recombinant dna part h

Contributors to Volume 217 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. GIOVANNA FERRO-Luzz1 AMES (32), De- partment of Molecular and Cell Biology, Division of Biochemistry, University of California, Berkeley, Berkeley, California 94720 SILV1A B~HRING (5), Institutfi~r Molekular- hiologie, Abteilung Molekulare Zellge- netik, D-Ill5 Berlin-Buch, Germany VLADIMIR [. BARANOV (9), RiboGene, Inc., Hayward, California 94545 CARL A. BATT (18), Department of Food Science, Cornell University, Ithaca, New York, 14853 JEAN-PAUL BEHR (41), Laboratoirede Chi. mie Gdndtique, Universitd Louis Pasteur, CNRS URA 1386, F-67401 lllkirch, France MARTIN W. BERCHTOLD (8), lnstitut fiir Pharmakologie und Biochemie, Universitiit Ziirich-lrchel, Ch-8057 Zurich, Switzerland MAX L. BIRNSTIEL (42), Research Institute of Molecular Pathology, A-I030 Vienna, Austria JOHN E. BOYNTON (37), Department of Bot- any, Duke University, Durham, North Carolina 27706 |RENA BRONSTEIN (29), Tropix, Inc., Bed- ford, Massachusetts 01730 LAKI BULUWELA (28), Department of BiD- chemistry, Charing Cross and Westmin- ster Medical School, London W6 8RF, England ZELING CAI (17), Department oflmmunol- ogy, Mayo Clinic, Rochester, Minnesota 55905 CELESTE CANTRELL (31), Department of Pharmacology, University of North Caro- ix lina, Chapel Hill, North Carolina 27599 RICHARD L. CATE (29), Biogen, Inc., Cam- bridge, Massachusetts 02142 KARL X. CHA1 (23), Department qf Bio- chemistry and Molecular Biology, Medi- cal University of South Carolina. Charleston, South Carolina 29425 JULIE CHAO (23), Department of Biochemis- try and Molecular Biology, Medical Uni- versity of South Carolina, Charleston, South Carolina 29425 LEE CHAO (23), Department of Biochemis- try and Molecular Biology, Medical Uni- versity of South Carolina, Charleston. South Carolina 29425 LIN CHEN (7), Department of Chemistry, Harvard University, Cambridge, Massa- chusetts 02138 YUNJE CHO (18), Field of Microbiology. Cornell University, Ithaca, New York 14853 CHRISTOPHER COLECLOUGH (11), Depart- ment of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 MATTHEW COTTEN (42), Research Institute of Molecular Pathology, A-I030 Vienna. Austria RICHARD G. H. COTTON (19), Olive Miller Protein Laborato~, Murdoch Institute. Royal Children's Hospital, Parkville Vic- toria 3052. Australia HENRY DANIELL (38), Department of Bot- any and Microbiology, Auburn Univer- sit3", Auburn, Alabama 36849 BIMALENDU DASMAHAPATRA (10), Depart- ment of Antiviral Chemotherapy, Schering-Plough Research Corporation, Bloomfield, New Jersey 07003 X CONTRIBUTORS TO VOLUME 217 NORMAN DAVIDSON (33), Division of Biol- ogy, California Institute of Technology, Pasadena, California 91125 ANTONIA DESTREE (39), Therion Biologics Corporation, Cambridge, Massachusetts 02142 V. J. DWARK! (43), Vical Inc., San Diego, California 92121 FRITZ ECKSTEIN (13), Abteilung Chemie, Max-Planck-lnstitut fiir Experimentelle Medizin, D-3400 GOttingen, Germany CHRISTIAN W. EHRENFELS (29), Biogen, Inc., Cambridge, Massachusetts 02142 J. VICTOR GARCIA (40), Department of Vi- rology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 NICHOLAS W. GILLHAM (37), Department of Zoology, Duke University, Durham, North Carolina 27706 ALEXANDER N. GLAZER (30), Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, Berke- ley, California 94720 MICHAEL M. GOTTESMAN (4), Laboratory of Cell Biology, National Cancer Insti- tute, National Institutes of Health, Be- thesda, Maryland 20892 RICHARD