Vol. 173, No. 12
JOURNAL OF BACTERIOLOGY, June 1991, p. 3918-3920
0021-9193/91/123918-03$02.00/0 Copyright © 1991, American Society for Microbiology
Overproduction and Purification of McrC Protein from Escherichia coli K-12 LING ZHENG' AND H. D. BRAYMERl 2*
Department of Microbiology, Louisiana State University, Baton Rouge, Louisiana 70803,1* and Pennington Biomedical Research Center, Baton Rouge, Louisiana 708082 Received 11 February 1991/Accepted 17 April 1991
The McrC protein, encoded by one of the two genes involved in the McrB restriction system, was produced in Escherichia coli cells by using a T7 expression system. Following sequential DEAE-Sepharose and hydroxylapatite column chromatography, the protein was purified to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified McrC protein agreed exactly with the one deduced from the DNA sequence by Ross et al. (J. Bacteriol. 171:1974-1981, 1989). lation of this protein. This arrangement would provide for a 1-nucleotide overlap between the stop codon for McrB transcripts and the initiation codon for the McrC transcript. However, Dila et al. (2) recently reported that the deduced McrC coding sequence overlapped the McrB transcripts by 26 nucleotides and had a frameshift of 1 bp from the triplet deduced by Ross et al. (8). They predicted a molecular size of 40 kDa for the McrC protein. Consequently, amino acid sequences of the McrC protein predicted by the two reports are completely different. The purification and sequence of the McrC protein should, therefore, provide the evidence for the correct amino acid sequence of this protein. In this paper, we report the overproduction and purification of the McrC protein from a T7 expression system. The N-terminal amino acid sequence of the purified McrC protein matched exactly with that of Ross et al. (8). A T7 promoter-containing plasmid, pT7-7 (12), was used to construct the overproducing plasmid, pZB7-mcrC (Fig. 1). The mcrC-containing DNA fragment was isolated from pRAB16 (7). This plasmid was first cleaved at the BamHI site within the pUC8 portion of pRAB16 that is adjacent to the EcoRV site on the right side of the map (Fig. 1). The BamHI-linearized pRAB16 was then restricted with EcoRI within the pUC8 portion that is adjacent to the EcoRV site on the left side of the map within the mcrB gene, and the McrC start codon is 80 nucleotides downstream from the EcoRV site (Fig. 1). The 1.6-kb fragment was ligated with EcoRI- and BamHI-digested pT7-7 (3). E. coli BL21 (DE3) (F- ompT rB- mB-) was used as the host for overproduction of the mcrC gene (11). The transformed strain, BL21(DE3)(pZB7-mcrC), was tested for its ability to overproduce McrC protein. The transformant was grown at 37°C on M9 medium supplemented with 1% methionine assay medium (Difco Laboratories). When cultures reached the mid-log to late-log phase (optical density at 600 nm = 0.4), isopropyl-p-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM, and the culture was incubated for 60 min at the same temperature. Since T7 RNA polymerase is not sensitive to rifampin, this antibiotic was added to 300 ,ug/ml to inhibit transcription from the host cell chromosome DNA. After the addition of rifampin, culture growth was continued for 110 min at 37°C. To analyze the newly synthesized proteins, 0.6 ,uCi of [35S]methionine (Du Pont) per ml of medium was added for another 10 min at 37°C. An aliquot (0.5 ml) of
The McrB (for methylated cytosine restriction) restriction system in Escherichia coli K-12 is responsible for the inac-
tivation and degradation of foreign DNA containing methylated cytosine, such as 5-hydroxylmethylcytosine (shmC), 5C-methylcytosine, and 4N-methylcytosine (1, 5). The common recognition sequence for the McrB system is G'C (5). McrB, formerly known as RglB, was discovered because of difficulties encountered in cloning the genes for site-specific modification methylases associated with restriction-modification systems in E. coli K-12 strains (5). Since DNA of many organisms, both procaryotic and eucaryotic, contains methylated cytosine, the McrB methyl-specific restriction system can significantly hinder DNA-cloning procedures in E. coli (8). The McrB locus has been physically mapped at about 99 min of E. coli chromosome DNA (5), and it is in a cluster of other restriction modification systems, such as the hsd genes encoding type I restriction-modification enzymes and the mrr gene encoding the methyladenine-specific restriction