absorption onto oligo(dT)-cellulose.: Different mRNA size fractions were obtained by use of a SDS-PAGE system as described by Wierenga et a l . 3 The mRNA fractions were injected into oocytes of Xenopus laevis as described by Gordon et al. 4 After incubation, the eggs were homogenized 2 S. Nagata, H. Taira, A. Hall, L. Johnsrud, M. Streuli, J. Ecs6di, W. Boll. K. Cantell, and C. Weissmann, Nature (London) 284, 316 0980). 3 B. Wierenga, J. Mulder, A. van der Ende, A. Bruggeman, G. Ab, and M. Gruber, Eur. J. Biochem. 89, 67 (1978). 4 j. B. Gordon, C. D. Lane, M. Woodlans, and G. Marbaix, Nature (London) 233, 177 (1971).
[62]
P,A T
IFN-cd
443
500 E
.-_"
i)i))))il)i)i)i!
400
D
.g
300
u
o
200 100
°_ i
|
4
8
|
12
16
|
2O
time after infection (hour) FIG. 1. Time course of IFN activity and 1FN mRNA production. At different times, the culture medium was decanted and replaced by fresh medium. The IFN activity was assayed in the withdrawn culture fluid (columns). The relative amount of IFN mRNA in cells was determined by the oocyte assay 4 ( ). For experimental details, see text.
and assayed for interferon activity. A single peak of IFN mRNA activity (which contained about 8/zg of RNA) was found at a position of the gel corresponding with a sedimentation coefficient of about 14 S. Construction and Identification of cDNA Clones Containing Rat Interferon a Sequences About 14/xg of 14 S mRNA yielded 250 ng of double-stranded cDNA after a reverse transcriptase reaction. The double-stranded cDNAs were size fractionated on a 5% polyacrylamide gel and 125 ng of material ranging from 800 to 1200 bp was obtained from the gel by electroelution. This cDNA was inserted into the PstI site of pBR322 DNA (400 ng) by the G-C tailing procedure of Villa-Komaroff et al. 5 After transformation of E. coli HB 101, 15,000 tetracycline resistant colonies were screened with a 690 bp HindIII-EcoRI fragment of the chromosomal mouse IFN-o~I DNA as a probe. Three transformants hybridized to the same extent with this probe. To determine whether the cDNA clones contained a complete coding region, DNA sequencing was carried out according to the procedure of Maxam and Gilbert. 6 The nucleotide sequence showed that the three cDNA clones contained rat IFN-a sequences when compared with the chromosomal mouse IFN-o~I gene (Fig. 2). However, they were incomplete with respect to the coding region of this gene. Since the 3' noncoding L. Villa-Komaroff, A. Efstratiadis, S. Broome, P. Lomedico, R. Tizard, S. P. Naker, W. L. Chick, and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 75, 3727 (1978). 6 A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 74, 560 (1977).
444
GENE ISOLATION AND EXPRESS|ON Hindm
EcoRl
LIIIHIIII
leader
[62]
l--
noncodingregion
coding region
Rat alpha IFN eDNA clones I
t
1T
~
I
m
.
coding ~
region
•
p olyCA)
•
poly(A)
•
poly(A)
noncoding
region
FIG. 2. Size determination of the three different rat I F N - a c D N A clones. On top is shown the structural organization of the chromosomal mouse IFN-al gene that was used as a probe. Below are shown the relative positions of the three rat I F N - a c D N A clones (all identical, same subtype).
regions of homologous genes generally diverge more strongly than the coding regions, the 3' noncoding regions were subjected to sequence determination. The analysis revealed that the three cDNA clones were of the same origin with respect to the 14 S mRNA. Isolation and Characterization o f a R a t IFN-o~ Chromosomal Gene
A rat gene library cloned in bacteriophage h harboring D N A fragments with an average size of 15 kb was used for the isolation of the structural gene of rat 1FN-o~. This library provided by Sargent et a l . 7 w a s screened by in situ plaque hybridization with nick-translated rat IFN-~I cDNA clone I. A set of hybrid phages of the library was affixed to nitrocellulose filters and probed with this 32p-labeled c D N A fragment. Eighteen positive phage clones were found among 450,000 and isolated by repeated plaque purification. Hybridization experiments with 32p-labeled cDNA from clone III (containing only the 3' noncoding region of the respective IFN-~ gene) revealed the presence of two phage clones which hybridized specifically to the probe. The DNAs from both phages were mapped by restriction enzyme analysis and found to contain identical parts (15.5 kb) of the rat genome, including the rat IFN-a subtype.
7 T. D. Sargent, J. R. W u , J. M. Sala-Trepat, R. B. Wallace, A. A. Reges, and J. Bonner, Proc. Natl. Acad. Sci. U.S.A. 76, 3256 (1979).
[62]
RAX IFN-al
445
Construction of a Plasmid Encoding Rat IFN-a Digestion of the 15.5 kb insert of the hybrid phage with Eco RI revealed fragments of 8.0, 2.9, 2.5, and 2.1 kb. Only the 2.5 kb fragment hybridized to the cDNA probe. This 2.5 kb EcoRI fragment was subcloned into the EcoRI site of pBR322. Recombinant plasmid DNA was prepared by the cleared lysate method, 8 digested by EcoRI and the 2.5 kb fragment was isolated by polyacrylamide gel electrophoresis. The 2.5 kb segment was cleaved by an appropriate restriction enzyme and the fragments were labeled at the 5' ends with [y-32p]ATP and cleaved with a second restriction enzyme to yield single-labeled fragments which served as templates for the determination of the DNA sequence by the method of Maxam and Gilbert. 6 The locations of the cleavage sites by means of a number of restriction enzymes is given in detail in an earlier publication. 9 The complete nucleotide sequence of rat IFN-al DNA is illustrated in Fig. 3. The sequence shows that the coding sequence of the rat IFN-al gene contains an open translational reading frame which codes for a protein of 192 amino acids, most likely comprised of a leader or signal peptide and the mature interferon protein. A sequence 5' TATTTAA 3' (Goldberg-Hogness box 1°) was identified at 100 nucleotides upstream from an ATG start codon, suggesting that initiation of transcription occurs about 30 nucleotides downstream from this sequence. More conclusive identification of the coding sequence of the rat IFN-cd gene was provided by a comparison with the nucleotide sequence of the chromosomal mouse IFN-aI gene. Jj This gene codes for a mature IFN-c~ containing 166 amino acids preceded by a 23 amino acid long leader peptide. Because all human 12,13 and mouse T M IFN-c~ genes code for cysteine at position 1 of the mature protein and since this gene family is highly conserved among vertebrates, the mature rat IFN-cd
8 H. C. Birnboim and J. Doly, Nucleic Acids Res. 7, 1513 (1979). 9 R. Dijkema, P. Pouwels, A. de Reus, and H. Schellekens, Nucleic Acids Res. 12, 1227 (1984). to F. Gannon, K. O'Hare, F. Perrin, J. P. LePennec, C. Benoist, M. Cochet, R. Breathnach, A. Royal, A. Garapin, B. Cami, and P. Chambon, Nature (London) 278, 428 (1979). H G. D. Shaw, W. Boll, H. Taira, N. Mantel, P. Lengyel, and C. Weissmann, Nucleic Acids Res. 11, 555 (1983). ~2 D. V. Goeddel, D. W. Leung, T. J. Dull, M. Gross, R. M. Lawn, R. McCandliss, P. H. Seeburg, A. U llrich, E. Yelverton, and P. W. van Gray, Nature (London) 290, 20 (1981). 13 S. Pestka, Arch. Biochern. Biophys. 221, 1 (1983). 14 B. Daugherty, D. Martin-Zanca, B. Kelder, K. Collier, T. C. Seamans, K. Hotta, and S. Pestka, J. Interferon Res. 4, 635 (1984).
