Table 11-3A. Synthetic Genes. Genes from a Single Cloned Synthetic Fragment
| Gene | Lenght of separately cloned synthetic fragment (b-p.) | Design of gene sequence (selection of codons; design of synthetic fragment ends; restriction sites (RS) | Length of synthetic oligonucleotides necessary to design the gene; method of their chemical synthesis and purification | Method of obtaining DNA fragment from synthetic oligonucleotides for cloning | Cloning vector |
Expression vector; regulatory portion of the gene and yield of expression product | Reference |
| Somatostatin | 56 | Initiation and stop codons; sticky ends for cloning at different RS | 8 oligonucleotides 10-15 b.p. long; synthesis by phosphotriester method in solution | Two-step ligation: I. Assembly of 2 DNA blocks from 4 oligonucleotides; II. Assembly of target DNA duplex from 2 blocks | pBR322 | pBR322; lac-promoter; yield of chimeric protein with long b-galactosidase fragment - 0.03 % of total E. coli cell protein | Itakura K., Hirose T. et al. Science, 1977, 192, pp. 10561063 |
| a1-Thymosine | 98 | Codons often encountered in highly expressed E. coli genes; sticky ends for cloning at different RS | 16 oligonucleotides 10-15 b.p. long; synthesis by phosphotriester method in solution; separation by anion-exchange HPLC and PAGE in 7 M urea | Two-step ligation: I. Assembly of 4 DNA blocks from 4 oligonucleotides; II. Assembly of target DNA duplex from 4 blocks | pBR322 | pBR322; lacUVS-promoter; yield of chimeric protein with long P-galactosidase fragment - 294 mg per 24 g of E. coli; 4.5 mg of al-thymosine after BrCN hydrolysis | Wetzel R., Heyneker H. L. Biochemistry, 1980, 19, pp. 6096-6104 |
| Human insulin, chains A and B |
I 77 II 104 |
Initiation and double stop codons; sticky ends for cloning at different RS | 12 and 18 oligonucleotides 10-15 b.p. long; synthesis by phosphotriester method in solution; separation by anion-exchange HPLC | Two-step ligation: I. Assembly of 5 DNA blocks from 5-8 oligonucleotides; II. Assembly of 2 target DNA duplexes from 2-3 blocks | pBR322 | pBR322; lac-promoter; yield of chimeric proteins with long P-galactosidase fragment - 10 mg per 24 g of wet E. coli cells | Goeddel O. V, Kleid O. G. et al. Proc. Natl. Acad. Sci. USA, 1979, 79, pp. 106-110 |
| Salmon calcitonin-33Gly | 113 | Codons often encountered in highly expressed E. coli genes; sticky ends for cloning at different RS; unique RS in the middle of gene for subsequent cassette mutagenesis | 2 oligonucleotides 109 and 117 b.p. long; synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | Hybridization of 2 extralong oligonucleotides by formation of DNA duplex with sticky ends | M13mp 18am |
ND | Hein E, Jansen H. W, Uhlmann E., Nucleotides, 1988, 7, pp. 497-510 |
| Salmon calcitonin-33Gly | 113 | One oligonucleotide 140 b.p. long; synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | 3'-terminal part of oligonucleotide is self-complementary, addition of polymerase to complementary chain and treatment with restriction endonuclease to form sticky ends | M13mp 18am |
ND | Uhlmann E., Hein E, Nucl. Acids Symp. Ser, 1987, 18, pp. 237-240 | |
| b-Urogastron | 170 | Codons of highly expressed yeast genes; initiation and double stop codons; sticky ends for cloning at different RS | 16 oligonucleotides 12-33 b.p. long; synthesis by phosphoramidite method; separation by PAGE in 7 M urea | Two-step ligation: I. Assembly of 3 DNA blocks from 3-7 oligonucleotides; II. Assembly of target DNA duplex from 3 blocks | pBR328 | pJDB219; GAPDH-promoter; b-urogastrone yield - 30 mg per liter of yeast culture at a culture density of 2 650O.U./ml | Urdea M. S., Merryweather J. P. et al., Proc. Natl. Acad. Sci. USA, 1983,80,pp. 7461-7465 |
