Table 11-3B. Synthetic Genes. Genes from Several Subcloned Synthetic Fragments
| Gene | Total gene length (b.p.) | 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 |
| b-Urogastrone | 174 | A.87 B.87 |
Codons often encountered in highly expressed E. coli genes | 23 oligonucleotides 11-16 b.p. long; synthesis by phosphotriester method in solution | A. Four-step ligation and pAT153 treatment of resulting dimer of fragment A with restrictase. B. Three-step ligation and treatment of resulting dimer of fragment B with restrictase | pAT153 pUB110 |
trp-promoter; fragment trpE (7 aa of N end); chimeric product yield - 2-3 mg/l of E. coli culture | Smith, J., Cook, E. et al Nucl. Acids Res., 1982, 10, pp. 4467-4482 |
| Artificial protein | 190 | A.83 B.93 C.64 |
Codons often encountered in highly expressed E. coli genes; RS flanked at synthetic fragment ends by additional G/C pairs, permitting subcloned fragments to be joined after treatment with appropriate restrictases and cloning at different RS; verification of hybridization of oligonucleotides before polymerase reaction | 6 oligonucleotides 38-53 b.p.long; synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | A,B,C. Addition of Polkl to duplex made up of 2 partially complementary oligonucleotides, treatment with restrictases to produce necessary sticky ends for ligation into vector | pKK233-2 | pGF211N; tac- and bla-promoters; pB1052; tac-promoter. atpE gene translation initiation region | Biernat J., Köster H. Protein Eng., 1987, 1, pp. 345-351,353-358 |
| Chirudin | 226 | A.99 B.70 C.84 |
Shine-Dalgarno sequence, initiation and stop codons; unique RS to cut out and join subcloned fragments | 3 oligonucleotides 49, 60 and 84 b.p. long, 11 oligonucleotides 12-31 b.p. long; synthesis by phosphoramidite method on automatic synthesizer (long nucleotides) and manually (short ones); separation by PAGE in 7 M urea and reversed phase HPLC | A. Addtion of AMV revertase to duplex made up of pVH27 two partially complementary oligonucleotides, cloning with the aid of linkers B,C.One-step ligation | pUC18 pVH27 |
pWH701, pMR3; pL-promoter; yield - 28 ng per 1 578O.U. of E. coli cells | Bergmann C., Dodt J. et al. Biol. Chem. Hoppe-Seyler, 1986 367, pp. 731-740 |
| Eglin C | 232 | A.123 B.106 |
Codons often encountered in highly expressed E. coli genes; initiation and stop codons; RS flanked by additional b.p. at 5'end of fragment A and 3' end of fragment B, RS to cut out and join fragments A and B | 6 oligonucleotides 34-61 b.p. long; synthesis by solid-phase method; manually; separation by reversed phase HPLC and PAGE in 7 M urea | A. Addition of PolkI to 2 duplexes made up of 2 pairs of partially complementary oligonucleotides, ligation of resulting duplexes at blunt ends. B. Addition of PolkI to duplex made up of two partially complementary oligonucleotides | pBR322 | pBR322; trp-promoter; yield- 3 x 105 molecules per E. coli cell | Rink H., Liersch M. et al. Nucl. Acids Res., 1984, 12, pp. 6369-6387 |
| Third factor of human anaphylatoxin complement system C3a |
263 | A.89 B.88 C.96 |
Codons often encountered in highly expressed E. coli genes, as many unique RS as possible; lac-gene ribosome binding site, initiation and stop codons | 3 oligonucleotides 122-125 b.p. long; synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | Three-step bridge mutagenesis: in each step, vector is linearized by Bsml restrictase and hybridized with a synthetic oligonucleotide whose ends are complementary to those of vector (18 b.p.) and transform E. coli without any manipulations in vitro | pUC9 | ND | Hayden M. A., Mandecki W DNA, 1988, 7, pp. 571-577 |
| Transforming rat growth factor (TGF-a) | 267 | A.140 B.60 C.120 |
Preferable codons for higher eucaryotes. gene sequence for preTGF-a |
16 oligonucleotides 22-40 b.p. long; synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | A. Addition of Polkl to 2 duplexes made up of
2 pairs of partially complementary oligonucleotides, ligation of resulting duplexes at
blunt ends B. 4 inserts each 14-16 b.p. long, by site-specific mutagenesis, into cloned
sequence of fragment A C. One-step ligation of 4 pairs of complementary oligonucleotides |
M13mp18 | pSW272 (retrovirus vector); yield - 0.3 ng/1 ml of medium | Watanabe S., Lazar, E., Sporn M. B. Proc. Natl. Acad. Sci. USA, 1987, 84, pp.1258-1262 |
| Human proinsulin | 277 | A.140 B.63 |