P. HAUGLAND (30), Molecular Probes, Inc., Eugene, Oregon 97402 STEFEAN N. Ho (17), Department of Pa- thology, Stanford University Medical School, Stanford, California 94305 BERND HOFER (12), Abteilung Mikrobiolo- gie, Gesellschaft far Biotechnologische Forschung, D-3300 Braunschweig, Ger- many CHRISTA HORICKE-GRANDPIERRE (6), Ab- teilung Genetische Grundlagen der Pflan- zenziichtung, Max-Planck-lnstitut fiir Ziichtungsforschung, D-5000 KOIn 30, Germany ROBERT M. HORTON (17), Department of Biochemistry, Gortner Laboratories, Uni- versity of Minnesota, St. Paul, Minnesota 55108 MICKEY C-T. Hu (33), Department of Ex- perimental Hematology, Amgen, Inc., Amgen Center, Thousand Oaks, Califor- nia 91320 TIM C. HUFFArER (21), Section of Bio- chemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853 HENRY D. HUNT (17), Department of Im- munology, Mayo Clinic, Rochester, Min- nesota 55905 ANDREW C. JAMIESON (18), Melvin Calvin Laboratory, University of California, Berkeley, California 94730 R. JILK (22), Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madi- son, Wisconsin 53706 SUSAN E. KANE (4), City of Hope National Medical Center, Duarte, California 91010 PETR KARLOVSKY (24), Institute of Plant Pathology, University of GOttingen, D-3400 Gdttingen, Germany DAVID C. KASLOW (20), Molecular Vaccine Section, Laboratory of Malaria Re- search, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 M. P. KREBS (22), Department of Chemis- try, Massachusetts Institute of Tech- nology, Cambridge, Massachusetts 02139 BIRGIT Kt)HLEIN (12), Max-Planck-lnstitut far Experimentelle Endocrinologie, D-3000 Hannover, Germany ERIC LAI (31), Department of Pharmacol- ogy, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 ANDRE LIEBER (5), Abteilung Molekulare Zellgenetik, lnstitut fiir Moleku- larbiologie, D-1115 Berlin-Buch, Ger- many JEAN-PHILIPPE LOEFFLER (41), lnstitut de Physiologie, CNRS URA 1446, F-67084 Strasbourg, France CONTRIBUTORS TO VOLUME 217 Xi CARMEL M. LYNCH (40), Targeted Genetics Corporation, Seattle, Washington 98101 CHRISTOPH MAAS (6), Abteilung Genetische Grundlagen der Pflanzenziichtung, Max- Planck-lnstitut fiir Ziichtungsforschung, D-5000 KOln 30, Germany KURTIS D. MACFERRIN (7), Department of Chemistry, Harvard University, Cam- bridge, Massachusetts 02138 KAYO MAEDA (1), European Molecular Bi- ology Laboratory, Hamburg Outstation, D-2000 Hamburg, Germany ANNA MASR (39), Integrated Genetics, Inc., Framingham, Massachusetts 01701 J. C. MAKRIS* (22), Lawrence Livermore National Laboratory, Livermore, Cali- fornia, 94551 ROBERT W. MALONE (43), Department of Pathology, University of California, Davis Medical Center, Sacramento, Cal- ifi~rnia, 95817 RICHARD A. MATHIES (30), Department of Chemistry, University of California, Berkeley, Berkeley, California 94720 GAIL P. MAZZARA (39), Therion Biologics Corporation, Cambridge, Massachusetts 02142 A. DUSTY MILLER (40), Program in Molecu- lar Medicine, The Fred Hutchinson Can- cer Research Center, Seattle, Washing- ton 98104 DANIEL G. MILLER (40), Program in Molec- ular Medicine, The Fred Hutchinson Can- cer Research Center, Seattle, Washing- ton 98104 CESAR MILSTEIN (28), Medical Research Council Laboratory of Molecular Biol- ogy, Cambridge CB2 2QH, England OWEN J. MURPHY (29), Tropix, Inc., Bed- ford, Massachusetts 01730 P. L. NORDMANN (22), Department of Mi- crobiology, Biozentrum, University of Ba- sel, CH-4056 Basel, Switzerland DAVID B. OLSEN (13), Merck Sharp and Dohme, Research Laboratories, West Point, Pennsylvania 