THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 5, Issue of March 10, pp. 2662-2!39.1985 Printed In U.S.A.

Amplification and Purification of the Bacteriophage Mu Encoded B Transposition Protein*

(Received for publication, March 5, 1984)

George Chaconas, Greg GloorS, and Janet L. Miller4 From the Cancer Research Laboratory and Departments of Biochemistry and Microbiology and Immurwlogy, University of Western Ontario, London, Ontario, N6A 587, Canada

The A and B proteins encoded by the temperate bacteriophage Mu are involved in the high efficiency DNA transposition reaction which is the distinguishing feature of this phage. The genes encoding these early proteins were cloned in an expression vector under the control of the bacteriophage X leftward promoter. Un- der optimal conditions gpB was overproduced to ac- count for 15% of the total cellular protein. The protein was purified to near homogeneity as determined by silver staining. Sequence analysis of the N terminus confirmed the identity of the purified protein as gpB. Proteolytic processing of the B protein does not occur at the amino terminus; the terminal methionine residue is quantitatively deformylated. The protein, which was found to be basic and a general DNA binding protein, was insoluble at low ionic strength in the absence, but not in the presence, of DNA. The B protein also dis- played a tendency to aggregate at high ionic strength where it was soluble in the absence of DNA. In addi- tion, the protein was characterized as to its amino acid composition and extinction coefficient at 280 nm. The purified protein is active in a soluble in vitro transpo- sition-replication system.

The temperate bacteriophage Mu is a giant transposon which utilizes DNA transposition as a means of propagating its genetic material (Toussaint and RBsibois, 1983; Kleckner, 1981). The integrative replication of Mu DNA is a process which closely resembles the transposition of other prokaryotic transposable elements except that in the case of Mu the in vivo reaction occurs at a much higher frequency. Bacterio- phage Mu is also unique among transposons in that the type of transposition end products generated appears to be devel- opmentally regulated (Chaconas et al., 1981b, 1983); infecting DNA integrates to give primarily simple insertions while induction of a prophage results predominantly in the forma- tion of replicon fusions.

Although Mu is a large double-stranded DNA virus (38 kilobases), as is the case with other transposons only the ends of the phage genome are required for transposition and rep- lication to occur (Toussaint and Rksibois, 1983). Furthermore, no transposition occurs with either end alone (Chaconas et

Institute and the Medical Research Council of Canada. The costs of *This work was supported by grants from the National Cancer

publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.

This paper is dedicated to the memory of Dr. Ahmad I. Bukhari who died unexpectedly in November 1983. His friendship, encourage- ment, and enthusiasm for bacteriophage Mu will always be remem- bered.

$ Research Student of the National Cancer Institute of Canada. Recipient of a studentship from the Medical Research Council of

Canada.

at., 1981b) or with both ends when they are not in their proper orientation (Schumm and Howe, 1981). A partial reaction apparently does not occur when only one end is present,’ and the reactive DNA at the ends of the Mu genome remains to be defined.

The proteins involved in Mu DNA transposition are spec- ified by both the host (Toussaint and Faelen, 1974) and phage genomes. The products of the Mu early genes A and B appear to be directly involved in the integrative replication process. Mu A- mutants show no integration and B- mutants show a 10-100-fold reduction in transposition (ODay et al., 1978; Faelen et al., 1978; Harshey, 1983). Physical association of mini-Mu plasmids with the host chromosome also does not occur in the absence of the A or B protein (Chaconas et al., 1980,1981~) and significant levels of Mu DNA replication do not occur in A - or B- mutants (Wijffelman and Lotterman, 1977; Waggoner et al., 1981). The presence of other early Mu genes has also been reported to stimulate Mu DNA replica- tion; however, these genes are not essential and may have only an indirect involvement (Waggoner et al., 1981; Goosen et al., 1982).

The mechanism by which Mu DNA transposition and rep- lication occurs remains obscure. A contributing factor to this lack of knowledge is the difficulty involved in studying the Mu A and B proteins because of their extremely low intracel- lular concentrations; during Mu development these proteins are difficult to detect by two-dimensional polyacrylamide gel electrophoresis of total extracts from radioactively labeled Escherichia coli cells? Furthermore, establishment of in vitro systems for Mu DNA transposition has only recently been accomplished. Higgins et al. (1983a, 1983b) have reported Mu DNA replication in vitro on cellophane discs, and during the preparation of this manuscript Mizuuchi (1983) published the first soluble in vitro transposition system. We now report the expression of the Mu A and B proteins under the control of the bacteriophage X leftward promoter carried on a multicopy plasmid. The Mu B protein is amplified to represent 15% of the total cellular protein and we present the first report of the purification of this protein.