| Pancreatic secretory inhibitor of human trypsin | 184 | Cloning with 3-sticky and 5-blunt ends | 25 oligonucleotides 7-16 b.p. long; synthesis by phosphotriester method on segments; separation by PAGE in 7 M urea or TLC on cellulose | Two-step ligation: I. Assembly of 2 DNA blocks from 11 and 13 oligonucleotides; II. Assembly of target DNA duplex from 2 blocks | pGV451, pUC8 |
pIN-III-ompA; IpplacZpo-promoter; leader sequence of protein from outer membrane of E. coli, yield of properly processed peptide - 2.5-10 mg/ml of E. coli supernatant | Maywald E, Böldicke T et al. Gene, 1988, 68, pp. 357-369 |
| Tyr-tRNASup | 200 | Natural sequence; sticky ends for cloning at one RS | 39 oligonucleotides 4-13 b.p. long; synthesis by phosphodiester method in solution; separation by anion-exchange chromatography | Multiple-step ligation: I. Assembly of 7 DNA blocks from 3-7 oligonucleotides; II. Step-by-step assembly of target duplex from DNA blocks | F-80 | Bacteriophage; synthetic gene suppressed amber mutation | Khorana H. G. Science, 1979, 203, pp. 614-624 |
| Somatomedin-C- C-L0-51 Thr |
226 | Codons often encountered in highly expressed E. coli genes; Thr is substituted for 51Met to separate somatomedin from BrCN leader sequence | 18 oligonucleotides 16-32 b.p. long; synthesis by solid-phase phosphoramidite method; separation by PAGE in 7 M urea | One-step ligation; assembly of target fragment from 9 DNA duplexes with sticky ends (from 9 oligonucleotide pairs) | M13mp9 | pCFM414; trp-promoter; leader sequence (8 aa)-N-end of b-galactosidase | Peters M. A., Lau E. P. et al Gene, 1985, 35, pp. 83-89 |
| Somatomedin-C | 234 | Codons often encountered in highly expressed E. coli genes; sticky ends for cloning at different RS | 23 oligonucleotides 10-23 b.p. long; synthesis by solid-phase phosphoramidite method; separation by anion-exchange HPLC | One-step ligation; assembly of target duplex from 23 oligonucleotides | M13mp8 | ND | Sproat B. S., Gait M. J. Nucl. Acids Res., 1985, 15, pp. 2959-2977 |
| Cow colostrum trypsin inhibitor | 239 | HindIII CP at 5'end of gene for solid-phase ligation of synthetic gene to vector; polylinker at 3' end for removal of gene ligated to vector from support | 19 oligonucleotides 28-36 b.p. long; synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | Solid-phase assembly; multiple step-by-step extension of DNA duplex by ligation of hybridized oligonucleotides (3-4) to an oligonucleonucleotide immobilized on polymer support. At the last step, a vector linearized by appropriate restrictases is ligated to the resulting DNA duplex | M13mp8 | ND | Hostomsky Z., Smrt J. Nucl. Acids Res., 1987, 15, pp. 4849-4856 |
| Ubiquitin | 240 | 16 nucleotide substitutions in natural sequence to create RS dividing gene into 8 modules for subsequent cassette mutagenesis; sticky ends for cloning at different RS | 8 oligonucleotides 50-64 b.p. long; synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | One-step ligation; assembly of target DNA duplex from 4 oligonueleotide pairs | pUC | pM627N-S; pl-promoter; yield - 10 - 15 % of total E. coli protein | Ecker D. J., Butt T.R. et al. J. Biol. Chem., 1987, 262, pp. 3524-3527, 14213-14221 |
| Neutrophileac- tivating factor |