19486 * Deceased. HENRig 0RUM (2), Department of Biochem- istry B, The Panum Institute, Research Center for Medical Biotechnology, Uni- versity of Copenhagen, DK-2200 Copen- hagen N, Denmark GARY V. PADDOCK (25), Department of Mi- crobiology and Immunology, Medical University of South Carolina, Charleston, South Carolina 29425 R. PADMANABHAN (14), Department of Bio- chemistry and Molecular Biology, Uni- versity of Kansas Medical Center. Kan- sas City, Kansas 66013 THOMAS L. PAULS (8), lnstitutfiir Pharma- kologie und Biochemie, Universitdt Zt~- rich-lrchel, CH-8057 Zurich, Switzerland LARRY R. PEASE (17), Department of Immu- nology, Mayo Clinic', Rochester, Minne- sota 55905 HUNTINGTON POTTER (34), Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115 LAgs K. POULSEN (2), Department of Mi- crobiology, Denmark Technical Univer- sity, DK-2800 Lyngby, Denmark ANNEMARIE POUSTKA (26, 27), lnstitut j'~r Virusforschung, Angewandte Tumorviro- logie, Deutsches Krebsforschungszen- (rum, D-6900 Heidelberg, Germany JEFFREY K. PULLEN (17), Department of Immunology, Mayo Clinic, Rochester. Minnesota 55905 MARK A. QUESADA (30), Department of Chemistry, University of California, Berkeley, Berkeley, California 94720 DAVID J. RAWLINGS (20), Howard Hughes Medical Institute, University of Califor- nia, Los Angeles, Los Angeles, California 90024 W. S. REZNIKOFF (22), Department of Bio- chemistry, College of Agricultural and Life Sciences, University of Wisconsin- Madison, Madison, Wisconsin 53706 J. A. RUSSELL (36), Department of Horticul- tural Sciences, New York State Agricul- xii CONTRIBUTORS TO VOLUME 217 tural Experiment Station, Cornell Univer- sity, Geneva, New York 14456 HAYS S. RYE (30), Department of Molecular and Cell Biology, Division of Biochemis- try and Molecular Biology, University of California, Berkeley, Berkeley, California 94720 JENNIFER A. SALEEBA (19), Department of Biological Science, Dartmouth College, Hanover, New Hampshire 03755 VOLKER SANDIG (5), lnstitutfiir Molekular- biologie, Abteilung Molekulare Zellge- netik, D-1115 Berlin-Buch, Germany J. C. SANFORD (36), Department of Horti- cultural Sciences, New York State Agri- cultural Experiment Station, Cornell Uni- versity, Geneva, New York 14456 JON R. SAYERS (13), School of Biological Science, University of North Wales, Ban- gor, Gwynedd, Wales LL57 2DG JEFF SCHELL (6), Abteilung Genetische Grundlagen der Pflanzenziichtung, Max- Planck-lnstitut fiir Ziichtungsforschung, D-5000 KOln 30, Germany STUART L. SCr~REIRER (7), Department of Chemistry, Harvard University, Cam- bridge, Massachusetts 02138 JAMIE K. SCOTT (15), Division of Biological Sciences, University of Missouri, Colum- bia, Missouri 65211 GEORG SCZAKIEL (1), Angewandte Tumor- virologie, Deutsches Krebsforschungs- zentrum, D-6900 Heidelberg, Germany VENKATAKRISHNA SHYAMALA (32), Chiron Corporation, Emeryville, California 94608 JOHN R. SIMON (35), Department of Biologi- cal Chemistry and Laboratory of Bio- medical & Environmental Sciences, Uni- versity of California School of Medicine, Los Angeles, California 90024 F. D. SMITH (36), Department of Horticul- tural Sciences, New York State Agricul- tural Experiment Station, Cornell Univer- sity, Geneva, New York 14456 GEORGE P. SMITH (15), Division of Biologi- cal Sciences, University of Missouri, Co- lumbia, Missouri 65211 WOLFGANG SOMMER (5), lnstitut far Mole- kularbiologie, Abteilung Molekulare Zellgenetik, D-Ill5 Berlin-Buch, Ger- many ALEXANDER S. SPIRIN (9), Institute of Pro- tein