enzyme (4). Two McrB genes, mcrB and mcrC, have been cloned and sequenced (6, 8, 9). The mcrB gene encodes 51and 33-kDa polypeptides which share the same C-terminal amino acid sequence. The mcrC gene encodes a 39-kDa polypeptide. The 51-kDa McrB polypeptide and the 39-kDa McrC polypeptide have been postulated to be necessary for the sequence-specific restriction of 5-methylcytosine DNA. It has also been postulated that the 33-kDa polypeptide might play a role in regulating activity of the 51-kDa polypeptide, since the 33-kDa polypeptide may compete with the 51-kDa polypeptide in DNA and protein binding abilities. However, it has not been demonstrated how these polypeptides are involved in McrB restriction. As a first step to gain insight into the function of those gene products and thereby the mechanism of McrB restriction, we started the purification of each of these polypeptides. Furthermore, there exist conflicting reports on the amino acid sequences of these polypeptides deduced from nucleotide sequence. For instance, Ross et al. (8) originally reported that the putative starting codon for McrC translation was a GTG triplet at the nucleotide position 1514. A purinerich region which is 7 bases upstream of this codon was suggested to serve as a ribosome binding site for the trans*
Corresponding author. 3918
NOTES
VOL. 173, 1991
Ecur I
hadS
Region , mcrC
mcrB
a
3919
OR F-51
| ORF-39 I
1kb
ORF-33
_
E 0
I*
= Ie ; z .. I.aI tozz AI . . .
_
_
_
I
U0
a
_
a
.xz
-I-
21
,
,
l0
_
s
_
_
*a
--
0
0bU
A
weI
mla
pZB7-mcrC
I
FIG. 1. Restriction map of the area from the E. coli K-12 chromosome showing the location of the mcrB and mcrC genes (8) as well as the adjacent hsdS gene (10). The 1.6-kb segment of DNA (bottom) was ligated with a T7 promoter-containing plasmid, pT7-7, used to construct pZB7-mcrC as described in the text.
harvested cells was centrifuged and washed twice with 1 ml of cold 50 mM phosphate buffer (pH 7.0). About 100,000 cpm per lane was subject to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% polyacrylamide), and the labelled polypeptides were detected by autoradiography. Prelabelled "'C protein molecular weight markers (Bethesda Research Laboratories) were used as standards. The result (Fig. 2, lane B) shows that pZB7-mcrC produced a strong band corresponding to a protein of 39 kDa, in agreement with the results obtained by Ross et al. (7). A 4-kDa band near the bottom of lane B was a fusion protein from the plasmid, pT7-7, and a small portion of the mcrB gene. On the basis of the amount of protein observed by Coomassie staining and SDS-PAGE (Fig. 3, lane C), the level of expression of McrC protein from pZB7-mcrC was estimated to be 20% of total cellular proteins. The enhanced McrC protein synthesis facilitated its purification. McrC protein was purified from the overproduction system. Cells from 1 liter of M9 culture were resuspended in 20 ml of buffer A containing 50 mM Tris (pH 8.0), 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10% (vol/vol) glycerol. The cells were lysed in a French press at 12,000 lb/in2. The lysates were centrifuged at 4°C at 12,000 x g for 20 min to separate the soluble material from the inclusion bodies and other insoluble material. McrC protein was found by SDS-PAGE to be in the supernatant. The supematant was then passed through a DEAE-Sepharose column (Sigma) which was equilibrated with 50 mM Tris (pH 7.5) and 50 mM NaCl. The column was washed with a linear NaCl gradient from 0.05 to 0.5 M, and fractions were analyzed by SDS-PAGE. The McrC protein was identified by its coelution with radioactively labelled McrC protein. McrC protein was found to elute at around 0.17 to 0.20 M NaCl, and most of the major contaminants were removed (Fig. 3, lane D). Fractions with McrC protein were pooled and then loaded onto a hydroxylapatite column (Bio-Rad) equilibrated with 50 mM phosphate buffer (pH 7.5). The column was washed with a phosphate gradient
A
B
966843-
29-
18-
14
-
FIG. 2. Autoradiograph of a SDS-12% polyacrylamide gel depicting plasmid-encoded proteins which were labelled with [355]methionine in a maxicell system. The host protein syntheses were inhibited with rifampin. Lane A, plasmid pT7-7 without mcrC; lane B, pZB7-mcrC. The arrow indicates the position of the 39-kDa McrC protein. The M, values (103) correspond to the following protein markers (top to bottom): phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, P-lactoglobulin, and lysozyme.