EXPERIMENTAL PROCEDURES3

G. Chaconas and A. I. Bukhari, unpublished results. * M. DuBow and A. I. Bukhari, personal communication. 3Portions of this paper (including “Experimental Procedures,”

Figs. 4-6, and Footnote 4) are presented in miniprint at the end of this paper. The abbreviations used are: SDS, sodium dodecyl sulfate; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PEI, polyethyleneimine; MSH, 8-mercaptoethanol; PTH, phenylthiohy- dantoin; HPLC, high performance liquid chromatography. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. “-691, cite the authors, and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

2662

Purification of the Mu B Protein 2663

RESULTS AND DISCUSSION

Construction of Recombinant Plasmids-Recombinant plas- mids were constructed using the plasmid vector pKC30 which carries the bacteriophage X leftward promoter. Expression in this system can be regulated by a temperature-sensitive re- pressor, and the great promoter strength coupled with high plasmid copy makes this system useful for amplification of many proteins in E. coli (Rosenberg et al., 1983). The Mu DNA used in the plasmid constructions was derived from the mutant phages (Fig. 1) Mucts62-13/4 (Goosen et al., 1982) and Mucts62XcamS (Chow and Bukhari, 1978). The former strain carries an Is1 insertion about 225 base pairs after the end of the Mu B gene. Cleavage of this DNA with BalI results in the liberation of a fragment carrying the 3' end of the ner gene followed by the A and B genes in their entirety. Several plasmids were derived from insertion of BalI fragments of the phage DNA into pKC30 and are shown in Fig. 2. The most important of these is pGC511 which has the Mu A and B genes inserted in the proper orientation downstream from PL. The 3' end of the ner gene is present on the inserted DNA and provides translational stops in all three reading frames to eliminate the possibility of a fusion between the X N protein

0 IO

and the Mu A protein. Analogous plasmids carrying only the Mu A gene were generated by the same rationale using the mutant phage Mucts62XcamS which carries a Tn9 insertion about 250 base pairs after the start of the B gene. These plasmids are also shown in Fig. 2.

Functional A or A and B genes are carried on the plasmids with inserts in the proper orientation as demonstrated by the following criteria:

1) Complementation of Mu Aam and B a n mutant phages in spot tests.

2) A high level of in uiuo transposition of a mini-Mukan catalyzed by the A and B proteins provided in trans from pGC511 and a low but significant level of mini-Muhn trans- position with gpA alone provided from pGG05 (Chaconas et al., 1984).

3) Amplification of the Mu A and B proteins and the ability of extracts from the overproducing strains to stimulate Mu DNA transposition in uitro (see Fig. 8).

Control plasmids also carrying the A or A and B genes but in the wrong orientation were also recovered. It was found, however, that low levels of transposition could be promoted by these plasmids in uiuo, probably because of readthrough into A and B from the 6-lactamase promoter of pBR322

20 30 40

0 5 IO P

FIG. 1. Restriction map of Mucts62-13/4 and Mucts62Xcam6 phage DNA. The upper restriction maps are of the entire genomes, drawn to the scale at the top of the figure. The lower restriction maps show the left ends blown up to the scale at the bottom of the figure. The thin lines denote Mu DNA and the thick lines the internal sequences of Tn9. The striped boxes indicate IS1 sequences and the wauy lines denote host DNA found at the ends of the Mu phage DNA. Beneath the enlarged restriction map is a diagram of the position of the Mu Pi and P. promoters, and the position of the Mu genes transcribed. The arrows indicate the direction of transcription. The position of the IS1 insertion in Mucts62-13/4 is taken from Goosen et al. (1982) and the Tn9 insertion in Mucts62Xcam5 from Chow and Bukhari (1978). The orientation of the insertions is based upon our unpublished data. The positions of Mu promoters, genes, and restriction enzyme sites are taken from Priess et d. (1982), Schumann et al. (1980), and Kahmann et al. (1977). The positions of the restriction sites in IS1 are taken from Ohtsubo and Ohtsubo (1978). The scales are calibrated in kilobases.