Research, Academy of Sciences, 142292 Pushchino, Moscow Region, Rus- sia HANS-HENNING STEINBISS (6), Abteilung Genetische Grundlagen der Pflanzen- ziichtung, Max-Planck-lnstitut far Ziich- tungsforschung, D-5000 KOln 30, Ger- many MICHAEL STRAUSS (5), Max-Planck Group of the Humboldt University, Max- Delbriick Center for Molecular Medicine, D-I 115 Berlin-Buch, Germany MICHAEL P. TERRANOVA (7), Department of Chemistry, Harvard University, Cam- bridge, Massachusetts 02138 RICHARD TIZARD (29), Biogen, Inc., Cam- bridge, Massachusetts 02142 REINHARD TOPFER (6), Abteilung Genetis- che Grundlagen der Pflanzenziichtung, Max-Planck-lnstitut far Ziichtungsfors- chung, D-5000 KOln 30, Germany SHIGEZO Ut)AKA (3), Department of Food Science and Technology, Faculty of Agri- culture, Nagoya University, Nagoya 464, Japan GREGORY L. VERDINE (7), Department of Chemistry, Harvard University, Cam- bridge, Massachusetts 02138 INDER M. VERMA (43), Molecular Biology and Virology Laboratory, The Salk Insti- tute, San Diego, California 92186 JOHN C. VOYTA (29), Tropix, Inc., Bedford, Massachusetts O1730 ERNST WAGNER (42), Research Institute of Molecular Pathology, A-I030 Vienna, Austria MARY M. Y. WAYE (16), Department of Bio- chemistry, Chinese University of Hong Kong, Hong Kong M. WEINREICH (22), Department of Bio- chemistry, College of Agricultural and Life Sciences, University of Wisconsin- Madison, Madison, Wisconsin 53706 CONTRIBUTORS TO VOLUME 217 Xlll T. WIEGAND (22), Department of Biochem- istry, College of Agricultural and Life Sci- ences, University of Wisconsin-Madison, Madison, Wisconsin 53706 LAI-CHu Wc (28), Davis Medical Center, Departments of Medical Biochemistry and Internal Medicine, The Ohio State University, Columbus, Ohio 43210 HIDEO YAMAGATA (3), Department of Food Science and Technology, Faculty of Agri- culture, Nagoya University, Nagoya 464, Japan C. YUNG YU (28), Departments of Pediat- rics and Medical Microbiology and lm- munology, The Ohio State University and Children's Hospital Research Founda- tion, Columbus, Ohio 43205 STEPHEN YUE (30), Molecular Probes, Inc., Eugene, Oregon 97402 Q X. ZHANG (14), Department of Biochem- istry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66103 L J. ZHAO (14), Department of Biochemis- try and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66103 [1] E. coli EXPRESSION PLASMID pPLEX 3 [1] Vector pPLEX for Expression of Nonfusion Polypeptides in Escherichia coli By GEORG SCZAKIEL and KAYO MAEDA Introduction Escherichia coli bacteria are a powerful tool for the production of heterologous proteins in large quantities, which is of general experimental importance in many fields of natural sciences, for example, in biochemical and biophysical studies. The functional genes coding for polypeptides of interest are introduced stably into E. coli bacteria by E. coli vectors (e.g., plasmids, bacteriophages, cosmids, and phagemids). The expressed polypeptides originate from a unique type of coding DNA and thus, in E. coli from nonspliceable mRNAs, the peptide sequence of expressed molecules is defined exactly, that is, they are monoclonal. For many studies, monoclonal polypeptides are of great advantage in comparison with protein preparations from natural sources, which may consist of numerous closely related but not identical isoforms. Escherichia coli is one of the best studied organisms and many well-established methodolo- gies used in molecular biology can be applied to modify and handle vectors and coding sequences. 