Rat proinsulin codons, initiation and stop codons, fragment A; sticky ends for cloning at different RS, fragment B; cloning with the aid of MboII adapter | 42 oligonucleotides 11-17 b.p. long; synthesis by phosphotriester method in solution separation by reversed phase HPLC | A. Three-step ligation B. Addition of AMV revertase to duplex made up of 2 partially complementary oligonucleotides, cloning with the aid of adapters | pBR322 | pBR322; lac-promoter; 590 a.a. fragment of b-galactosidase; yield of chimeric protein - 41.7 mg per g of total E. coli protein | Brousseau R., Scarpulla R. et al Gene, 1982, 17, pp. 279-289 |
| Cytochrome B5 | 330 | A.96 B.143 C.90 |
Synthetic Shine-Dalgarno sequence, initiation and double stop codons; RS dividing the sequence into fragments A, B and C; fragments synthesized with corresponding sticky ends | 16 oligonucleotides 31-46 b.p. long; synthesis by phosphoramidite method on automatic snythesizer; separation by PAGE in 7 M urea | A, B, C. One-step ligation of 2, 4 and 2 pairs of complementary oligonucleotides, respectively | pUC13 | pUC13; lac-promoter; yield - 8 % of total E. coli cell protein (cells stained red) | Von Bodman S. B., Schuller M. A. et al. Proc. Natl. Acad. Sci., USA, 83, pp. 9443-9447 |
| Bovine hepatitis B virus protein | 560 | A.349 | Based on natural sequence with 56 b.p. substitutions to create 34 RS; double stop codon, synthetic transcription termination signal | 22 oligonucleotides 36-64 b.p. long; synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | A. Two-step ligation B. One-step ligation C. 2 complementary synthetic oligonucleotides |
pLT1 | pLT1; pl-promoter; Shine-Dalgarno sequence; yield - 1.2-4 % of total soluble E. coli protein | Nassal M. Gene, 1988, 66, pp. 279-294 |
| Human Growth Hormone | 584 | A.415 B.176 |
Codons of E. coli elongation factor Tu gene | 78 oligonucleotides 10-25 b.p. long; synthesis by solid-phase phosphotriester block method; separation by anion-exchange HPLC | A, B. Two-step ligation fragment A from
5 blocks B. I. Assembly of 3 DNA blocks from 5-6 oligonucleotide pairs; II. Assembly of fragment B from 3 blocks |
pBR322 | pBR322; trp-promoter and Shine-Dalgarno sequence; yield - 169 Rg/ml of E. coli culture | Ikehara M., Ohtsuka, E. et al. Proc. Natl. Acad. Sci. USA, 1984, 81, pp. 5956-5960 |
| Transducin (guanosine- binding protein |
1076 | A.637 B.451 |
38 unique RS, consensus sequence CCACC before initiation codon | 44 oligonucleotides 39-59 b.p. long; synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | A, B. Two-step ligation 1. Assembly of 2 DNA blocks from 5-6 oligonucleotide pairs; II. Ligation of 2 DNA blocks with vector | pT2 | pMT-2, main late adenovirus promoter; level of expression in COS-1 cells - 0.3 % of total cell protein | Sakmar, T. P., Khorana, H. G. Nucl. Acids Res., 1988, 16, pp. 6361-6372 |
| Bovine prochimosin |
12a 1146 12b 1146 |
A.87 B.89 C.307 D.267 E.150 F.246 A.468 B.348 C.318 |
12a: Codons of highly expressed yeast genes, RS for subcloning, a total of 25 % of nucleotide substitutions as compared to natural sequence; | 12a: 28 oligonucleotides 59-102 b.p. long; 12b: 26 oligonucleotides 71-102 b.p. long; synthesis by phosphoramidite method on automatic synthesizer; separation by PAGE in 7 M urea | 12a: A, B, D, E, E Ligation of 1-3 pairs of oligonucleotides with linearized vector; 12a: C. Assembly of a fragment from 4 pairs of oligonueleotides, ligation with a vector after separation by electrophoresis in native PAGE; 12b: A, B. Assembly of fragments from 5 and 4 pairs of oligonueleotides, respectively; separation by electrophoresis and ligation with linearized vector in low-melting agarose gel; 12b: C. Two-step ligation, separation of two intermediate DNA blocks and target fragment by electrophoresis in low-melting agarose gel | M13mp18 M13mp19 |
pMV-1; promoter and signal sequences of melibiose | Wosnick M. A., Barnett R. W et al. Gene, 1987, 60, pp. 115-127 |
| Tissue type plasminogen activator t-PA | 1730 | A.710 B.650 C.290 D.134 |
Codons of highly expressed yeast genes, insertion of RS at equal intervals, gene encodes pre-pro-t-PA | 101 oligonucleotides 17-37 b.p. long; synthesis of 6 oligonucleotides at a time by phosphoramidite method on specially designed automatic synthesizer; separation by PAGE in 7 M urea | A, B, C. Two-step ligation; I. Assembly of DNA blocks from 5 oligonucleotides; II. Assembly of fragments from 4-8 blocks | pAT153 | Vector with origin pBR322 and SV40; long terminal repeats RSV; polyadenylation and splicing sites SV40; expression in cells COS-7; yield - 20 ng t-PA in 1 ml of culture supernatant | Bell L. O., Smith J.C. et al. Gene 1988, 63, pp. 155-163 |
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