3920
NOTES
J. BACTERIOL.
A
B
0
C
.. ..
Dila et al. (2) was the initiation point for McrC translation. Ross et al. (8) predicted that the start codon would be a GTG triplet at the nucleotide position of 1,514 bases and the size of the deduced polypeptide would be 39 kDa. On the other hand, Dila et al. (2) suggested that the translation of McrC would start 25 nucleotides upstream of the GTG as by Ross et al. (8), and that the mcrC gene might produce two peptides with molecular sizes of 40 and 38 kDa. In our present maxicell labelling experiment, the mcrC gene produced only one peptide of about 39 kDa (Fig. 2, lane B). Furthermore, our N-terminal amino acid sequence of the purified McrC protein confirms the prediction of Ross et al. that the initiation codon of the McrC peptide overlaps with the termination codon of McrB by 1 nucleotide.
E
......
4
36
4 29
2'0
FIG. 3. Purification of the McrC protein. Lane A, molecular weight markers (top to bottom): bovine albumin, egg albumin, glyceraldehyde-3-phosphate dehydrogenase, carbonic anhydrase, trypsin inhibitor, and a-lactalbumin; lane B, crude lysate of host cell, DL21(DE3), harboring pT7-7 plasmid without mcrC; lane C, crude lysate of host cell harboring pZB7-mcrC; lane D, pooled fraction of DEAE-Sepharose-purified McrC protein; lane E, hydroxylapatite-purified McrC protein. The arrow indicates the position of the 39-kDa McrC protein. Plurification steps were performed as described in the text.
from 0.1 to 0.2 M, and fractions
were analyzed by SDSprotein eluted at around 0.16 to 0.20 M phosphate. The protein purity was about 99%, as assessed by Coomassie blue staining and SDS-PAGE (Fig. 3, lane E). By using this scheme, we were able to purify about 10 mg of McrC protein from 1 liter of culture. To prove the purified protein is from the mcrC gene, its N-terminal amino acid sequence was determined. The purified McrC protein (50 jLg) was subjected to SDS-PAGE (12% polyacrylamide), and the protein was then electrophoretically transferred onto a polyvinylidine difluoride membrane (Millipore) by using 10 mM 3-[cyclohexylamino]-l-propanesulfonic acid buffer (pH 10) at a constant voltage of 80 V for
PAGE. The
20
mi.
The membrane
was
then stained with Coomassie
blue in 50% methanol for 2
min
methanol for 2
was
mm.
and destained with 50%
extensively washed with might interfere analysis. The McrC protein band
The blot
water to remove other contaminants which
with
subsequent sequence excised, and its N-terminal amino acid sequence-was analyzed by an automated gas-phase protein sequenator at the Department of Immunology and Microbiology, Baylor College of Medicine, Houston, Tex. The N-terminal amino acids of the purified McrC are M, E, Q, P, V, P, V, R, and N, which match exactly with the amino acid sequence
was
I,
deduced from mcrC DNA sequence
disagree
with that
by Dila
et al.
by Ross
et al.
(8) and
(2).
On the basis of the DNA sequence of the mcrC gene, the
major discrepancy between the reports by Ross
et al.