1,2 Polypeptides of interest can be expressed in E. coli as fusion proteins, usually extended at the amino terminus with prokaryotic portions intended to provide increased translational initiation, stability, solubility, alterna- tive purification protocols, and yield, or to allow secretion. Fusion proteins can be used for immunological studies, such as the production of antisera, or as antigens in enzyme-linked immunosorbent assay (ELISA) or Western analysis. However, their use in other studies, for example, those concern- ing enzymatic activities and three-dimensional structures, is limited, espe- cially in the latter case, where the expression of nonfusion proteins is desirable. The necessary elements that an expression plasmid should supply are an origin of replication, a dominant selection marker for plasmid propagation and maintenance, and transcriptional (promoter) and transla- L T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 2 F. M. Ausubel, R. Breut, R. E. Kingston, D, D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, "Current Protocols in Molecular Biology." Wiley, New York, 1987. Copyright © 1993 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 217 All rights of reproduction in any form reserved. 4 VECTORS FOR EXPRESSING CLONED GENES [1] tional initiation sites (Shine-Dalgarno sequence and start codon), as well as termination signals for translation and transcription. Transcription di- rected by strong promoters can down-regulate plasmid replication, which may result in the loss ofplasmid. For this reason transcription from strong promoters usually needs to be terminated by efficient transcriptional termi- nators, A number of other parameters for successful expression of heterolo- gous eukaryotic sequences in E. coli must be considered and tested: (1) DNA sequence and primary and secondary structure of the transcript in the vicinity of the start codon, 3 (2) codon usage, 4 (3) possible toxicity of expression products for E. coli, (4) posttranslational modifications, (5) RNA editing of eukaryotic sequences in the homologous system, 5'6 which does not occur in E. coli, and (6) evaluation of the ability of expressed portions of proteins to form defined structures. The techniques for pro- karyotic gene expression have been described in detail. 7 Principle of Method The expression vector pPLEX 8 contains all elements necessary for the expression of open reading frames in E. coli. For transcription the bacterio- phage h-derived strong PL P romoter9 and the t R terminator are used. The PL promoter can be regulated, that is, repressed or induced by the thermolabile h ci857 repressor,10 which is active at the permissive temperature of 28 ° but is inactive at 37 or 42 °. The gene coding for the ci857 repre s sor can be plasmid encoded or can be integrated into the host cell chromosome (e.g., E. coli strain NF 1). The translational control elements, that is, the ribosomal bind- ing site and stop codons in all three reading frames as well as unique cloning sites in between, are indicated in Fig. 1. Materials and Methods Escherichia coli Strains NF1 (K12) AH1 H): F- A(bio- uvrB) lacZam hNam7 Nam53 ci857 AH1 (cro-F-A-J-b2 ) 3 H. A. De Boer and A. S. Hui, this series, Vol. 185, p. 103. 4 p. M. Sharp and W H. Li, Nucleic Acids Res. 15, 1281 (1987). 5 L. Simpson and J. Shaw, Cell 57, 355 (1989). 6 A. M. Weiner and N. Maizels, Cell 61, 917 (1990). 7 D. V. Goeddel, this series, Vol. 185, p. 3. 8 G. Sczakiel, A. Wittinghofer, and J. Tucker, Nucleic" Acids Res. 15, 1878 (1987). 