# Patent

## Title
Method for producing cellulosic fibers and microcrystalline cellulose

## Abstract

The incubation is carried out in substantial accordance with the teaching of Example 3 except that Saccharomyces cerevisiae/pIT3210, rather than S. cerevisiae/pIT210, is used. The resultant induction of the YG100 gene transcriptional and translational activating sequence results in high level expression of human methionyl proinsulin. The desired proinsulin product is conventionally isolated by preparative HPLC and identified by radioimmunoassay and other tests.

## Background
## Claims

1. A method for inducing a high level expression in yeast of a nucleotide sequence that is under the transcriptional and translational control of the yeast YH100 gene, said method comprising autonomous replication or genomic integration, said plasmid comprising, in translational reading frame, YG100 gene, and polypeptide, growth, anaerobic growth, and aerobic growth and gene expression, subject to the limitation that said conditions for anaerobic growth are maintained for a time sufficient to cause induction of the transciptional and translational activating sequence of the yeast YG100 gene when said conditions for aerobic growth are restored.

2. The method of claim 1 wherein prior to culturing under conditions suitable for anaerobic growth, said transformed cell is cultured under conditions suitable for aerobic growth until about mid-logarithmic growth phase and wherein conditions suitable for anaerobic growth are maintained for about 12 hours to at or near stationary growth phase.

3. The method of claim 2 wherein the nucleotide sequence codes for bovine growth hormone or bovine pre-growth hormone.

4. The method of claim 2 wherein the selectable plasmid is capable of genomic integration.

5. The method of claim 2 wherein the nucleotide sequence that codes for a functional polypeptide is selected from the group consisting of sequence that code for bovine growth hormone, bovine pre-growth hormone, human growth hormone, human pre-growth hormone, porcine growth hormone, mammalian growth hormone, avian growth hormone, growth hormone releasing factor, human insulin A chain, human insulin B chain, human proinsulin, human pre-proinsulin, human and non-human interferon, urokinase, tissue plasminogen activator, interleukin I, interleukin II, beta-galactosidase, any hormone, any enzyme and any biactive polypeptide.

6. The method of claim 5 wherein the nucleotide sequence that codes for a functional polypeptide is selected from the group consisting of sequences that code for beta-galalctosidase and human proinsulin.

7. The method of claim 2 in which the plasmid is pIT210.

8. The method of claim 2 in which the plasmid is pIT2100.

9. The method of claim 2 in which the plasmid is pIT3210.

10. The method of claim 2 wherein the yeast cell is Saccharomyces.

11. The method of claim 2 wherein the transformed yeast cell is Saccharomyces cerevisiae.

12. The method of claim 2 wherein the transformed yeast cell is Saccharomyces cerevisiae DBY 689/pIT210.

13. The method of claim 2 wherein the transformed yeast cell is Saccharomyces cerevisiae DBY689/pIT3210.

14. A method for inducing high level expression in yeast of a nucleotide sequence that is under the transcriptional and translational control of the yeast YG100 gene, said method comprising culturing a tranformed yeast cell at or near stationary growth phase under conditons suitable for growth and gene expression, said transformed yeast cell comprising the selectable plasmid of claim 1.

15. The method of claim 10 wherein the nucleotide sequence that codes for a functional polypeptide is selected from the group consisting of sequences that code for bovine growth hormone, bovine pre-growth hormone, human growth hormone, human pre-growth hormone, procine growth hormone, mammalian growth hormone, avian growth hormone, growth hormone releasing factor, human insulin A chain, human insulin B chain, human proinsulin, human pre-proinsulin, human and non-human interferon, urokinase, tissue plasminogen activator, interleukin I, interleukin II, beta-galactosidase, any hormone, any enzyme and any bioactive polypeptide.

16. The method of claim 15 wherein the nucleotide sequence that codes for a functional polypeptide is selected from the group consisting of sequences that code for beta-galactosidase and human proinsulin.

17. The method of claim 10 wherein the selectable plasmid is capable of genomic integration.

18. The method of claim 10 in which the plasmid is pIT210.

19. The method of claim 10 in which the plasmid is pIT2100.

20. The method of claim 10 in which the plasmid is pIT3210.

21. The method of claim 10 wherein the yeast cell is Saccharomyces.

22. The method of claim 10 wherein the yeast cell is Saccharomyces cerevisiae.

23. The method of claim 10 wherein the yeast cell is Saccharomyces cerevisiae DBY689/pIT210.

24. The method of claim 10 wherein the yeast cell is Saccharomyces cerevisiae DBY746/pIT3210.

25. The method of claim 1 wherein said tranformed cell is cultured at or near stationary growth phase prior to culturing under conditions suitable for anaerobic growth.

26. The method of claim 1 wherein said anaerobically-grown cells are cultured at or near stationary growth phase under conditons suitable for aerobic growth and gene expression.

27. The method of claim 1 wherein the culture temperature for aerobic and anaerobic growth is about 37.degree. C.

28. The method of claim 2 wherein the culture temperature for aerobic and anaerobic growth is about 37.degree. C.

29. The method of claim 17 wherein the culture temperature for aerobic and anaerobic growth is about 37.degree. C.

30. The method of claim 18 wherein the culture temperature for aerobic and anaerobic growth is about 37.degree. C.

31. The method of claim 22 wherein the selectable DNA is plasmid PIT210.

32. A selectable plasmid that is capable of autonomous replication or genomic integration, said plasmid comprising, in translational reading frame, YG100 gene, and polypeptide.

33. The selectable plasmid of claim 24 which is plasmid pIT210.

34. The selectable plasmid of claim 24 which is plasmid pIT2100.

35. The selectable plasmid of claim 24 which is plasmid pIT3210.

36. The transformed yeast cell of claim 1 which is Saccharomyces.

37. The transformed yeast cell of claim 1 which is Saccharomyces cerevisiae.

38. The transformed yeast cell of claim 1 which is Saccharomyces cerevisiae/pIT3210.

39. The transformed yeast cell of claim 1 which is Saccharomyces cerevisiae DBY689/pIT3210.

40. A method for inducing high level expression in yeast of a nucleotide sequence that is under the transcriptional and translational control of the yeast YG100gene, said method comprising culturing a yeast cell transformed with the selectable plasmid of claim 32 under anaerobic conditions until late or stationary growth phase and then culturing said anaerobically grown cells under conditions suitable for aerobic growth and gene expression.

41. The method of claim 40 wherein the culture temperature for anaerobic and aerobic growth is about 37.degree. C.