Summary
A specific 1,596 bp HincII fragment ('skc) from the chromosome of Streptococcus equisimilis contains an active streptokinase (SK) gene (skc) lacking, in addition to the expression signals, codons 1 through 39 of wild-type skc but retaining the remainder of the skc coding sequence together with the transcription terminator. Using this fragment as an indicator gene, we constructed two types of vectors which in appropriate hosts resulted in the synthesis of SK fusion proteins after insertional activation of 'skc. The first type are open reading frame (ORF) vectors in which 'skc was inserted into pUC18 out of frame with respect to lacZ' expression signals and 'skc such that the skc reading frame was restored resulted in the production of tripartite proteins which exhibited SK activity. The second type of vector, which functioned in both gram-positive and gram-negative bacteria, used the streptococcal speA expression and secretion signals in front of the ORF to activate 'skc insertionally. Using a large fragment from the chymosin gene as the target sequence, the usefulness of these vectors for studying foreign gene expression in streptococci as well as Escherichia coli was demonstrated.
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References
Amourache L, Vijayalakshmi MA (1984) Affinity chromatography of kid chymosin on histidyl-Sepharose. J Chromatogr 303:285–290
Berman ML (1983) Vectors for constructing hybrid genes. Bio Techniques 1:178–183
Bode W, Huber R (1976) Induction of the bovine trypsinogentrypsin transition by peptides sequentially similar to the N-terminus of trypsin. FEBS Lett 68:231–236
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Friedrich KH, Lütticken R (1984) A rapid screening method for streptococcal plasmids yielding plasmid DNA suitable for restriction enzyme analysis. FEMS Lett 25:183–186
Gasson MJ (1983) Plasmid complements of Streptococcus lactis NCD0712 and other lactic streptococci after protoplast curing. J Bacteriol 154:1–9
Gerlach D, Köhler W, Knoll H, Moravek L, Weeks C, Ferretti JJ (1987) Purification and characterization of Streptococcus pyogenes erythrogenic toxin type A produced by a cloned gene in Streptococcus sanguis. Zentralbl Bakteriol IA 266:347–358
Harris TJR, Lowe PA, Thomas PG, Eaton MAW, Millican TA, Patel TP, Bose CC, Carey NH, Doel MT (1982) Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin. Nucleic Acids Res 10:2177–2187
Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197
Johnson DA, Gantsch JW, Sportsman RK, Elder JH (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Techn 1:3–8
Koenen M, Griesser HW, Müller-Hill B (1985) Immunological detection of chimeric beta-galactosidases expressed by plasmid vectors. In: Glover DM (ed) DNA cloning: a practical approach, vol I. IRL Press, Oxford, Washington DC, pp 89–100
Kok J, van Dijl JM, van der Vossen JMBM, Venema G (1985) Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis. Appl Environ Microbiol 50:94–101
Kyhse-Andersen J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10:203–209
Laemmli MK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Laplace F, Klessen C, Malke H (1987) pLKM6181. In: Ferretti JJ, Curtiss III R (eds) Streptococcal genetics. Am Soc Microbiol. Washington DC, p 271
LeBlanc DJ, Hassell FP (1976) Transformation of Streptococcus sanguis by plasmid deoxyribonucleic acid from Streptococcus faecalis. J Bacteriol 128:347–355
Lennox ES (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190–206
Liebscher DH, Lipoldova M, Smrt J, Schwertner S, Wambut R, Speter W, Pohlmann C, Pfeifer M, Hahn V, Rapoport TA, Rosenthal S, Zadrazil Z (1985) Molecular cloning and identification of cDNA recombinants coding for bovine prochymosin in E. coli. Folia Biol 31:81–92
Malke H (1986) Codon usage in streptococci. J Basic Microbiol 26:587–595
Malke H, Ferretti JJ (1984) Streptokinase: cloning, expression, and excretion by Escherichia coli. Proc Natl Acad Sci USA 81:3557–3561
Malke H, Roe B, Ferretti JJ (1985) Nucleotide sequence of the streptokinase gene from Streptococcus equisimilis H46A. Gene 34:357–362
Malke H, Lorenz D, Ferretti JJ (1987) Streptokinase: expression of altered forms. In: Ferretti JJ, Curtiss III R (eds) Streptococcal genetics. Am Soc Microbiol, Washington DC, pp 143–149
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Schmidt KH, Schleenvoigt G, Köhler W (1987) Extraction of group C streptococcal IgG-binding receptor and characterization of the active peptides by Western blotting and isoelectric focusing. Zentralbl Bakteriol IA 265:430–438
Silhavy TJ, Beckwith JR (1985) Uses of lac fusions for the study of biological problems. Microbiol Rev 49:398–418
Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Terzaghi BE, Sandine WE (1975) Impoved medium for lactic streptococci and their bacteriophages. Appl Microbiol 29:807–813
Towbin H, Gordon J (1984) Immunoblotting and dot immunoblotting —current status and outlook. J Immunol Methods 72:313–340
Vieira J, Messing J (1982) The pUC plasmids, and M13mp7-derived systems for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Weaver KE, Clewell DB (1987) Transposon Tn917 delivery vectors for mutagenesis in Streptococcus faecalis. In: Ferretti JJ, Curtiss III R (eds) Streptococcal genetics. Am Soc Microbiol, Washington DC, pp 17–21
Weeks CR, Ferretti JJ (1984) The gene for type A streptococcal exotoxin (erythrogenic toxin) is located in bacteriophage T12. Infect Immun 46:531–536
Weeks CR, Ferretti JJ (1986) Nucleotide sequence of the type A streptococcal exotoxin (erythrogenic toxin) gene from Streptococcus pyogenes bacteriophage T12. Infect Immun 52:144–150
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
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Klessen, C., Schmidt, K.H., Ferretti, J.J. et al. Tripartite streptokinase gene fusion vectors for gram-positive and gram-negative procaryotes. Mol Gen Genet 212, 295–300 (1988). https://doi.org/10.1007/BF00334699
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DOI: https://doi.org/10.1007/BF00334699