9 E. Remaut, P. Stanssens, and W. Fiers, Gene 15, 81 (1981). l0 M. Lieb, J. Mol. Biol. 16, 149 (1966). ii H U. Bernard, E. Remaut, M. V. Hershfield, H. K. Das, D. R. Helinski, C. Yanowsky, and N. Franklin, Gene 5, 59 (1979). [1] E. coli EXPRESSION PLASMID pPLEX 5 (130) NCOI SalI HindIII HpaI BclI I CCATr~GTC GAC AAG CTT AC;TTAACTOATCA (o) ~ Stul / // Pvu[ / / I- "\ \ \ (3450)"~ ( { pPLEX / /¢~/ PstI l\ \ i fO / / (~ Ms 2 Shine- Datgarno Sequence: ® EcoR 1 GAATTCCGAC TGCGAGCTTA TTGTTAAGGC AATGCAAGGT CTCCTAAAAG ATGGAAACCC GATTCCCTCA GCAATCGCAG CAAACTCCGG CATCTACTAA TAGACGCCGG CCATTCAAAC ATGAGGATTA CCCATGG Nco 1 %t R Sequence: TAAATAACCC CGCTCTTACA CATTCCAGCC CTGAAAAAGG Nsi I GCATCAAATT AAACCACACC TATGGTGTAT GCATTTATTT GCATACATTC AATCAATTGT TATCTAAGGA AATACTTACA TATG FIG. 1. Structure of the E. coli expression plasmid pPLEX and sources of sequence elements: A, MS 2 Shine-Dalgarno (S.D.) sequence [G. Simons, E. Remaut, B. Allet, R. Devos, and W. Fiers, Gene 28, 55 (1984)]; B, htR fragment; C, galactokinase gene [C. Debouck, A. Riccio, D. Schlumperli, K. McKenney, J. Jeffers, C. Hughes, and M. Rosen- berg, Nuclei(" Acids Res. 13, 1841 (1985)]; D, fragment from pPLc245 containing the ,kpL promoter [E. Remaut, P. Stanssens, and W. Fiers, Nucleic Acids Res. 11, 4677 (1983)]. Note that BclI is sensitive to Dam methylation. In order to use the BclI site pPLEX must be grown in a dam- E. coli strain. An additional AccI site located on the pBR322 sequence that is present in the original plasmid pPLEX but was filled in with Klenow fragment and nucleotide triphosphates, that is, it was destroyed in pPLEXAcc • (J. Tucker, unpublished observations, 1986.) 6 VECTORS FOR EXPRESSING CLONED GENES [1] W6 (origin not known): su-, cI (wild type) unc195912: lacI Q lacL8 thr-1 ara-14 leuB6 A(gpt-proA) 62 lacY1 1on-22 supE44 galK2 h- sulA27 hisG4 rpsL31 xyl-5 mtl-1 thi-1 Cloning Methods of recombinant DNA technology are essentially performed following the protocols of Maniatis et al.1 For cloning pPLEX-derived constructs we use E. coli strain W6, containing the h wild-type cI repressor integrated into its chromosome. The wild-type repressor is able to shut off the PL promoter efficiently, thus allowing stable replication and high copy numbers of recombinant pPLEX-derived constructs. In principle an E. coli strain harboring the thermolabile ci857 repressor is also suitable at the permissive temperature of 28°; however, the clearly decreased growth rate at this temperature seems to be a disadvantage. For induction of the PL promoter, E. coli host strains NFI and unc1959, both containing a cI857-carrying plasmid, are used. Transformation of E. coli cells is performed following the CaCI2 method 13 for W6 and NF1 or the protocol developed by Hanahan 14 for DH2/6. The transformation yields for 1 /xg of pPLEX DNA with freshly prepared bacteria are in the range of 5 × 105 for W6 and 1 × 106 for NF1. The transformation frequency after storage of transformation-competent cells in 5% (v/v) glycerol at -70 ° is decreased by a factor of approximately 10. Induction of hPL Promoter The protocol for the induction of the hPL promoter of E. coli strains carrying pPLEX constructs is depicted schematically in Fig. 2. As an alternative way of induction of the LMM expression plasmid pEXLMM74 a temperature shift to 42 ° may be performed for 15 min with subsequent incubation at 37 ° for 4 hr. To raise the temperature quickly to 42 ° for large volumes (e.g., 10 liter), an appropriate amount of fresh medium preheated to 60 ° is added. On induction, suppression of the htR terminator results in transcription of a bicistronic mRNA consisting of the heterologous open reading frame and the coding sequence for galactokinase. Thus, an in- crease in galactokinase activity monitors efficient hpL-directed tran- scription. 