(8) and
We thank Xuemin Wang and Eric C. Achberger for their discussion and Richard Cook of the Baylor College of Medicine for his assistance with the N-terminal amino acid sequence determination. REFERENCES 1. Butkus, V., S. Klimasauskas, L. Petrauskiene, Z. Maneliene, A. Lebionka, and A. Janulaitis. 1987. Interaction of AluI, Cfr6l, and PvuII restriction-modification enzymes with substrates containing either N4-methylcytosine or 5-methylcytosine. Biochim. Biophys. Acta 909:201-207. 2. Dila, D., E. Sutherland, L. Moran, B. Slatko, and E. A. Raleigh. 1990. Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12. J. Bacteriol. 172:4888-4900. 3. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 4. Raleigh, E. A., R. Trimarchi, and H. Revel. 1989. Genetic and physical mapping of the mcrA (rglA) and mcrB (rglB) loci of Escherichia coli K12. Genetics 122:279-296. 5. Raleigh, E. A., and G. Wilson. 1986. Escherichia coli K-12 restricts DNA containing 5-methylcytosine. Proc. Natl. Acad. Sci. USA 83:9070-9074. 6. Ross, T. K., E. C. Achberger, and H. D. Braymer. 1987. Characterization of the Escherichia coli modified cytosine restriction (mcrB) gene. Gene 61:277-289. 7. Ross, T. K., E. C. Achberger, and H. D. Braymer. 1989. Identification of a second polypeptide required for McrB restriction of 5-methylcytosine-containing DNA in Escherichia coli K12. Mol. Gen. Genet. 216:402-407. 8. Ross, T. K., E. C. Achberger, and H. D. Braymer. 1989. Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus. J. Bacteriol. 171:1974-1981. 9. Ross, T. K., and H. D. Braymer. 1987. Localization of a genetic region involved in McrB restriction by Escherichia coli K-12. J. Bacteriol. 169:1757-1759. 10. Sain, B., and N. E. Murray. 1980. The hsd (host specificity) genes of E. coli K-12. Mol. Gen. Genet. 180:35-46. 11. Studier, F. W., A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorif. 1990. Gene expression technology. Methods Enzymol. 185:60-89. 12. Tabor, S., and C. C. Richardson. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82:1074-1078.
JOURNAL OF BACTERIOLOGY, June 1991, p. 3918-3920
0021-9193/91/123918-03$02.00/0 Copyright © 1991, American Society for Microbiology
Overproduction and Purification of McrC Protein from Escherichia coli K-12 LING ZHENG' AND H. D. BRAYMERl 2*
Department of Microbiology, Louisiana State University, Baton Rouge, Louisiana 70803,1* and Pennington Biomedical Research Center, Baton Rouge, Louisiana 708082 Received 11 February 1991/Accepted 17 April 1991
The McrC protein, encoded by one of the two genes involved in the McrB restriction system, was produced in Escherichia coli cells by using a T7 expression system. Following sequential DEAE-Sepharose and hydroxylapatite column chromatography, the protein was purified to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified McrC protein agreed exactly with the one deduced from the DNA sequence by Ross et al. (J. Bacteriol. 171:1974-1981, 1989). lation of this protein. This arrangement would provide for a 1-nucleotide overlap between the stop codon for McrB transcripts and the initiation codon for the McrC transcript. However, Dila et al. (2) recently reported that the deduced McrC coding sequence overlapped the McrB transcripts by 26 nucleotides and had a frameshift of 1 bp from the triplet deduced by Ross et al. (8). They predicted a molecular size of 40 kDa for the McrC protein. Consequently, amino acid sequences of the McrC protein predicted by the two reports are completely different. The purification and sequence of the McrC protein should, therefore, provide the evidence for the correct amino acid sequence of this protein. In this paper, we report the overproduction and purification of the McrC protein from a T7 expression system. The N-terminal amino acid sequence of the purified McrC protein matched exactly with that of Ross et al. (8). A T7 promoter-containing plasmid, pT7-7 (12), was used to construct the overproducing plasmid, pZB7-mcrC (Fig. 1). The