Rot IFN chromosomol clone 6 ond 11
t
II11
EcoRI
I
Pvu]I
I
t
Pvu]I
t
Eco RI
L___._ GCA TTC AGA AAG TAA AAT TAG TGr AAA CCC ATT rAA GAC ACA rCC ACA CAG GAT
GGT
T~CCAG mRN A AGC
ACC TAG ACT GGA AGG ATT AGG ACC AAA CAG ACC CAA GGA CCA
~ A ~ pre-RIF CAAGCA
TTG GCC ACA TTT GCC
Ala Arg Leu Cys Ala Phe Leu Met S e t Leu CGG CTC TGT GCT TTC TTG ATG TCC CTG
matureR I F
Val Val Val Set Tyr Trp Set Ala Cys Cys Leu GIF GTG GTG GTG AGC TAC TGG TCA GCC TGC TGT CTA GGA
~GA~
Asp Leu Pro His Thr CTG CCT CAT ACT
His ASh Leu Arg Asn Lys Arg Val Phe Thr Leu Leu A l a Gln Met Arg Arg Leu CAT AAC CTC AGG AAC AAG AGA GTC TTC ACA CTC CTG GCA CAA ATG AGG AGA CTC
Set Pro Val Set Cys Leu Lys Asp Arg Lys Tyr Phe Gly Phe Pro Leu GIu Lys TCC CCT GTC TCA TGC CTG AAG GAC AGA AAG TAC TTT GGG TTC CCT TTG GAG AAG
Val Asp Gly Gin Gin lle Gin Lys Ala Gin Ala lie Pro Val Leu His Glu Leu GTG GAT GGC CAG CAG ATC CAG AAG GCT CAA GCT ATC CCT GTC CTG CAT GAG CTG
T h r Gin Gln l i e Leu S e r Leu Phe Thr S e r Lys Glu S e r S e r T h r Ala T r p Asp ACC CAG CAG ATC CTC AGC CTC TTC ACA TCA AAG GAG TCA TCT ACT GCT TGG GAT
Ala Thr Leu Leu Asp Set Phe Cys ASh Asp Leu Gin Gin Gin Leu Set Gly Leu GCA ACC CTC CTA GAC TCA TTC TGT AAT GAC CTC CAG CAG CAG CTG AGT GGT CTG
Gin Ala Cys Leu Met Gln Gin Val Giy Val Gin Glu Set Pro Leu Thr Gin Glu CAA GCC TGT CTG ATG CAG CAG GTA GGG GTG CAG GAA TCT CCC CTG ACC CAG GAA
Asp S e t Leu Leu A l a Val Arg Glu T y r Phe His Arg I l e Thr Val T y r Leu Arg GAC TCC CFA CTG GCT GTG AGO GAA TAC TTC CAC AGA A~C ACT GTG TAC CTG AGA
[~cDNA crone
Glu Ash Lys His S e r Pro Cys Ala T r p Glu Va[ Val Lys Ala Giu VaI Trp Arg GAG AAT AAA CAC AGC CCC TGT GCC TGG GAG GTG GTC AAA GCA GAA GTC TGG AGA
Ala Leu S e t S e t S e r AIn Asn Leu Met Gly Arg Leu Arg GILJ Glu Arg Ash G[U GCC CTG FCT TCC TCA GCC AAC TTG ATG GGA AGA CTG AGA GAA GAA AGA /kAY GAG
TCC
GCC ACA FFG GAG AGC AC[' CCG
FIG. 3. A partial nucleotide fragment of rat I F N - a l in a plasmid. T h e a m i n o a c i d seq u e n c e was deduced from the nucleotide sequence. Indicated are the putative starts of the IFN m R N A , pre-IFN-al protein, and mature I F N - a l protein.
[62]
RAT IFN-al
447
protein most probably starts with the cysteine residue at position 199 (box in Fig. 3) and predicts a protein with 169 amino acids. The mouse and rat IFN-al mature proteins are closely related in their amino acid sequences; 135 of 166 amino acids occupy identical positions (81% homology), whereas the DNA sequences showed 90% homology (for detailed information, see Ref. 9). To determine whether an IFN-like protein was produced, the coding sequence of the mature rat IFN-al protein was inserted into the EcoRI site of plasmid pMBL604. This vector contains a hybrid tetracycline-tryptophan promoter and a synthetic Shine-Dalgarno (SD) sequence. 9 For that purpose, the 2.5 kb EcoRI fragment was digested with HphI to remove the nucleotide sequence which codes for the first 17 amino acids (of 22) of the signal peptide. After electrophoresis on 5% polyacrylamide gels, the HphI fragment was isolated from excised slices and treated with Bal31 to remove the remaining 6 amino acid signal codons. An additional ATG start codon (in the form of a synthetic DNA adapter) was ligated to the population of Bal31 treated molecules and then cloned into the EcoRI site of pMBL604. Transformants of E. coli JA2219 which became ampicillin resistant were investigated for their capacity to produce substances with interferon activity. Two of 500 transformants were found to exhibit antiviral activity. However, both produced relatively low levels of interferon activity, about 200,000 units per liter of culture (at OD600 equal to 1.0). DNA analysis revealed that in the plasmid of the highest producer the initiation codon (AUG) is directly followed by the UGU (Cys) codon of mature IFN. The construction and features of the recombinant plasmid 13.1 are schematically depicted in Fig. 4. Subcloning of the Chromosomal Rat IFN-~I Gene In order to further increase the production level of rat IFN-o~ in bacteria, other constructions were carried out. The plasmid vectors used in these experiments were pBR322 derived multicopy vectors carrying the powerful major leftward promoter PL of bacteriophage lambda. This promoter is controlled by a temperature-sensitive repressor protein cI whose gene is located on the bacterial chromosome of a specific host strain (E. coli M5219). The PL promoter gives rise to efficient transcription at 42 °, while the promoter is shut off by this thermosensitive repressor at 28 °. The vectors carry a 247 base pair fragment containing the leftward operator and promoter and code for the first 114 nucleotides of the PL transcript. 15There is no translation initiation signal present on this transcript. 15 E. Remaut, P. Stanssens, and W. Fiers, Gene 15, 81 (1981).
