Summary
In order to produce tuna (Thunnus thynnus) growth hormone (GH), expression plasmid (pUES13S) carrying tuna GH cDNA was constructed using a vector (pKK223-3), in which the replication origin was replaced with that of pUC19. The expression of the tuna GH cDNA was greatly affected by the distance between a Shine-Dalgarno (SD) sequence and the initiation codon (ATG) and was most efficient when the distance was adjusted to 13 base pairs (bp). The amount of tuna GH produced by Escherichia coli JM109 with pUES13S was more than 12.5% of the total cytosolic proteins and the product was immunologically identified to be tuna GH (mol. wt. 21 000) by Western blot analysis using tuna GH specific immunoglobulin G (IgG). Another plasmid (pUES13S-2) containing tandemly polymerized tuna GH cDNA was constructed, to improve the productivity of tuna GH. When E. coli JM109 carrying pUES13S-2 was incubated at 40°C, the amount of tuna GH produced reached about 20% of the total cytosolic proteins.
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References
Amann E, Brosius J (1985) ‘ATG vectors’ for regulated high-level expression of cloned genes in Escherichia coli. Gene 40:183–190
Appleyard RK (1954) Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12. Genetics 39:440–445
Beisiegel V, Schneider WJ, Brown MS, Goldstein JL (1982) Immunoblot analysis of low density lipoprotein receptors in fibroblasts from subjects with familial hypercholesterolemia. J Biol Chem 257:13150–13156
Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472
Brent R, Ptashne M (1981) Mechanism of action of the lexA gene product. Proc Natl Acad Sci USA 78:4204–4208
Brosius J, Holy (1984) Regulation of ribosomal RNA promoters with a synthetic lac operator. Proc Natl Acad Sci USA 81:6929–6933
Gheysen D, Iserentant D, Derom C, Fiers W (1982) Systematic alteration of the nucleotide sequence preceding the translation initiation codon and the effects on bacterial expression of the cloned SV40 small-t antigen gene. Gene 17:55–63
Hanahan D (1983) Study on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Low B (1968) Formation of merodiploids in matings with a class of Rec- recipient strains of Escherichia coli K-12. Proc Natl Acad Sci USA 60:160–167
Messing J, Crea R, Seeburg PH (1981) A system for shotgun DNA sequencing. Nucleic Acids Res 9:309–321
Miki T, Yasukochi T, Nagatani H, Furuno M, Orita T, Yamada H, Imoto T, Horiuchi T (1987) Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: an application to overproduction of chicken lysozyme. Protein Eng 1:327–332
Nagao M, Nakagawa S, Ishii A, Sasaki R, Tanaka H, Chiba H (1988) Expression of bovine 158-1 cDNA in Escherichia coli. Agric Biol Chem 52:191–200
Saito A, Sekine S, Okada Y, Saito M, Ito S, Hirano T (1985) Molecular cloning and expression of eel growth hormone cDNA, in Abstract of the 8th International Congress on Molecular Biology held at Tokyo, Japan (ed: The Molecular Biology Society of Japan); p 177
Sancer A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sato N, Watanabe K, Murata K, Sakaguchi M, Karyiya Y, Kimura S, Nonaka M, Kimura A (1988a) Molecular cloning and nucleotide sequence of tuna growth hormone cDNA. Biochim Biophys Acta 949:35–42
Sato N, Murata K, Watanabe K, Hayami T, Kariya U, Sakaguchi M, Kimura S, Nonaka M, Kimura A (1988b) Growth promoting activity of tuna growth hormone and expression of tuna growth hormone cDNA in Escherichia coli. Biotechnol Appl Biochem 10:385–393
Sekine S, Mizukami T, Nishi T, Kuwana Y, Saito A, Sato M, Ito S, Kawauchi H (1985) Cloning and expression of cDNA for salmon growth hormone in Escherichia coli. Proc Natl Acad Sci USA 82:4306–4310
Shine J, Dalgarno L (1975) Determinant of cistron specificity in bacterial ribosomes. Nature 254:34–38
Twigg AJ, Sherratt D (1980) Trans-complementable copy-number mutants of plasmid ColE1. Nature 283:216–218
Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Watabiki N, Tanaka M, Masuda N, Yamakawa M, Yoneda Y, Nakajima K (1988) cDNA cloning and primary structure of yellow tail (Seriora quinqueradiata) pregrowth hormone. Gen Comp Endocrinol 70:401–406
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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Sato, N., Hayami, T., Murata, K. et al. Expression of tuna growth hormone cDNA in Escherichia coli . Appl Microbiol Biotechnol 30, 153–159 (1989). https://doi.org/10.1007/BF00264004
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DOI: https://doi.org/10.1007/BF00264004