mcrC-containing DNA fragment was isolated from pRAB16 (7). This plasmid was first cleaved at the BamHI site within the pUC8 portion of pRAB16 that is adjacent to the EcoRV site on the right side of the map (Fig. 1). The BamHI-linearized pRAB16 was then restricted with EcoRI within the pUC8 portion that is adjacent to the EcoRV site on the left side of the map within the mcrB gene, and the McrC start codon is 80 nucleotides downstream from the EcoRV site (Fig. 1). The 1.6-kb fragment was ligated with EcoRI- and BamHI-digested pT7-7 (3). E. coli BL21 (DE3) (F- ompT rB- mB-) was used as the host for overproduction of the mcrC gene (11). The transformed strain, BL21(DE3)(pZB7-mcrC), was tested for its ability to overproduce McrC protein. The transformant was grown at 37°C on M9 medium supplemented with 1% methionine assay medium (Difco Laboratories). When cultures reached the mid-log to late-log phase (optical density at 600 nm = 0.4), isopropyl-p-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM, and the culture was incubated for 60 min at the same temperature. Since T7 RNA polymerase is not sensitive to rifampin, this antibiotic was added to 300 ,ug/ml to inhibit transcription from the host cell chromosome DNA. After the addition of rifampin, culture growth was continued for 110 min at 37°C. To analyze the newly synthesized proteins, 0.6 ,uCi of [35S]methionine (Du Pont) per ml of medium was added for another 10 min at 37°C. An aliquot (0.5 ml) of
The McrB (for methylated cytosine restriction) restriction system in Escherichia coli K-12 is responsible for the inac-
tivation and degradation of foreign DNA containing methylated cytosine, such as 5-hydroxylmethylcytosine (shmC), 5C-methylcytosine, and 4N-methylcytosine (1, 5). The common recognition sequence for the McrB system is G'C (5). McrB, formerly known as RglB, was discovered because of difficulties encountered in cloning the genes for site-specific modification methylases associated with restriction-modification systems in E. coli K-12 strains (5). Since DNA of many organisms, both procaryotic and eucaryotic, contains methylated cytosine, the McrB methyl-specific restriction system can significantly hinder DNA-cloning procedures in E. coli (8). The McrB locus has been physically mapped at about 99 min of E. coli chromosome DNA (5), and it is in a cluster of other restriction modification systems, such as the hsd genes encoding type I restriction-modification enzymes and the mrr gene encoding the methyladenine-specific restriction enzyme (4). Two McrB genes, mcrB and mcrC, have been cloned and sequenced (6, 8, 9). The mcrB gene encodes 51and 33-kDa polypeptides which share the same C-terminal amino acid sequence. The mcrC gene encodes a 39-kDa polypeptide. The 51-kDa McrB polypeptide and the 39-kDa McrC polypeptide have been postulated to be necessary for the sequence-specific restriction of 5-methylcytosine DNA. It has also been postulated that the 33-kDa polypeptide might play a role in regulating activity of the 51-kDa polypeptide, since the 33-kDa polypeptide may compete with the 51-kDa polypeptide in DNA and protein binding abilities. However, it has not been demonstrated how these polypeptides are involved in McrB restriction. As a first step to gain insight into the function of those gene products and thereby the mechanism of McrB restriction, we started the purification of each of these polypeptides. Furthermore, there exist conflicting reports on the amino acid sequences of these polypeptides deduced from nucleotide sequence. For instance, Ross et al. (8) originally reported that the putative starting codon for McrC translation was a GTG triplet at the nucleotide position 1514. A purinerich region which is 7 bases upstream of this codon was suggested to serve as a ribosome binding site for the trans*
Corresponding author. 3918
NOTES
VOL. 173, 1991
Ecur I
hadS
Region , mcrC
mcrB
a
3919
OR F-51
| ORF-39 I
1kb
ORF-33
_
E 0
I*
= Ie ; z .. I.aI tozz AI . . .