12 Obtained from B. Bachman, E. coli Genetic Stock Centre, New Haven, Connecticut. I3 M. Mandel and A. Higa, J. Mol. Biol. 53~ 159 (1970). 14 D. Hanahan, J. Mol. Biol. 166, 557 (1983). [1] E. coli EXPRESSION PLASMID pPLEX 7 Grow 1 ml overnight culture of E. coli strain NF1 transformed with pPLEX construct in medium (standard I or L-broth supplemented with 100 p.g/ml ampiciUin) at 28 ° $ Inoculate 1 ml of fresh medium with 10/xl of dense overnight culture Incubate for 1 hr at 28 ° $ Divide culture in two 0.5-ml aliquots / \ 4 hr, 28 ° 4 hr, 28 ° (uninduced control) (induced control) l 1 Protein analysis Protein analysis FIG. 2. Protocol for the induction of the PL promoter-driven expression cassette of pPLEX. In analysis of expression products by SDS-polyacrylamide gel electrophoresis induced cultures have higher cell densities, i.e., protein concentrations, than do control cultures grown at 28 ° . Analysis of Expression Products Soluble Protein Fraction. Escherichia coti cells are harvested by cen- trifugation (30 sec, room temperature, 7000 rpm, Eppendorf centrifuge) and the cell pellet is resuspended with 1 ml 50 mM Tris-HCl (pH 7.4). After centrifugation the pellet is resuspended vigorously in 0.5 ml lysis buffer containing 50 mM Tris-HCl (pH 7.4), 0.5 mM dithioerythritol (DTE), 0. I mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM ethyl- enediaminetetraacetic acid (EDTA). Lysozyme (3/zl, 10 mg/ml in 10 mM Tris-HCl pH 8.0, 1 mM EDTA) is added and the mixture is maintained for 10 to 20 min at room temperature. Sodium deoxycholate (3/zl, 40 mg/ml in water) is added and the solution is kept for 15 min at room temperature. After centrifugation (15 min, 4 °, Eppendorf centrifuge) soluble proteins are contained in the clear supernatant. Sodium Dodecyl Sulfate-Soluble Proteins. Escherichia coli cells are spun down by centrifugation (30 sec, room temperature, 7000 rpm, Eppendorf centrifuge), the cell pellet is resuspended once with 1 ml of 50 mM Tris-HC1 (pH 7.4), and cells are centrifuged again (30 sec, room [...]... may result from the digestion depending on whether target sequences for the enzymes are present in the insert or not) Those vectors that contain the correct DNA insert are then analyzed to distinguish between the possibility that translation initiates at the lacZ translation start site or within the insert The rationale behind this analysis is shown schematically in Fig 3 First, the selected vectors... in plFF8 has a more pronounced effect on the Lac ÷ phenotype Consistent with this notion, insertion of a 1.6-kb pal cDNA fragment in plFF8 produced light blue colonies that, however, turned deep blue when the lacZ t~ gene was substituted by the entire lacZ gene This suggests that the functional limits of plFF8 can be expanded by insertion of the entire lacZ gene On the other hand, when using the indirect... requires that the lacZ-encoded part of the fusion protein retain enzymatic activity In plFF8, the foreign DNA is inserted into the small lacZ ~ gene, which must successfully complement the host encoded product of the lacZ AM 15 gene to produce the Lac ÷ phenotype Thus, compared to other ORF vectors that usually