GENEISOLATIONANDEXPRESSION
448 Eco RI
.
Kpn I ~ l r p bD
tit
pRIF-a I
'
~%~
y" ) t Mst
AMP~/'"""....u..
[62]
] Hph'I
=
Bal I
Bal 31
adapter
,,
Eco
T 4 ligase Eco RI
RI
T 4 ligase
Eco RI Kpn
I
~
RIF
Ec° RI
FIO.4. Schematic illustration of the construction of the rat IFN-~I expression plasmid p13.1.Amp indicates the region coding for/3-1actamase. The operator/promotor region is indicated by a P. SD stands for the Shine-Dalgarno ribosomal binding site. Three different hybrid plasmids were constructed (as outlined in Fig. 5). At first, an 1400 FnuDII fragment containing the complete sequence coding for the mature rat IFN-~ protein plus the synthetic ribosomal binding site of the original vector pMBL604 was isolated by polyacrylamide gel electrophoresis. This fragment was subsequently converted to a BamHI bounded fragment which was ligated to purified BamHI-cut pPLc236 vector D N A (p13.2). The other two plasmids used (pAT1 and pAT55) were kindly provided by Dr. W. Fiers. These plasmids are derivatives of pPLc236 and contain a synthetic Shine-Dalgarno (SD) sequence. Plasmid pAT55 differs from pAT~ by a deletion of 26 bp between the promoter/operator region and the
[62]
RAT IFN-al
449
SD site. Lysates from cultures of E. coli cells carrying pAT55 produce high levels of Hu-IFN-fl activity, whereas no activity was found in extracts of E. coli carrying pAT~ (Dr. W. Fiers, personal communication). Plasmid p13.3 was constructed by replacing the PstI-Sbal fragment (containing the hybrid tetracycline-tryptophan promoter and the ribosomal binding site) by a 1250 bp XbaI-PstI fragment from pAT harboring the PL promoter/operator region with SD site as shown in Fig. 5. The other subclone p13.4 was constructed in the same way, except that the fragment with the PL promoter was obtained from pAT55. To determine whether rat interferon was produced, lysates of transformants were prepared and assayed for antiviral activity. The results clearly showed that all three clones exhibited antiviral activity. However, in no case was the level exceptionally higher than the amount produced by the original construction pl3.1 (see Table I).
Preparation o f Bacterial Extracts Overnight cultures were diluted 1 : 50 with L-broth supplemented with 60/xg/ml ampicillin. Cultures of 500 ml were grown in l-liter Erlenmeyer flasks at 37° (clone p13.1) or 28° (clones p13.2-4) in a rotary shaking waterbath (100 rpm). When cells of clone p 13.1 reached an optical density of about 1.0 at 600 nm they were rapidly cooled on ice, harvested by
TABLE I RAT IFN LEVELS IN CELLS OF E. Coli HARBORING DIFFERENT EXPRESSION PLASMIDS"
Strain
Plasmid
I F N activity (units/liter culture) ( x 105)
JA 221 M5219 M5219 M5219
p13.1 p13.2 p13.3 p13.4
1.7 1.3 1.3 2.4
" Cells (containing p13.1) were cultured in rich m e d i u m (L broth) and harvested w h e n the culture reached an A6~ of about 1.0. Cultures with cells containing PL plasmids (p13.2-4) were heat induced after reaching an A600 of about 0.3. The induction time was 150 rain. The strains JA 2219 and M5219 t~ have been described.
450
GENE ISOLATION AND EXPRESSION Eco RI /
BAM HI
Eco R]
[62]
Eco RI DI]
Kpn I
rt AMP
Pst
"
'
~
Pst Pvu [I
/ Fnu D TI
I k,,..
Pvu I~I
I
ligase
I
I
I•
I I
I
I
/~, digestion BAM HI
BAM HI
linker molecules
/
CCGGATCCGG
Eco R I / B A M
HI
Pst " ~ , ~ Pvu II
FIo. 5. Schematic illustrations of the construction of two expression plasmids: p l 3.2 (A) and p13.3 (B). PLc denotes the position of the PL promoter. Construction and features of plasmid p13.1 is shown in Fig. 4.
centrifugation, and washed once in 200 ml of phosphate-buffered saline (PBS). The other clones were induced after reaching an A600 of about 0.3 by a temperature shift from 28 to 42 °. The incubation was continued for 150 min at the elevated temperature. They were then chilled, harvested, and washed once with PBS. Cells from the different clones were resuspended in 5 ml of PBS supplemented with 10 mM EDTA and 1 mg/ml lysozyme. The mixtures were incubated for 1 hr at 0 ° and subsequently sonicated by 10 pulses of 30 sec at 30 sec intervals. The resulting suspension, which was designated a crude extract, was clarified by centrifugation at 30,000 g for 30 min, yielding a low-speed supernatant. This suspension was centrifuged at 150,000 g for 3 hr at 4 °, yielding a ribosome-free supernatant (S-100). This S-100 fraction was the starting material for purification.
[62]
RAT
B
IFN-al
451
Met Cys
Met Cys
AAGGAGGFCTLxbaIjAGAFGTGT
Xba
Pst
T AGGAAT,TCTAGACATGTGT~xbaI_ [
I
Xba
[ ~
I
Pst ] I
Xba I + PstI
Xba I + Pst I / AMP
1
Xba I •~
PLc
RIF
/ Xba
I
AMP
I
PstI
L
Xba I
Pst~ij
Xba
I
Xba I
~
Xba I
Pst I ~ FIG. 5B.