2664 Purification of the Mu B Protein

(Chaconas et d , 1984). For this reason another control plas- A by-product of the cloning work on the A and B genes was mid (pGC333) was recovered which contained no ineertion the plasmid pGC180 which carries the Mu c gene (immunity but had a small deletion which knocked out the X N gene. repressor) under the control of PL.

0 5 IO

X sequence, IS- I sequence, - M u sequence, = pBA322 sequence, G I , H I 1 junction

FIG. 2. Restriction maps of recombinant plasmids derived from pKC30. Plasmids pGC511, pGC240, and pGC180 contain B d fragments of Mucts62-13/4 phage DNA cloned into the HpaI site of the plasmid vector pKC30. Plasmid pGC333 is a derivative of pKC30 with a deletion of approximately 165 base pairs (indicated by A) at the HpaI site within the X N gene. Plasmids pGGO4, pGGO6, and pGGO6 contain &rlI fragments of MuetsG2XcamS phage DNA in the HpaI site of pKC30. The solid dot in pGC180 indicates the junction of X DNA with host sequences found at the left end of Mu. Beneath each restriction map is a diagram of the position of the X PL promoter in each plasmid and the position of the Mu genes transcribed. The arrows indicate the direction of transcription. Plasmid pGC180 contains the Mu repressor (e) gene. The positions of restriction sites in the pBR322 sequences are taken from Sutcliffe (1978) and the position of PL and the restriction sites in the X DNA are from Sanger et aL (1982). Other information was taken from the sources indicated in the legend to Fig. 1. The scale at the bottom is calibrated in kilobases.

Purification of the Mu B Protein 2665

0 25 50 75 1 0 0 125 M

.92.5

.66.2

.4 5.0

-31.0

-21.5

-14.4

Time after induction (minuter)

FIG. 3. Kinetics of synthesis of the M u B protein directed by the X leftward promotor in pGC511. Strain GC115 which carries this plasmid was grown in LB broth containing 100 pg/ml of ampicillin a t 32 "C to a density of about 6 X 10s cells/ml. The culture was shifted to 43 "C, and aliquots were removed for analysis a t the indicated times. A, Coomassie blue stain of the acrylamide gel used to analyze the protein samples. Slot M contains molecular mass markers. The molecular masses are given in kilodaltons. E, quantifi- cation of the percentage of the total protein comprising gpE by densitometric analysis of the gel shown in pane l A .

9 2 . 5 4 6 6 . 2 4 4 5 . 0 4

31 .O - 21.5-

14.4-

M 1 2 3 4 5 ,-, . -,-

Amplification of the Mu B Protein-Thermal induction of the leftward promoter on pGC511 (Fig. 3) resulted in the dramatic appearance of a protein the size of gpB (33,000 daltons, Giphart-Gassler et d., 1981) on sodium dodecyl SUI- fate-polyacrylamide gels. The 33,000-dalton polypeptide was not observed with the control plasmids pGC333 or pGC240 and was therefore assumed to be the B protein. The amplifi- cation leveled off with gpB comprising almost 15% of the total cellular protein. Under these conditions we have not detected the A protein. This is of interest since in vivo experiments demonstrated the presence of a functional A gene which was transcribed from PL. Furthermore, the A gene is transcribed before the B gene in the same operon, so the message for gpA must be present. It is possible that the A protein is not amplified to the same extent as B because it is rapidly degraded; Pato and Reich (1982) have reported a half- life of approximately 3 min for the A protein. It is also possible that translational inefficiency and/or translational control mechanisms are responsible for the low yields of gpA in this system. Using lower percentage gels and with pGG04 and pGG05 which carry A but not B we have detected amplified gpA by Coomassie blue staining, but at levels much lower than for gpB (Chaconas et aL, 1984).