carry the entire lacZ gene, it may be expected that insertion of foreign ORF DNA fragments... destroys the lacZ reading frame, allowing the desired recombinant to be selected by its L a c - phenotype Second, purified vector from the selected L a c - clone is cleaved with BssHII, extracted with phenol and chloroform, precipitated with ethanol, and religated using T4 DNA ligase (Section 2, procedure 2.3) This ligation shuffles the BssHIIexcised insert/vector fragments, with the result that a subset... important that the vector sample is totally cleaved at this step because residual uncleaved pIFF8 will give rise to false positives in subsequent transformation/plating (both the desired recombinant pIFF8 vector as well as the pIFF8 vector itself have a Lac + phenotype) After cleavage with BssHII, the vector is treated with calf intestinal phosphatase (CIP) to remove the terminal 5'-phosphate groups This... gel The purified fragment was then dephosphorylated with calf intestinal phosphatase, labeled with [7-32p]ATP and T4 DNA kinase, and digested with PstI, which cleaves the labeled fragment into two unequal halves Finally, the labeled products were separated by electrophoresis in a polyacrylamide sequencing gel and autoradiographed Two sets of bands corresponding to cleavage at all BssHII sites in the... vectors utilize the fact that the lacZ gene-encoded fl-galactosidase enzyme is usually active when an additional polypeptide is inserted near its N terminus Thus, when an open reading frame DNA fragment is inserted near the 5' end of the lacZ gene, the correct fusion (a tripartite gene) will have a Lac ÷ phenotype whereas the incorrect fusions will be L a c - To confer the Lac ÷ phenotype, the DNA insert... that the inserted DNA contains an ORF, the first step is to prepare a vector minipreparation from each of the selected clones For this we use the alkaline lysis method, which is both rapid and reliable 7 Next, purified vectors are digested with restriction enzymes followed by electrophoresis in agarose or polyacrylamide gels to determine which of the vectors contain the correct insert Digestion with... the logarithmic phase of growth form hexagonal arrays on the cell surface During the early stationary phase of growth, the protein layers begin shedding concomitantly with a prominent increase in protein secretion 5 During the stationary growth phase, cells continue to synthesize and secrete the cell wall proteins These proteins do not stay on the cell surface, but instead accumulate in the medium as... The DNA having the nucleotide sequence shown is inserted between the NruI and HindlII sites of pBR322 The sequence from the 5' terminus to the Pst! site encodes the COOH-terminal portion of the MWP signal peptide Downstream from the cleavage site of the signal peptide is a multicloning site (MCS), the same as that inserted in pNU210 (Fig 1) The ampicillin resistance gene on pBR322 was replaced by that . near the 5' end of the lacZ gene, the correct fusion (a tripartite gene) will have a Lac ÷ phenotype whereas the incorrect fusions will be Lac To confer the Lac ÷ phenotype, the DNA insert. well as the pIFF8 vector itself have a Lac + phenotype). After cleavage with BssHII, the vector is treated with calf intestinal phosphatase (CIP) to remove the terminal 5'-phosphate groups This ligation shuffles the BssHII- excised insert/vector fragments, with the result that a subset of inserts are brought in frame with the lacZ gene. Clones containing these vectors can then