Method of making proteins in transformed yeast cells
A yeast cell, yeast expression technology, applied in microorganism-based methods, peptide/protein components, chemical instruments and methods, etc., can solve problems such as bacterial antibiotic resistance hazards
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Embodiment 1
[0055] The yeast plasmid pAK729 constructed to express insulin precursor (an N-terminally extended B(1-29)-Ala-Ala-Lys-A(1-21) insulin precursor, see WO 97 / 22706) contains two ApaLI enzyme restriction sites ApaLI (4477) and ApaLI (5723) (see attached figure 1 ). These restriction sites are located on each side of the AMP marker gene. Removal of the 1246 nucleotides between the two AplaLI sites in pAK729 will remove the AMP marker gene and some additional E. coli-derived plasmid DNA.
[0056]The pAK729 plasmid was digested with ApaLI restriction enzyme, subjected to agarose gel electrophoresis, separated, re-ligated and then transformed into competent Saccharomyces cerevisiae cells (MT663, see EPB0163529) to obtain transformed yeast strain NN729.1-ΔAMP. The modified expression plasmid was re-isolated from the yeast strain NN729.1-ΔAMP and the DNA sequence was checked after PCR, and then the DNA region characterized by the deletion was subcloned. Similarly, the DNA sequence encodin...
Embodiment 2
[0059] The restriction sites XhoI (5676) and XhoI (5720) of the enzyme were introduced into the pAK729 plasmid by PCR. The selected DNA sequence of the resulting pAK729.5 plasmid was then checked. The restriction plasmid map of pAK729.5 is shown in Figure 2. The DNA fragment between the restriction enzyme sites XhoI (5676) and XhoI (5720) can be deleted from the plasmid pAK729.5 in which 44 nucleotides in the AMP gene are deleted.
[0060] The plasmid pAK729.5 was digested with XhoI restriction enzyme, subjected to agarose gel electrophoresis, separated, re-ligated, and then transformed into competent Saccharomyces cerevisiae cells, thereby obtaining the yeast transformant NN729.5-ΔAMP. The modified expression plasmid was re-isolated from the yeast strain NN729.5-ΔAMP and the DNA sequence was checked after PCR, and then the DNA region characterized by deletion was subcloned. Similarly, the DNA sequence encoding the insulin precursor was tested on the plasmid DNA re-isolated from t...
Embodiment 3
[0063] The restriction site AatII (4982) of the enzyme was introduced into the pAK729 plasmid by PCR. The selected DNA sequence of the resulting pAK729.6 plasmid was then checked. The restriction plasmid map of pAK729.6 is shown in Figure 3. In pAK729.6, the DNA fragment between the restriction enzyme sites AatII (4982) and AatII (5978) can be deleted, thereby removing 996 nucleotides from the plasmid. This can remove all AMP genes and promoters.
[0064] The pAK729.6 plasmid was digested with DNA restriction enzyme AatII, subjected to agarose gel electrophoresis, separated, re-ligated and then transformed into competent MT663 Saccharomyces cerevisiae cells. The modified expression plasmid was re-isolated from the yeast strain NN729.6-ΔAMP and the DNA sequence was checked after PCR, and then the DNA region characterized by deletion was subcloned. Similarly, the DNA sequence encoding the insulin precursor was tested on the plasmid DNA re-isolated from the yeast strain NN729.6-ΔAMP....
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