Purification of Recombinant Rat IFN-~ with Polyclonal Antibodies Directed against Recombinant Human IFN-~2 The interferon activity of the different clones was neutralized by antiserum from rabbits immunized with recombinant human IFN-a2. As expected from these experiments, the bacterially derived rat IFN-~ could be purified by use of immobilized polyclonal antibodies directed against human IFN-o~2. S-100 lysates from clone p13.1 were passed through a column to which the purified IgG fraction from rabbit anti Hu-IFN-a2 serum was bound. The procedure for the preparation of the affinity
452
GENE ISOLATION AND EXPRESSION
[62]
TABLE II PURIFICATION OF BACTERIALLY DERIVED RAT I F N - a BY A COLUMN CONTAINING IMMOBILIZED ANTI-Hu-IFN-a2"
Purification step
Volume (ml)
IFN (units/ml)
S-100 lysate Antibody columneluate
7 20
1.5 × l04 2-4 × 103
Recovery Protein (%) (mg/ml) 100 38-72
9.8 0.005
The columnsize was 7 ml; the elutionbufferwas 0.1 M glycine•HCI, pH 2.3. column is described in detail in the chapter which deals with naturally derived rat interferon, l The bacterial rat interferon was retained and could be eluted by a 0.1 M glycine.HCl buffer of pH 2.3. In this way, recombinant rat IFN-al was purified to a specific activity of at least 4 × 105 units/ mg protein. However, owing to variation in the recovery (see Table II) on immunochromatography and because there is uncertainty about the protein determination (because of the extremely low protein concentration), this value is a conservative estimate. The eluate (20 ml) from the affinity column was supplemented with 1 ml of a BSA solution containing 500/zg/ ml protein. The solution was lyophilized and analyzed by PAGE in the presence of SDS. The gel slices were extracted in a buffer containing 0.5% (w/v) N-laurylsarcosine and I% (v/v) 2-mercaptoethanol and assayed for antiviral activity. These experiments showed that we are dealing with a protein with a mobility corresponding to a Mr of about 16,000. Comments Because unambiguous detection of an interferon specific protein band by SDS-polyacrylamide gel electrophoresis was not yet possible, the exact production level of interferon protein in the clones is unknown. However, deduced from several electrophoretic analyses on SDSPAGE, an amount exceeding 0.5 p.g/mg of cellular protein is not likely. We can only speculate about the cause of the low expression in these clones. One of the reasons could be that secondary structure formation of the mRNA may prevent the efficient expression of the gene, but it is also possible that the IFN mRNA is unstable and is rapidly broken down in the cell. Another aspect of interest in the stability of this protein in the presence of different chemical substances (see Table III). Methods of cell lysis with these detergents revealed that treatment of the bacteria with 6 M guanidine.HCl results in a 3-fold higher interferon yield compared to SDS treatment or uhrasonic disruption. Also, the presence of 2-mercapto-
[63]
RAT IFN-3' RECOVERY O F
TABLE Ill RAT IFN A C T I V I T Y
453 FROM C L O N E
13.1
BY
TREATMENT WITH DIFFERENT CHEMICALS a
Chemical substance
IFN activity (units/liter culture) ( x l0 t)
Control (no additions) 6 M guanidine • HCI 0.5% N-laurylsarcosine 0.1% sodium dodecyl sulfate (SDS) 9 M urea 1% Nonidet P-40 0.08% sodium deoxycholate I% Triton X-100
1.7 4.1 3.5 1.2 1.6 1.5 0.9 1.4
"
Cell pellets of 1.0 liter cultures were resuspended in 6 ml of 20 mM Tris. HCI (pH 7.0) containing 1% (v/v) 2mercaptoethanol and the different detergents mentioned in the table. After an incubation time of 24 hr at 4°, cells were sonicated and centrifuged at 20,000 g. The supernatants were assayed for IFN activity.
ethanol is essential for the recovery of activity from the cells. This illustrates that the level of rat IFN-a is dependent upon the extraction procedure and that the absence of a reducing agent might lead to an inactive aggregated form of interferon, as already suggested by SDS gel electrophoresis of naturally derived rat interferon.~ The finding that rat IFN-al reacts with antibodies against human IFN-a2 indicates that certain interferon subtypes of rat and human origin have strong antigenic similarities. Acknowledgments We thank C. Weissmann for generously providing the chromosomal mouse IFN-al probe.
[63] C l o n i n g , E x p r e s s i o n , a n d Purification o f
Rat IFN-7
By R E I N D I J K E M A , PETER H , VAN DER M E I D E , MARTIN D U B B E L D , M A R T I N CASPERS, JACQUELINE W U B B E N , a n d H U U B SCHELLEKENS This chapter deals with the molecular cloning of the chromosomal rat IFN-y gene from a rat gene library by use of heterologous hybridization with the human IFN-3, c D N A as a probe and the characterization of its Copyright (~3 1986 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 119
All rights of reproduction in any fl~rm reserved.
RAT IFN-al
441
Concluding Comments Hybridization of the murine genomic library to the 32P-labeled human alpha interferon gene (642 bp AvaII-pLeIFLrA25 fragment) resulted in strong positive signals with virtually no background. The probe chosen represented over 90% of the coding region and contained no poly(A) sequences and no introns. Since we were cross-hybridizing between species and since the extent of the homology between human and mouse interferon genes was unknown, the stringency of the hybridization solution was lowered from a standard concentration of 50% formamide to a lower stringency of 40% formamide. This method proved successful in the isolation of a genomic murine alpha interferon gene. After the nucleotide sequence of the gene was determined, it was possible to construct an effective expression vector. It should be noted that since the gene isolated was a genomic clone rather than a cDNA clone, it could possibly contain introns. An open reading frame of 190 amino acids was determined from the sequence. By comparison of the nucleotide sequence with that of the nucleotide sequence of the human alpha interferon genes, TM it was concluded that the gene contained no introns, but contained a signal peptide of 23 amino acids followed by a cysteine which corresponded to Cys 1 of the human gene. Therefore, cysteine was concluded to be the first amino acid of the mature protein and the expression vector was appropriately designed and constructed as described above. 7 14 S. Pestka, Arch. Biochem. Biophys. 221, l (1983).
[62] C l o n i n g , E x p r e s s i o n , a n d P u r i f i c a t i o n o f R a t I F N - a l
By
PETER H. VAN DER M E I D E , REIN DIJKEMA, MARTIN CASPERS, KITTY VIJVERBERG, and H U U B SCHELLEKENS
This chapter describes the introduction of a structural gene for a rat alpha interferon subtype (rat IFN-a) into Escherichia coli to obtain an efficient expression system for the production of large quantities of rat interferon. This would provide a more economic way of production than does the system employing a rat continuous cell linO and also make it possible to study the properties of a single gene product. DNA comple1 p. H. van der Meide, J. Wubben, K. Vijverberg, and H. Schellekens, this volume [31].