244 | Natural sequence, sticky ends for cloning at different RS; RS for subsequent cassette mutagenesis of 5' end | 6 oligonucleotides 7-83 b.p. long; synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | One-step ligation; assembly of target DNA duplex from 3 oligonucleotide pairs | pBSM13 | pIL402; trp-promoter; isolation of NAF from 516 g of E. coli; quantity not specified | Lindley I., Aschauer H. et al. Proc. Natl. Acad. Sci. USA, 1989, 85, pp. 9199-9203 |
| Transactivating protein of human immunodefi- ciency virus |
287 | Preferential codons either in E. coli or in yeast; sticky ends for cloning at different RS; some RS are included for cassette mutagenesis; inverted repeats and other secondary structure elements in gene are excluded | 2 oligonucleotides 139 and 144 b.p. long and 3 oligonucleotides (11, 14 and 16 b.p.); synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | In vivo repair of gap-duplex resulting from ligation of 2 long oligonucleotides to vector using 3 additional template oligonucleotides | pUC18 | pKV461; hCMW-promoter; splicing and polyadenylation signals from SV40; gene biologically active in HeLa cells | Adams S. E., Johnson, I. 0. et al Nucl. Acids Res., 1988, 16, pp. 4287-4298 |
| Human lysozyme | 418 | Codons often encountered in highly expressed yeast genes; Shine-Dalgarno sequence | 56 oligonucleotides 11-15 b.p. long; synthesis by solid-phase phosphotriester block method; separation by reversed phase HPLC | Three-step ligation; I. Assembly of 9 DNA blocks from 4-9 oligonucleotides; II. Assembly of 3 gene parts from DNA blocks; III. Assembly of target DNA duplex | pBR322 | pMY12-6; pL-promoter; yield of lysozyme with N-terminal Met - a few per cent of total E. coli cell protein | Muraki M., Jigami Y. et al. Agric. Biol. Chem., 1986, 50, pp. 713-723 |
| Interferon | 453 | Codons for Arg-CGT and Leu-CI'G; sticky ends for cloning at different RS | 66 oligonucleotides 12-16 b.p. long; synthesis by solid-phase chlorophosphite method; separation by anion-exchange and reversed phase HPLC | Two-step ligation; 1. Assembly of 6 DNA blocks from 9-14 oligonucleotides; II. Assembly of target DNA duplex from 6 DNA blocks | pBR322 | pBR322; bla- and T5--promoter; yield - 16.3 % of total E. coli cell protein | Jay E., Rommens J. et al. Proc. Natl. Acad. Sci. USA, 1984, 81, pp. 2290-2294 |
| Bovine pancreatic phospholipase A2 (PLA2) | 456 | Codons of highly expressed yeast genes; sticky ends for cloning at different RS | 22 oligonucleotides 33-48 b.p. long; synthesis by phosphoramidite method on automatic synthesizer; separation by reversed phase HPLC and PAGE in 7 M urea | Two-step ligation; 1. Assembly of 3 DNA blocks from 3-4 oligonucleotide pairs; 11. Assembly of target DNA duplex from 3 DNA blocks | pUC119 | pAT405; PHO5-promoter; yield - 2.8 mg/ml of yeast supernatant of active form of PLA2 | Tanaka T., Kimuro S., Ota Y Gene, 1989, 64, pp. 257-264 |
| Interferon-a2 | 511 | Codons often encountered in highly expressed E. coli genes; 20 % nucleotide substitutions, as compared to natural gene; sticky ends for cloning at different RS | 68 oligonucleotides 10-16 b.p. long; synthesis by solid-phase phosphotriester method; separation by anion-exchange HPLC | Three-step ligation; I. Assembly of 13 DNA blocks from 5-8 oligonucleotides; II. Assembly of 3 gene parts from 4-5 DNA blocks; III. Assembly of target duplex from 3 parts | pPM60 | pAT153; trp-promoter; yield - 5 x 107 units of IFN-a2 per liter of E. coli culture at a culture density of 1 O.U./ml | Edge M. D., Greene A. R. et al. Nucl. Acids Res., 1983, 11, pp. 6419-6435 |
Go to Table 11-3B Synthetic Genes. Genes from Several Subcloned Synthetic Fragments
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