_
_
_
I
U0
a
_
a
.xz
-I-
21
,
,
l0
_
s
_
_
*a
--
0
0bU
A
weI
mla
pZB7-mcrC
I
FIG. 1. Restriction map of the area from the E. coli K-12 chromosome showing the location of the mcrB and mcrC genes (8) as well as the adjacent hsdS gene (10). The 1.6-kb segment of DNA (bottom) was ligated with a T7 promoter-containing plasmid, pT7-7, used to construct pZB7-mcrC as described in the text.
harvested cells was centrifuged and washed twice with 1 ml of cold 50 mM phosphate buffer (pH 7.0). About 100,000 cpm per lane was subject to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% polyacrylamide), and the labelled polypeptides were detected by autoradiography. Prelabelled "'C protein molecular weight markers (Bethesda Research Laboratories) were used as standards. The result (Fig. 2, lane B) shows that pZB7-mcrC produced a strong band corresponding to a protein of 39 kDa, in agreement with the results obtained by Ross et al. (7). A 4-kDa band near the bottom of lane B was a fusion protein from the plasmid, pT7-7, and a small portion of the mcrB gene. On the basis of the amount of protein observed by Coomassie staining and SDS-PAGE (Fig. 3, lane C), the level of expression of McrC protein from pZB7-mcrC was estimated to be 20% of total cellular proteins. The enhanced McrC protein synthesis facilitated its purification. McrC protein was purified from the overproduction system. Cells from 1 liter of M9 culture were resuspended in 20 ml of buffer A containing 50 mM Tris (pH 8.0), 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10% (vol/vol) glycerol. The cells were lysed in a French press at 12,000 lb/in2. The lysates were centrifuged at 4°C at 12,000 x g for 20 min to separate the soluble material from the inclusion bodies and other insoluble material. McrC protein was found by SDS-PAGE to be in the supernatant. The supematant was then passed through a DEAE-Sepharose column (Sigma) which was equilibrated with 50 mM Tris (pH 7.5) and 50 mM NaCl. The column was washed with a linear NaCl gradient from 0.05 to 0.5 M, and fractions were analyzed by SDS-PAGE. The McrC protein was identified by its coelution with radioactively labelled McrC protein. McrC protein was found to elute at around 0.17 to 0.20 M NaCl, and most of the major contaminants were removed (Fig. 3, lane D). Fractions with McrC protein were pooled and then loaded onto a hydroxylapatite column (Bio-Rad) equilibrated with 50 mM phosphate buffer (pH 7.5). The column was washed with a phosphate gradient
A
B
966843-
29-
18-
14
-
FIG. 2. Autoradiograph of a SDS-12% polyacrylamide gel depicting plasmid-encoded proteins which were labelled with [355]methionine in a maxicell system. The host protein syntheses were inhibited with rifampin. Lane A, plasmid pT7-7 without mcrC; lane B, pZB7-mcrC. The arrow indicates the position of the 39-kDa McrC protein. The M, values (103) correspond to the following protein markers (top to bottom): phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, P-lactoglobulin, and lysozyme.
3920
NOTES
J. BACTERIOL.
A
B
0
C
.. ..
Dila et al. (2) was the initiation point for McrC translation. Ross et al. (8) predicted that the start codon would be a GTG triplet at the nucleotide position of 1,514 bases and the size of the deduced polypeptide would be 39 kDa. On the other hand, Dila et al. (2) suggested that the translation of McrC would start 25 nucleotides upstream of the GTG as by Ross et al. (8), and that the mcrC gene might produce two peptides with molecular sizes of 40 and 38 kDa. In our present maxicell labelling experiment, the mcrC gene produced only one peptide of about 39 kDa (Fig. 2, lane B). Furthermore, our N-terminal amino acid sequence of the purified McrC protein confirms the prediction of Ross et al. that the initiation codon of the McrC peptide overlaps with the termination codon of McrB by 1 nucleotide.
E
......