M E T H O D S IN E N Z Y M O L O G Y , V O L . 119
Copyright It) 1986 by Academic Press, Inc. All rights of reproduction in any hwm reserved.
442
GENE ISOLATION AND EXPRESSION
[62]
mentary to rat interferon mRNA isolated from Ratec cells induced by Sendai virus was cloned and amplified in E. coli. A purified cDNA fragment identified by hybridization with the mouse a2 gene was then used as a probe for the isolation of the chromosomal genes from a rat gene library. In this way, a 2.5 kb EcoRI fragment encoding a complete rat IFN-a gene (rat IFN-al) was isolated and placed under the control of a hybrid tetracycline-tryptophan promoter into the EcoRI site of pMBL604 (a derivative plasmid of PBR322). E. coli strains carrying this recombinant plasmid synthesized biologically active interferon. The procedure for the production and purification of this type of interferon will be described. Also attempts to increase production with other host/vector systems are described and some characteristics of rat IFN-al will be discussed. Cloning of the Chromosomal Rat IFN-a Gene
Preparation, Purification, and Identification of l F N m R N A from Induced Ratec Cells For the construction of cDNA clones, total cytoplasmic RNA was isolated from Ratec cells actively synthesizing interferon. To establish at what time after virus infection a maximum amount of mRNA coding for rat interferon was present in the cells, poly(A) ÷ RNA was isolated at different times after Sendai infection and translated in Xenopus laevis oocytes (see below). The pattern of IFN activity in this assay revealed an optimum at about l0 hr after infection (see Fig. 1). At that time, total RNA (from 101° cells) was isolated from the cells by extraction with a buffer containing l0 mM Tris.HCl (pH 7.9), 1 mM EDTA, 150 mM NaCl, and 0.65% Nonidet P40 for 5 min at 4°. After centrifugation, the supernatant was mixed with an equal volume of a buffer containing 20 mM Tris.HC1 (pH 7.9), 20 mM EDTA, 300 mM NaCl, 7 M urea, and 1% SDS. The mixture was then extracted twice with phenol. The poly(A) ÷ RNA fraction (about 250/xg) was isolated by repeated batchwise absorption onto oligo(dT)-cellulose.: Different mRNA size fractions were obtained by use of a SDS-PAGE system as described by Wierenga et a l . 3 The mRNA fractions were injected into oocytes of Xenopus laevis as described by Gordon et al. 4 After incubation, the eggs were homogenized 2 S. Nagata, H. Taira, A. Hall, L. Johnsrud, M. Streuli, J. Ecs6di, W. Boll. K. Cantell, and C. Weissmann, Nature (London) 284, 316 0980). 3 B. Wierenga, J. Mulder, A. van der Ende, A. Bruggeman, G. Ab, and M. Gruber, Eur. J. Biochem. 89, 67 (1978). 4 j. B. Gordon, C. D. Lane, M. Woodlans, and G. Marbaix, Nature (London) 233, 177 (1971).
[62]
P,A T
IFN-cd
443
500 E
.-_"
i)i))))il)i)i)i!
400
D
.g
300
u
o
200 100
°_ i
|
4
8
|
12
16
|
2O
time after infection (hour) FIG. 1. Time course of IFN activity and 1FN mRNA production. At different times, the culture medium was decanted and replaced by fresh medium. The IFN activity was assayed in the withdrawn culture fluid (columns). The relative amount of IFN mRNA in cells was determined by the oocyte assay 4 ( ). For experimental details, see text.
and assayed for interferon activity. A single peak of IFN mRNA activity (which contained about 8/zg of RNA) was found at a position of the gel corresponding with a sedimentation coefficient of about 14 S. Construction and Identification of cDNA Clones Containing Rat Interferon a Sequences About 14/xg of 14 S mRNA yielded 250 ng of double-stranded cDNA after a reverse transcriptase reaction. The double-stranded cDNAs were size fractionated on a 5% polyacrylamide gel and 125 ng of material ranging from 800 to 1200 bp was obtained from the gel by electroelution. This cDNA was inserted into the PstI site of pBR322 DNA (400 ng) by the G-C tailing procedure of Villa-Komaroff et al. 5 After transformation of E. coli HB 101, 15,000 tetracycline resistant colonies were screened with a 690 bp HindIII-EcoRI fragment of the chromosomal mouse IFN-o~I DNA as a probe. Three transformants hybridized to the same extent with this probe. To determine whether the cDNA clones contained a complete coding region, DNA sequencing was carried out according to the procedure of Maxam and Gilbert. 6 The nucleotide sequence showed that the three cDNA clones contained rat IFN-a sequences when compared with the chromosomal mouse IFN-o~I gene (Fig. 2). However, they were incomplete with respect to the coding region of this gene. Since the 3' noncoding L. Villa-Komaroff, A. Efstratiadis, S. Broome, P. Lomedico, R. Tizard, S. P. Naker, W. L. Chick, and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 75, 3727 (1978). 6 A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 74, 560 (1977).
444
GENE ISOLATION AND EXPRESS|ON Hindm
EcoRl
LIIIHIIII
leader
[62]
l--
noncodingregion
coding region
Rat alpha IFN eDNA clones I
t
1T
~
I
m
.
coding ~
region
•
p olyCA)
•
poly(A)
•
poly(A)
noncoding
region
FIG. 2. Size determination of the three different rat I F N - a c D N A clones. On top is shown the structural organization of the chromosomal mouse IFN-al gene that was used as a probe. Below are shown the relative positions of the three rat I F N - a c D N A clones (all identical, same subtype).
regions of homologous genes generally diverge more strongly than the coding regions, the 3' noncoding regions were subjected to sequence determination. The analysis revealed that the three cDNA clones were of the same origin with respect to the 14 S mRNA. Isolation and Characterization o f a R a t IFN-o~ Chromosomal Gene
A rat gene library cloned in bacteriophage h harboring D N A fragments with an average size of 15 kb was used for the isolation of the structural gene of rat 1FN-o~. This library provided by Sargent et a l . 7 w a s screened by in situ plaque hybridization with nick-translated rat IFN-~I cDNA clone I. A set of hybrid phages of the library was affixed to nitrocellulose filters and probed with this 32p-labeled c D N A fragment. Eighteen positive phage clones were found among 450,000 and isolated by repeated plaque purification. Hybridization experiments with 32p-labeled cDNA from clone III (containing only the 3' noncoding region of the respective IFN-~ gene) revealed the presence of two phage clones which hybridized specifically to the probe. The DNAs from both phages were mapped by restriction enzyme analysis and found to contain identical parts (15.5 kb) of the rat genome, including the rat IFN-a subtype.