Purification of the Mu B Protein-Starting from strain GC115 which harbors the overproducing plasmid pGC511 we purified the Mu B protein to near homogeneity. Since there was no available assay for gpB, the purification was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. During preliminary work to devise a purification protocol we found that gpB was very soluble at high concentrations in low ionic strength buffer (50 mM NaCl) as long as there was DNA present in the extract. As soon as the DNA was removed from the extract, however, the protein became insoluble with some precipitation occurring even in 0.5 M NaCl. The precipitation was found to be irreversible unless urea or guanidine HC1 was used for solubilization, suggesting that the protein had dena- tured. Variation in pH or the presence of detergents, divalent metal ions, dimethyl sulfoxide, glycerol, ATP, deoxynucleo- side triphosphates, or monophosphates did not change the solubility properties of gpB. The insolubility of the protein at low ionic strength created an obvious problem with respect to

6 -r I

COOMASSIE BLUE SILVER FIG. 7. Sodium dodecyl sulfate-polyacrylamide gel analysis of gpB purification stages. The 12% gel

with a 6% stack contained about 8 pg of B protein per slot. Lane I , total cells; lane 2, supernatant of cell lysate; lane 3, resuspended streptomycin sulfate pellet; lane 4, pooled fractions from hydroxylapatite chromatography; lane 5, pooled fractions from DEAE-Sepharose chromatography; lane 6, pooled fractions from CM-Sepharose chromatography; lane M, standards whose molecular masses are indicated in kilodaltons. The gel was first stained with Coomassie blue and subsequently stained with silver to accentuate any trace contaminants. The apparent molecular mass of the purified protein is 33,000 daltons as determined from this gel.

2666 Purification of the Mu B Protein

the use of ion exchange chromatography in the purification procedure. We therefore unsuccessfully attempted to obtain a clean preparation of B protein using DNA binding at low salt and a variety of steps at 0.5 M NaCl or above (hydroxylapatite chromatography, hydrophobic chromatography, dye matrix chromatography, and gel filtration). We finally resorted to denaturation of the protein in 6.2 M urea to eliminate the solubility problems in the final two steps of the purification. The final purification procedure described under “Experimen- tal Procedures” results in the recovery of 5-7 mg of protein from 1 liter of cells. The entire purification can be completed in 8 h and the final preparation is nearly homogeneous as can be seen in Fig. 7. Only a few trace contaminants can be visualized by an overdeveloped silver strain.

Because denaturation in urea was used during the purifi- cation procedure we were concerned that the renatured pro- tein might be inactive or have altered properties. It is note- worthy, however, that the total time during which the protein was in contact with highly purified urea was less than 4 h. Carbamylation of amino groups in proteins is a slow reaction requiring much longer times, and carbamylation of thiol groups, although rapid, is readily reversible above neutral pH (Cohen and Oppenheimer, 1977). Furthermore, several pro- teins involved in DNA metabolism with solubility problems similar to that of gpB have been recently purified using solubilization in urea. Both the bacteriophage X 0 and P proteins (Tsurimoto and Matsubara, 1982) and the Tn3 re- solvase (Krasnow and Cozzarelli, 1983) retained their ability to stimulate X DNA replication or site specific recombination, in uitro after removal of the urea. In the case of the Mu B protein the solubility properties of the native protein are restored upon renaturation out of urea. In addition, similar chromatographic behavior on gel filtration and hexyl-Sepha- rose was observed. These findings demonstrate a structural similarity between gpB before and after purification in urea. We also assayed the purified B protein by in uitro comple- mentation in the soluble transposition-replication system of Mizuuchi (1983). Fig. 8 shows that the purified protein rena- tured from urea is as active in the complementation assay as

ope ( n d

FIG. 8. In vitro complementation of the bacteriophage Mu B gene product by the streptomycin sulfate fraction (0) or purified B protein (0). The concentration of gpB in the strepto- mycin sulfate fraction was determined by Coomassie blue staining of SDS-polyacrylamide gels containing the streptomycin fraction and known amounts of purified gpB. The 12.5-pl reactions contained 210 pmol of Mu DNA nucleotide and [a-’*P]d’M’P at 220 cpm/pmol of total deoxynucleoside triphosphate. All reactions contained both host and Mu A + extracts. Background DNA synthesis in the absence of gpB was 2.2 pmol, which was subtracted from each plotted value. Assays were performed as described under “Experimental Proce- dures.”

TABLE I Amino acid Composition of gpB

The number of residues for each amino acid has been determined based upon a chain length of 312 residues (Miller et al., 1984). Each value represents the average of two determinations (except Cys and TV).