4
36
4 29
2'0
FIG. 3. Purification of the McrC protein. Lane A, molecular weight markers (top to bottom): bovine albumin, egg albumin, glyceraldehyde-3-phosphate dehydrogenase, carbonic anhydrase, trypsin inhibitor, and a-lactalbumin; lane B, crude lysate of host cell, DL21(DE3), harboring pT7-7 plasmid without mcrC; lane C, crude lysate of host cell harboring pZB7-mcrC; lane D, pooled fraction of DEAE-Sepharose-purified McrC protein; lane E, hydroxylapatite-purified McrC protein. The arrow indicates the position of the 39-kDa McrC protein. Plurification steps were performed as described in the text.
from 0.1 to 0.2 M, and fractions
were analyzed by SDSprotein eluted at around 0.16 to 0.20 M phosphate. The protein purity was about 99%, as assessed by Coomassie blue staining and SDS-PAGE (Fig. 3, lane E). By using this scheme, we were able to purify about 10 mg of McrC protein from 1 liter of culture. To prove the purified protein is from the mcrC gene, its N-terminal amino acid sequence was determined. The purified McrC protein (50 jLg) was subjected to SDS-PAGE (12% polyacrylamide), and the protein was then electrophoretically transferred onto a polyvinylidine difluoride membrane (Millipore) by using 10 mM 3-[cyclohexylamino]-l-propanesulfonic acid buffer (pH 10) at a constant voltage of 80 V for
PAGE. The
20
mi.
The membrane
was
then stained with Coomassie
blue in 50% methanol for 2
min
methanol for 2
was
mm.
and destained with 50%
extensively washed with might interfere analysis. The McrC protein band
The blot
water to remove other contaminants which
with
subsequent sequence excised, and its N-terminal amino acid sequence-was analyzed by an automated gas-phase protein sequenator at the Department of Immunology and Microbiology, Baylor College of Medicine, Houston, Tex. The N-terminal amino acids of the purified McrC are M, E, Q, P, V, P, V, R, and N, which match exactly with the amino acid sequence
was
I,
deduced from mcrC DNA sequence
disagree
with that
by Dila
et al.
by Ross
et al.
(8) and
(2).
On the basis of the DNA sequence of the mcrC gene, the
major discrepancy between the reports by Ross
et al.
(8) and
We thank Xuemin Wang and Eric C. Achberger for their discussion and Richard Cook of the Baylor College of Medicine for his assistance with the N-terminal amino acid sequence determination. REFERENCES 1. Butkus, V., S. Klimasauskas, L. Petrauskiene, Z. Maneliene, A. Lebionka, and A. Janulaitis. 1987. Interaction of AluI, Cfr6l, and PvuII restriction-modification enzymes with substrates containing either N4-methylcytosine or 5-methylcytosine. Biochim. Biophys. Acta 909:201-207. 2. Dila, D., E. Sutherland, L. Moran, B. Slatko, and E. A. Raleigh. 1990. Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12. J. Bacteriol. 172:4888-4900. 3. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 4. Raleigh, E. A., R. Trimarchi, and H. Revel. 1989. Genetic and physical mapping of the mcrA (rglA) and mcrB (rglB) loci of Escherichia coli K12. Genetics 122:279-296. 5. Raleigh, E. A., and G. Wilson. 1986. Escherichia coli K-12 restricts DNA containing 5-methylcytosine. Proc. Natl. Acad. Sci. USA 83:9070-9074. 6. Ross, T. K., E. C. Achberger, and H. D. Braymer. 1987. Characterization of the Escherichia coli modified cytosine restriction (mcrB) gene. Gene 61:277-289. 7. Ross, T. K., E. C. Achberger, and H. D. Braymer. 1989. Identification of a second polypeptide required for McrB restriction of 5-methylcytosine-containing DNA in Escherichia coli K12. Mol. Gen. Genet. 216:402-407. 8. Ross, T. K., E. C. Achberger, and H. D. Braymer. 1989. Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus. J. Bacteriol. 171:1974-1981. 9. Ross, T. K., and H. D. Braymer. 1987. Localization of a genetic region involved in McrB restriction by Escherichia coli K-12. J. Bacteriol. 169:1757-1759. 10. Sain, B., and N. E. Murray. 1980. The hsd (host specificity) genes of E. coli K-12. Mol. Gen. Genet. 180:35-46. 11. Studier, F. W., A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorif. 1990. Gene expression technology. Methods Enzymol. 185:60-89. 12. Tabor, S., and C. C. Richardson. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82:1074-1078.