7 T. D. Sargent, J. R. W u , J. M. Sala-Trepat, R. B. Wallace, A. A. Reges, and J. Bonner, Proc. Natl. Acad. Sci. U.S.A. 76, 3256 (1979).
[62]
RAX IFN-al
445
Construction of a Plasmid Encoding Rat IFN-a Digestion of the 15.5 kb insert of the hybrid phage with Eco RI revealed fragments of 8.0, 2.9, 2.5, and 2.1 kb. Only the 2.5 kb fragment hybridized to the cDNA probe. This 2.5 kb EcoRI fragment was subcloned into the EcoRI site of pBR322. Recombinant plasmid DNA was prepared by the cleared lysate method, 8 digested by EcoRI and the 2.5 kb fragment was isolated by polyacrylamide gel electrophoresis. The 2.5 kb segment was cleaved by an appropriate restriction enzyme and the fragments were labeled at the 5' ends with [y-32p]ATP and cleaved with a second restriction enzyme to yield single-labeled fragments which served as templates for the determination of the DNA sequence by the method of Maxam and Gilbert. 6 The locations of the cleavage sites by means of a number of restriction enzymes is given in detail in an earlier publication. 9 The complete nucleotide sequence of rat IFN-al DNA is illustrated in Fig. 3. The sequence shows that the coding sequence of the rat IFN-al gene contains an open translational reading frame which codes for a protein of 192 amino acids, most likely comprised of a leader or signal peptide and the mature interferon protein. A sequence 5' TATTTAA 3' (Goldberg-Hogness box 1°) was identified at 100 nucleotides upstream from an ATG start codon, suggesting that initiation of transcription occurs about 30 nucleotides downstream from this sequence. More conclusive identification of the coding sequence of the rat IFN-cd gene was provided by a comparison with the nucleotide sequence of the chromosomal mouse IFN-aI gene. Jj This gene codes for a mature IFN-c~ containing 166 amino acids preceded by a 23 amino acid long leader peptide. Because all human 12,13 and mouse T M IFN-c~ genes code for cysteine at position 1 of the mature protein and since this gene family is highly conserved among vertebrates, the mature rat IFN-cd
8 H. C. Birnboim and J. Doly, Nucleic Acids Res. 7, 1513 (1979). 9 R. Dijkema, P. Pouwels, A. de Reus, and H. Schellekens, Nucleic Acids Res. 12, 1227 (1984). to F. Gannon, K. O'Hare, F. Perrin, J. P. LePennec, C. Benoist, M. Cochet, R. Breathnach, A. Royal, A. Garapin, B. Cami, and P. Chambon, Nature (London) 278, 428 (1979). H G. D. Shaw, W. Boll, H. Taira, N. Mantel, P. Lengyel, and C. Weissmann, Nucleic Acids Res. 11, 555 (1983). ~2 D. V. Goeddel, D. W. Leung, T. J. Dull, M. Gross, R. M. Lawn, R. McCandliss, P. H. Seeburg, A. U llrich, E. Yelverton, and P. W. van Gray, Nature (London) 290, 20 (1981). 13 S. Pestka, Arch. Biochern. Biophys. 221, 1 (1983). 14 B. Daugherty, D. Martin-Zanca, B. Kelder, K. Collier, T. C. Seamans, K. Hotta, and S. Pestka, J. Interferon Res. 4, 635 (1984).
Rot IFN chromosomol clone 6 ond 11
t
II11
EcoRI
I
Pvu]I
I
t
Pvu]I
t
Eco RI
L___._ GCA TTC AGA AAG TAA AAT TAG TGr AAA CCC ATT rAA GAC ACA rCC ACA CAG GAT
GGT
T~CCAG mRN A AGC
ACC TAG ACT GGA AGG ATT AGG ACC AAA CAG ACC CAA GGA CCA
~ A ~ pre-RIF CAAGCA
TTG GCC ACA TTT GCC
Ala Arg Leu Cys Ala Phe Leu Met S e t Leu CGG CTC TGT GCT TTC TTG ATG TCC CTG
matureR I F
Val Val Val Set Tyr Trp Set Ala Cys Cys Leu GIF GTG GTG GTG AGC TAC TGG TCA GCC TGC TGT CTA GGA
~GA~
Asp Leu Pro His Thr CTG CCT CAT ACT
His ASh Leu Arg Asn Lys Arg Val Phe Thr Leu Leu A l a Gln Met Arg Arg Leu CAT AAC CTC AGG AAC AAG AGA GTC TTC ACA CTC CTG GCA CAA ATG AGG AGA CTC
Set Pro Val Set Cys Leu Lys Asp Arg Lys Tyr Phe Gly Phe Pro Leu GIu Lys TCC CCT GTC TCA TGC CTG AAG GAC AGA AAG TAC TTT GGG TTC CCT TTG GAG AAG
Val Asp Gly Gin Gin lle Gin Lys Ala Gin Ala lie Pro Val Leu His Glu Leu GTG GAT GGC CAG CAG ATC CAG AAG GCT CAA GCT ATC CCT GTC CTG CAT GAG CTG
T h r Gin Gln l i e Leu S e r Leu Phe Thr S e r Lys Glu S e r S e r T h r Ala T r p Asp ACC CAG CAG ATC CTC AGC CTC TTC ACA TCA AAG GAG TCA TCT ACT GCT TGG GAT
Ala Thr Leu Leu Asp Set Phe Cys ASh Asp Leu Gin Gin Gin Leu Set Gly Leu GCA ACC CTC CTA GAC TCA TTC TGT AAT GAC CTC CAG CAG CAG CTG AGT GGT CTG
Gin Ala Cys Leu Met Gln Gin Val Giy Val Gin Glu Set Pro Leu Thr Gin Glu CAA GCC TGT CTG ATG CAG CAG GTA GGG GTG CAG GAA TCT CCC CTG ACC CAG GAA
Asp S e t Leu Leu A l a Val Arg Glu T y r Phe His Arg I l e Thr Val T y r Leu Arg GAC TCC CFA CTG GCT GTG AGO GAA TAC TTC CAC AGA A~C ACT GTG TAC CTG AGA
[~cDNA crone
Glu Ash Lys His S e r Pro Cys Ala T r p Glu Va[ Val Lys Ala Giu VaI Trp Arg GAG AAT AAA CAC AGC CCC TGT GCC TGG GAG GTG GTC AAA GCA GAA GTC TGG AGA
Ala Leu S e t S e t S e r AIn Asn Leu Met Gly Arg Leu Arg GILJ Glu Arg Ash G[U GCC CTG FCT TCC TCA GCC AAC TTG ATG GGA AGA CTG AGA GAA GAA AGA /kAY GAG
TCC
GCC ACA FFG GAG AGC AC[' CCG
FIG. 3. A partial nucleotide fragment of rat I F N - a l in a plasmid. T h e a m i n o a c i d seq u e n c e was deduced from the nucleotide sequence. Indicated are the putative starts of the IFN m R N A , pre-IFN-al protein, and mature I F N - a l protein.