Residues determined Residues predicted Amino acid by amino acid from DNA

composition sequences Asx 29.6 30 Thr 24.1 22 Ser 17.3 17 Glx 41.2 40 Pro 8.7 8 GlY 20.9 20 Ala 27.1 27 Cys“ 4.0 3 Val 18.0 18 Met 8.1 8 Ile 16.9 19 Leu 34.7 36 T Y ~ 4.9 5 Phe 8.1 8 Trpb 2.1 4 LYS 13.9 14 His 5.3 5 Arg 27.1 28

Determined as cysteic acid after performic acid oxidation. Determined after hydrolysis with methanesulfonic acid.

the streptomycin sulfate fraction which had not been dena- tured and renatured. The maximal level of Mu DNA synthesis under the assay conditions used was about 22 pmol which corresponds to 10.5% of the Mu DNA template and is in good agreement with the 5-10% efficiency observed by Mizuuchi (1983).

Besides assaying the ability of purified gpB to stimulate Mu DNA replication, we also analyzed the transposition end products generated in the in uitro system. Agarose gel electro- phoresis of the reaction products (data not shown) revealed no detectable difference in the products formed with the renatured protein or the streptomycin sulfate fraction; in both cases replicon fusions were the predominant end products. These experiments demonstrate that the Mu B protein puri- fied in urea by the protocol we have developed is functionally indistinguishable from an early purification fraction which has not been subjected to denaturation and renaturation.

Physical Characterization of the Mu B Protein-The iden- tity of purified gpB was verified by sequence analysis of the amino terminus. The first 5 residues were found to be Met- Asn-Ile-Ser-Asp which is the expected amino acid sequence based upon nucleotide sequence of the Mu B gene (Miller et aL, 1984). This indicates that no proteolytic processing occurs at the amino terminus. Furthermore, a 95% recovery of the terminal methionine indicates that the N terminus is defor- mylated.

The amino acid composition of gpB is shown in Table I. Our observed values are in good agreement with the expected composition based upon the nucleotide sequence (Miller et al., 1984). The protein was found to contain 51% polar amino acids which is within the normal range (47 f 6%) for non- membrane proteins (CapaIdi and Vanderkooi, 1972). In ad- dition, the protein contained more basic than acidic residues based upon an isoelectric point of about 9.0 in 8 M urea (data not shown). Based upon the DNA sequence the molecular mass of gpB was calculated to be 34,750 daltons. This is in good agreement with an apparent molecular mass of 33,000 daltons displayed on sodium dodecyl sulfate-polyacrylamide gels (Giphart-Gassler et al., 1981 and Fig. 6).

Purification of the Mu B Protein 2667

The extinction coefficients, c, at 280 nm were found to be 1.06, 1.18, and 1.25 for a 1 mg/ml solution as determined by dry weight, relative to absorbance at 205 nm, or by alkaline titration of the 5 tyrosine residues at 295-300 nm, respec- tively. The average of these three values was 1.16.

The purified B protein was stored in 1 M NaCl in order to avoid precipitation. Under conditions where the protein was soluble it had a tendency to aggregate as determined by gel filtration on Bio-Gel A-0.5m or A-1.5m. The unaggregated gpB (usually about 50% of the protein) chromatographed in the position expected for a monomeric form. The protein could be kept in solution at 50 mM NaCl if the solution contained about 1 weight equivalent of DNA. Attempts were made to quantitatively characterize the DNA binding; how- ever, uninterpretable results were obtained, probably due to the precipitation of unbound protein at low salt where DNA binding occurs. The Mu B protein did bind, however, with the same efficiency to both single-stranded and double-stranded DNA cellulose and did not demonstrate a significant prefer- ence for Mu DNA binding.

Experiments are now in progress to determine the role of gpB in the transposition process.

Acknowledgments-We would like to thank Drs. Roger McMacken, Martha Howe, Martin Marinus, Martin Rosenberg, and Helios Mu- rialdo for providing strains. Amino acid sequence determination was performed in Dr. The0 Hofmann’s laboratory at the University of Toronto by S. Rhee. (Support was provided to Dr. Hofmann for the sequenator facility from the J. P. Bickell Foundation.) Amino acid analysis was performed by C. Yu in Dr. Hofmann’s laboratory and by W. Chung at our university. We are grateful for this help in the analysis of the B protein. We would also like to thank Dr. Mike DuBow for communication of unpublished data. We are grateful to the late Dr. Harro Preiss for the predicted sequence of the N terminus of gpB based upon his nucleotide sequence data. The expert assistance of Dale Marsh and Linda Bonis in preparation of figures and typing of the manuscript, respectively, is gratefully acknowledged.

REFERENCES Birnboim, H. C., and Doly, J. (1979) Nuckic Acids Res. 7,1513-1523 Capaldi, R. A., and Vanderkooi, G. (1972) Proc. Natl. Acad. Sci. U.

Chaconas, G., de Bruijn, F. J., Casadaban, M. J., Lupski, J. R., Kwoh, T. J., Harshey, R. M., DuBow, M. S., and Bukhari, A. I. (1981a) Gene (Amst.) 13,37-46

Chaconas, G., Gloor, G., Miller, J. L., Kennedy, D. L., Giddens, E. B., and Nagainis, C. R. (1984) Cold Spring Harbor Symp. Quant.

Chaconas, G., Harshey, R. M., and Bukhari, A. I. (1980) Proc. Natl.

Chaconas, G., Harshey, R. M., Sarvetnick, N., and Bukhari, A. I.

Chaconas, G., Harshey, R. M., Sarvetnick, N., and Bukhari, A. I.

Chaconas,.G., Kennedy, D. L., and Evans, D. (1983) Virology 128 ,

Chow, L. T., and Bukhari, A. I. (1978) Gene (Amst.) 3,333-346 Cohen, S., and Oppenheimer, E. (1977) in The Chemistry of Cyanates

and Their Thio Derivatiues, Part 2, from The Chemistry of Func- tional Groups (Patai, S., ed) John Wiley and Sons, New York

S. A. 69,930-932

BWl. 49,279-284

Acad. Sci. U. S. A. 7 7 , 1778-1782

(1981b) J. Mol. Biol. 150 , 341-359

(1981~) Cold Spring Harbor Symp. Quant. BWl. 45,311-322

48-59

Edelhoch, H. (1967) Biochemistry 6,1948-1954 Faelen, M., Huisman, O., and Toussaint, A. (1978) Nature (Lond.)

27 1,580-582 Fuller, R. S., Kaguni, J. M., and Kornberg, A. (1981) Proc. Natl. Acad.

Giphart-Gassler, M., Reeve, J., and Van de Putte, P. (1981) J. Mol.

Goosen, T., Giphart-Gassler, M., and Van de Putte, P. (1982) Mol.

Grunstein, M., and Hogness, D. S. (1975) Proc. Natl. Acad. Sei. U. 8.

Harshey, R. (1983) P m . Natl. Acad. Sci. U. S. A. 80, 2012-2016 Higgins, N. P., Manlapaz-Ramos, P., Gandhi, R. T., and Olivera, B.

Higgins, N. P., Moncecchi, D., Manlapaz-Ramos, P., and Olivera, B.

Hirs, C. H. W. (1967) Methods Enzymol. 11, 197-199 Kahmann, R., Kamp, D., and Zipser, D. (1977) in DNA Insertion

Elements, Plasmids and Episomes (Bukhari, A. I., Shapiro, J. A., and Adhya, S. L., e&) pp. 335-339, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Sci. U. S. A. 78,7370-7374

BWl. 145,165-191

Gen. Genet. 186,135-139

A. 72,3961-3965

M. (1983a) Cell 33 , 623-628

M. (1983b) J. BWZ. Chem. 268,4293-4297

Kleckner, N. (1981) Annu. Rev. Genet. 16,341-404 Krasnow, M. A., and Cozzarelli, N. R. (1983) Cell 32 , 1313-1324 Kwoh, D. Y., Zipser, D., and Erdmann, D. S. (1980) Virology 101,

Laemmli, U. K. (1970) Nature (Lond.) 227,680-685 Miller, J. H. (1972) in Experiments in Molecular Genetics, Cold Spring

Harbor Laboratory, Cold Spring Harbor, New York Miller, J. L., Anderson, S. K., Fujita, D. J., Chaconas, G., Baldwin,

D. L., and Harshey, R. M. (1984) Nucleic Acids Res. 12,8627-8638 Mizuuchi, K. (1983) Cell 35, 785-794 O’Day, K., Schultz, D., Ericsen, W., Rawluk, L., and Howe, M. (1979)

VirolOgy.93,320-328 O’Day, K. J., Schultz, D. W., and Howe, M. M. (1978) in Microbiology

1978 (Schlessinger, D., ed) pp. 48-51, American Society for Micro- biology, Washington, D. C.