[62]
RAT IFN-al
447
protein most probably starts with the cysteine residue at position 199 (box in Fig. 3) and predicts a protein with 169 amino acids. The mouse and rat IFN-al mature proteins are closely related in their amino acid sequences; 135 of 166 amino acids occupy identical positions (81% homology), whereas the DNA sequences showed 90% homology (for detailed information, see Ref. 9). To determine whether an IFN-like protein was produced, the coding sequence of the mature rat IFN-al protein was inserted into the EcoRI site of plasmid pMBL604. This vector contains a hybrid tetracycline-tryptophan promoter and a synthetic Shine-Dalgarno (SD) sequence. 9 For that purpose, the 2.5 kb EcoRI fragment was digested with HphI to remove the nucleotide sequence which codes for the first 17 amino acids (of 22) of the signal peptide. After electrophoresis on 5% polyacrylamide gels, the HphI fragment was isolated from excised slices and treated with Bal31 to remove the remaining 6 amino acid signal codons. An additional ATG start codon (in the form of a synthetic DNA adapter) was ligated to the population of Bal31 treated molecules and then cloned into the EcoRI site of pMBL604. Transformants of E. coli JA2219 which became ampicillin resistant were investigated for their capacity to produce substances with interferon activity. Two of 500 transformants were found to exhibit antiviral activity. However, both produced relatively low levels of interferon activity, about 200,000 units per liter of culture (at OD600 equal to 1.0). DNA analysis revealed that in the plasmid of the highest producer the initiation codon (AUG) is directly followed by the UGU (Cys) codon of mature IFN. The construction and features of the recombinant plasmid 13.1 are schematically depicted in Fig. 4. Subcloning of the Chromosomal Rat IFN-~I Gene In order to further increase the production level of rat IFN-o~ in bacteria, other constructions were carried out. The plasmid vectors used in these experiments were pBR322 derived multicopy vectors carrying the powerful major leftward promoter PL of bacteriophage lambda. This promoter is controlled by a temperature-sensitive repressor protein cI whose gene is located on the bacterial chromosome of a specific host strain (E. coli M5219). The PL promoter gives rise to efficient transcription at 42 °, while the promoter is shut off by this thermosensitive repressor at 28 °. The vectors carry a 247 base pair fragment containing the leftward operator and promoter and code for the first 114 nucleotides of the PL transcript. 15There is no translation initiation signal present on this transcript. 15 E. Remaut, P. Stanssens, and W. Fiers, Gene 15, 81 (1981).
GENEISOLATIONANDEXPRESSION
448 Eco RI
.
Kpn I ~ l r p bD
tit
pRIF-a I
'
~%~
y" ) t Mst
AMP~/'"""....u..
[62]
] Hph'I
=
Bal I
Bal 31
adapter
,,
Eco
T 4 ligase Eco RI
RI
T 4 ligase
Eco RI Kpn
I
~
RIF
Ec° RI
FIO.4. Schematic illustration of the construction of the rat IFN-~I expression plasmid p13.1.Amp indicates the region coding for/3-1actamase. The operator/promotor region is indicated by a P. SD stands for the Shine-Dalgarno ribosomal binding site. Three different hybrid plasmids were constructed (as outlined in Fig. 5). At first, an 1400 FnuDII fragment containing the complete sequence coding for the mature rat IFN-~ protein plus the synthetic ribosomal binding site of the original vector pMBL604 was isolated by polyacrylamide gel electrophoresis. This fragment was subsequently converted to a BamHI bounded fragment which was ligated to purified BamHI-cut pPLc236 vector D N A (p13.2). The other two plasmids used (pAT1 and pAT55) were kindly provided by Dr. W. Fiers. These plasmids are derivatives of pPLc236 and contain a synthetic Shine-Dalgarno (SD) sequence. Plasmid pAT55 differs from pAT~ by a deletion of 26 bp between the promoter/operator region and the
[62]
RAT IFN-al
449
SD site. Lysates from cultures of E. coli cells carrying pAT55 produce high levels of Hu-IFN-fl activity, whereas no activity was found in extracts of E. coli carrying pAT~ (Dr. W. Fiers, personal communication). Plasmid p13.3 was constructed by replacing the PstI-Sbal fragment (containing the hybrid tetracycline-tryptophan promoter and the ribosomal binding site) by a 1250 bp XbaI-PstI fragment from pAT harboring the PL promoter/operator region with SD site as shown in Fig. 5. The other subclone p13.4 was constructed in the same way, except that the fragment with the PL promoter was obtained from pAT55. To determine whether rat interferon was produced, lysates of transformants were prepared and assayed for antiviral activity. The results clearly showed that all three clones exhibited antiviral activity. However, in no case was the level exceptionally higher than the amount produced by the original construction pl3.1 (see Table I).
Preparation o f Bacterial Extracts Overnight cultures were diluted 1 : 50 with L-broth supplemented with 60/xg/ml ampicillin. Cultures of 500 ml were grown in l-liter Erlenmeyer flasks at 37° (clone p13.1) or 28° (clones p13.2-4) in a rotary shaking waterbath (100 rpm). When cells of clone p 13.1 reached an optical density of about 1.0 at 600 nm they were rapidly cooled on ice, harvested by
TABLE I RAT IFN LEVELS IN CELLS OF E. Coli HARBORING DIFFERENT EXPRESSION PLASMIDS"
Strain
Plasmid
I F N activity (units/liter culture) ( x 105)
JA 221 M5219 M5219 M5219
p13.1 p13.2 p13.3 p13.4
1.7 1.3 1.3 2.4
" Cells (containing p13.1) were cultured in rich m e d i u m (L broth) and harvested w h e n the culture reached an A6~ of about 1.0. Cultures with cells containing PL plasmids (p13.2-4) were heat induced after reaching an A600 of about 0.3. The induction time was 150 rain. The strains JA 2219 and M5219 t~ have been described.
450
GENE ISOLATION AND EXPRESSION Eco RI /
BAM HI
Eco R]
[62]
Eco RI DI]
Kpn I
rt AMP
Pst
"
'
~
Pst Pvu [I
/ Fnu D TI
I k,,..