Ohtsubo, H., and Ohtsubo, E. (1978) Proc. Natl. Acad. Sci. U. S. A.

Pato, M. L., and Reich, C. (1982) Cell 2 9 , 219-225 Priess, H., Kamp, D., Kahmann, R., Brauer, B., and Delius, H. (1982)

Rosenberg, M., Ho, Y., and Shatzman, A. (1983) Methods Enzymol.

Salit, I. E., Vavougios, J., and Hofmann, T. (1983) Znfect. Immun.

Sanger, F., Coulson, A. R., Hong, G. F., Hill, D. F., and Petersen, G.

Schumann, W., Bade, E. G., Forgie, R. A., and Howe, M. M. (1980)

Schumm, J. W., and Howe, M. M. (1981) Virology 114,429-450 Scopes, R. K. (1974) Anal. Biochem. 59,277-282 Simpson, R. J., Neuberger, M. R., and Lius, T.-Y. (1976) J. Biol.

Sutcliffe, J. G. (1978) Coid Spring Harbor Symp. Quunt. Biol. 43,77-

Toussaint, A., and Faelen, M. (1974) Mol. Gen. Genet. 131,209-214 Toussaint, A., and RBsibois, A. (1983) in Mobile Genetic Ekments

Tsurimoto, T., and Matsubara, K. (1982) Proc. Natl. Acad. Sci. U. S.

van Vliet, F., Couturier, M., Desmet, L., Faelen, M., and Toussaint,

Waggoner, B., Pato, M., Toussaint, A., and Faelen, M. (1981) Virology

Wijffelman, C., and Lotterman, B. (1977) Mol. Gen. Genet, 151,169-

Wray, W., Boulikas, T., Wray, U. P., and Hancock, R. (1981) Anal.

419-431

75,615-619

Mol. Gen. Genet. 186,315-321

101,123-138

42,755-762

B. (1982) J. Mol. BWl. 162, 729-773

Virology 104,418-425

Chem. 251,1936-1940

90

(Shapiro, J. A., ed) pp. 105-158, Academic Press, New York

A. 79,7639-7643

A. (1978) Mol. Gen. Genet. 160 , 195-202

113,379-387

174

Biochem. 118,197-203

Continued on next page.

2668 Purification of the Mu B Protein SUPPLEUENTARY IUTERIAL TO

Amplification and Rtrification Of Bacteriophase Mu Encoded 1 Transposition Protein

George Chaeonae, Greg Gloor and Janet L. niller

FXPERIHL71TAL PROCEOllRES

Media - LB broth was prepared as deaeribed by Hiller (1972) except that N-2 a (Scheffield Products Kraft Ine Heophis, Tenn.) was uaed a. a aub- stitute for Bseto tryptone: LB agar &e# were aupplnented with glucose to a final concentration of 0.lX and with nDS0 and CaCII to a final concen- tration of 2.5 mu. Strain8 harboring plasmids'were alwa I) 8 r m in the presence of 100 unlml ampicillin (in liquid) or 100 ug1.Y carbenicillin (on plates).

:e from Bio Rad.

SLeD 11: Straptolycin nu1Dhata preeipitatlon - A aolutlon Of 5X .trcptOMycin sulphate In 50 mn NlCl (0.4 volumes) was added t o the .upernatant. Thhe

minute esntrifugation at 10,000 r a (9,500 re) in a Sorvall SS-34 rotor. mlution was miled by end over end rotadon for 15 minute. follasd by a 10

The B prorein precipitated with the MIA and the pellet and tube were bently rinszd with 10 m1 of 1.431 streptomyein'sulphate in 50 mu NaC1. The atrep- rmyein pellet wae resuspended In 10 m1 buffer A (25 mu imidazole - RC1. pH7.4 containing 10 mn E-mcrcaptoethmol and 1 mu EUTA) containing 0.8 H NaC1 by disruption in a gla.a-8lass homogenizer. Thhe hiah eoncentretion of NaC1 dissociated the gp! f r a the from the DNA and kept the protein in solu- tion upon subsequent r a o v a l of the M A . fie nuclele acid was precipitated b the addition of 0.06 volumes of 10X (VI") polyethslenaimlne - HC1. pH7.8. A e solution was mixed for 15 nlnutes by rotation followed by a low speed centrlfugarion LIS demeribed for the atreptomyein aulphate step. About 10 m1

material at 280 nU was uaually about 3.0. of supernatant fluid containing gp! wa# recovered. The absorbance of this

Srep 111: Hydroxylapatite chrmatouraph - The PEI supernaranc was directly

a. described in Figure 4. Thhe column was run with a preasure head of 30-40 applied to a hydroxylapatite column equflibatcd in buffer plus 0 . 8 H NaCl

elpitate in the f i n t peak fraction. Solid urea we.* added to this tuba en. Elvrion of gpt rich 7l4 urea often resulted in rhe appearance of a pre-

unci1 the protein went back into eolucion. Buffer B contained 25 on lnida- role - HC1, pH7.4 with 1 h U 8-merCaptoethanOl end 0.1 mu EDTA.

Step IV. OEAE-SepharOae chromatoaraph - The pooled fraction. from hydroxy- lapatice were applied directly to I &E-Sepharosc column equilibrated in buffer B plus 6.2 H urea as Indicated in Figure 5. The column wa. run with

bur the trailing edge contained contaminating protelns. a presaura head of 38-42 Q. The leading edge of the elution peak wa. clean

step V. Rl-Sepharose chromatonravh - Thhe pooled fractions from OE*E- Sepharose were diluted a. describe3 in Figure 6 and were applied to a cn- Sepharone column equilibrated in buffer B containinn 6.2 H urea and 25 m u NaCl. Thhe column was run with L preasuce head of 39-45 cm. Thhe front edge of the elution peak eometimes contained protein contaminants but the traii- ins edge wae clean. The peak fractions w ~ r e poled, solid Wac1 was added to 1.0 H and the purified gpl wae dialyzed exteneively against 30 mU Trls-HC1 pH7.8 plu. 10 mu 8-mercap~oethanol. I Wi EDTA and 1.0 U NaCl to remove the' urea and renature the protein.

The final yleld wan usually 5-7 ag of B protein from 1 1 liter culture m d the entire purification proeedure took asour R hours.

LOAD 0.8 M NoCl 0.0 M NoCl 7.0 M UREA

E

FRACTION NUMBER

E LOAD 50 NaCl 1 I

OD 1.2 0 - - 1.0

e N

g 0.8 - 2 0.8

- a 0 0.4 u)

- O P - 0- 5 - IO de IL I5

FRACTION NUMBER Flgure 5 DM-Sc broee column fhrmato raph proflle The ooled free- tion. f r a hydroxyfapatlte were applied dfreerry to a 115 I 7.5 an DEAE-

U urea and the B protein warn eluted with 50 mu NaCl in buffer B atill con- sepbrrose colun. m e column m o washed with 9.0 01 buffer B containing 6.2

tdnlng 6.2 N uraa. Practlone containing 2.9 m 1 were collected and ths pooled fractions are Indicated a, solid elrcles.

Flgure 5 DM-Sc broee column fhrmato raph proflle The ooled free- tion. f r a hydroxyfapatlte were applied dfreerry to a 115 I 7.5 an DEAE-

U urea and the B protein warn eluted with 50 mu NaCl in buffer B atill con- sepbrrose colun. m e column m o washed with 9.0 01 buffer B containing 6.2

tdnlnl 6.2 U UIC.. PracCions containin. 2.9 m1 were collected and the

60 mM NoCl 100 mM NoCl

0 1.2

2 0.6

2 0.4 m a 0.2

i i \ - w r 4 16 ;4 ;6

FRACTION NUMBER Figure 6. M-Sepharoae column chromatography profile. Thhe pooled fraction.

U urea and applied to a 1.5 x 6.0 ~ l l W-Sepharoae column. Thhe column was from DMB-Sepharone were added to an equal volume of buffer B containing 6.2

washed with 20 ml of 60 Wi NaC1 in buffer B containing 6.2 u urea. Thhe B protein YOS eluted with 100 mu NaCl in the same urea containing buffer. Fraction* Eontaining 2.9 "1 were collected up t o and includlng the 60 mu NaC1 maah. Thhe fraction size was decreased t o 1.9 m1 for the elution and the pwled fraerlons are indicated by solid circle..

Purification of the Mu B Protein 2669