Pvu I~I
I
ligase
I
I
I•
I I
I
I
/~, digestion BAM HI
BAM HI
linker molecules
/
CCGGATCCGG
Eco R I / B A M
HI
Pst " ~ , ~ Pvu II
FIo. 5. Schematic illustrations of the construction of two expression plasmids: p l 3.2 (A) and p13.3 (B). PLc denotes the position of the PL promoter. Construction and features of plasmid p13.1 is shown in Fig. 4.
centrifugation, and washed once in 200 ml of phosphate-buffered saline (PBS). The other clones were induced after reaching an A600 of about 0.3 by a temperature shift from 28 to 42 °. The incubation was continued for 150 min at the elevated temperature. They were then chilled, harvested, and washed once with PBS. Cells from the different clones were resuspended in 5 ml of PBS supplemented with 10 mM EDTA and 1 mg/ml lysozyme. The mixtures were incubated for 1 hr at 0 ° and subsequently sonicated by 10 pulses of 30 sec at 30 sec intervals. The resulting suspension, which was designated a crude extract, was clarified by centrifugation at 30,000 g for 30 min, yielding a low-speed supernatant. This suspension was centrifuged at 150,000 g for 3 hr at 4 °, yielding a ribosome-free supernatant (S-100). This S-100 fraction was the starting material for purification.
[62]
RAT
B
IFN-al
451
Met Cys
Met Cys
AAGGAGGFCTLxbaIjAGAFGTGT
Xba
Pst
T AGGAAT,TCTAGACATGTGT~xbaI_ [
I
Xba
[ ~
I
Pst ] I
Xba I + PstI
Xba I + Pst I / AMP
1
Xba I •~
PLc
RIF
/ Xba
I
AMP
I
PstI
L
Xba I
Pst~ij
Xba
I
Xba I
~
Xba I
Pst I ~ FIG. 5B.
Purification of Recombinant Rat IFN-~ with Polyclonal Antibodies Directed against Recombinant Human IFN-~2 The interferon activity of the different clones was neutralized by antiserum from rabbits immunized with recombinant human IFN-a2. As expected from these experiments, the bacterially derived rat IFN-~ could be purified by use of immobilized polyclonal antibodies directed against human IFN-o~2. S-100 lysates from clone p13.1 were passed through a column to which the purified IgG fraction from rabbit anti Hu-IFN-a2 serum was bound. The procedure for the preparation of the affinity
452
GENE ISOLATION AND EXPRESSION
[62]
TABLE II PURIFICATION OF BACTERIALLY DERIVED RAT I F N - a BY A COLUMN CONTAINING IMMOBILIZED ANTI-Hu-IFN-a2"
Purification step
Volume (ml)
IFN (units/ml)
S-100 lysate Antibody columneluate
7 20
1.5 × l04 2-4 × 103
Recovery Protein (%) (mg/ml) 100 38-72
9.8 0.005
The columnsize was 7 ml; the elutionbufferwas 0.1 M glycine•HCI, pH 2.3. column is described in detail in the chapter which deals with naturally derived rat interferon, l The bacterial rat interferon was retained and could be eluted by a 0.1 M glycine.HCl buffer of pH 2.3. In this way, recombinant rat IFN-al was purified to a specific activity of at least 4 × 105 units/ mg protein. However, owing to variation in the recovery (see Table II) on immunochromatography and because there is uncertainty about the protein determination (because of the extremely low protein concentration), this value is a conservative estimate. The eluate (20 ml) from the affinity column was supplemented with 1 ml of a BSA solution containing 500/zg/ ml protein. The solution was lyophilized and analyzed by PAGE in the presence of SDS. The gel slices were extracted in a buffer containing 0.5% (w/v) N-laurylsarcosine and I% (v/v) 2-mercaptoethanol and assayed for antiviral activity. These experiments showed that we are dealing with a protein with a mobility corresponding to a Mr of about 16,000. Comments Because unambiguous detection of an interferon specific protein band by SDS-polyacrylamide gel electrophoresis was not yet possible, the exact production level of interferon protein in the clones is unknown. However, deduced from several electrophoretic analyses on SDSPAGE, an amount exceeding 0.5 p.g/mg of cellular protein is not likely. We can only speculate about the cause of the low expression in these clones. One of the reasons could be that secondary structure formation of the mRNA may prevent the efficient expression of the gene, but it is also possible that the IFN mRNA is unstable and is rapidly broken down in the cell. Another aspect of interest in the stability of this protein in the presence of different chemical substances (see Table III). Methods of cell lysis with these detergents revealed that treatment of the bacteria with 6 M guanidine.HCl results in a 3-fold higher interferon yield compared to SDS treatment or uhrasonic disruption. Also, the presence of 2-mercapto-
[63]
RAT IFN-3' RECOVERY O F
TABLE Ill RAT IFN A C T I V I T Y
453 FROM C L O N E
13.1
BY
TREATMENT WITH DIFFERENT CHEMICALS a
Chemical substance
IFN activity (units/liter culture) ( x l0 t)
Control (no additions) 6 M guanidine • HCI 0.5% N-laurylsarcosine 0.1% sodium dodecyl sulfate (SDS) 9 M urea 1% Nonidet P-40 0.08% sodium deoxycholate I% Triton X-100
1.7 4.1 3.5 1.2 1.6 1.5 0.9 1.4
"
Cell pellets of 1.0 liter cultures were resuspended in 6 ml of 20 mM Tris. HCI (pH 7.0) containing 1% (v/v) 2mercaptoethanol and the different detergents mentioned in the table. After an incubation time of 24 hr at 4°, cells were sonicated and centrifuged at 20,000 g. The supernatants were assayed for IFN activity.
ethanol is essential for the recovery of activity from the cells. This illustrates that the level of rat IFN-a is dependent upon the extraction procedure and that the absence of a reducing agent might lead to an inactive aggregated form of interferon, as already suggested by SDS gel electrophoresis of naturally derived rat interferon.~ The finding that rat IFN-al reacts with antibodies against human IFN-a2 indicates that certain interferon subtypes of rat and human origin have strong antigenic similarities. Acknowledgments We thank C. Weissmann for generously providing the chromosomal mouse IFN-al probe.
[63] C l o n i n g , E x p r e s s i o n , a n d Purification o f
Rat IFN-7
By R E I N D I J K E M A , PETER H , VAN DER M E I D E , MARTIN D U B B E L D , M A R T I N CASPERS, JACQUELINE W U B B E N , a n d H U U B SCHELLEKENS This chapter deals with the molecular cloning of the chromosomal rat IFN-y gene from a rat gene library by use of heterologous hybridization with the human IFN-3, c D N A as a probe and the characterization of its Copyright (~3 1986 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 119
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