Protein production process

Details Protein production process Protein production process

1998-09-29

2001-03-20

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S195000, C435S252300, C435S320100, C435S254100, C435S255100, C435S255500, C435S255600, C536S023100, C536S023200, C530S350000

Reexamination Certificate

active

06204012

ABSTRACT:

BACKGROUND OF THE INVENTION
Methylotrophic yeasts are known to provide suitable expression systems for heterologous proteins.
Hansenula polymorpha
and
Pichia pastoris,
for example, are easy to handle host organisms for a wide variety of foreign genes (for reviews see Gelissen & Melber 1996 and Cregg & Madden 1988). The promoters for the enzymes involved in methanol metabolism in these organisms are particularly strong, and these promoters are generally used to control the heterologous expression of proteins (see EP 0173 378, EP 0299 108, EP 0183 071).
The specific carbon source used for cultivation of these organisms has an enormous influence on the regulation of methanol metabolism promoters. Until now, methanol and glycerol have been considered adequate substrates for the methylotrophic yeast expression system, while glucose has been considered inadequate (EP 299 108). During growth on methanol, key enzymes of methanol metabolism in
Hansenula polymorpha
are present in large amounts (Gelissen et al. (1994). Similarly, during growth on glycerol significant levels of the key enzymes methanol oxidase (MOX) and formate dehydrogenase (FMDH) can be detected. Derepression of the MOX and FMDH promoters permits expression of MOX and FMDH during growth on glycerol. However, these enzymes of methanol metabolism are absent in batch cultures if glucose is used as the carbon source (see Gelissen & Melber and EP 0299 108) (in glucose limited chemostat cultures of
Hansenula polymorpha
and Kloeckera sp. 2201, FMDH is only produced below growth rates of 0.1 h
−1
in very small amounts (Egli et al. 1980)). Thus glucose is considered a repressor of the methanol metabolism promoters. These promoters permit only very limited expression of their genes when glucose is the available environmental carbon source.
Therefore, so far nonrepressive carbon sources, e.g. glycerol or methanol, are the standard carbon sources for the heterologous expression of proteins in methylotrophic yeasts.
Hansenula polymorpha
is cultivated for the production of a wide variety of pharmaceutical proteins in a fed batch process either in the single carbon source mode with glycerol or in the two carbon source mode with glycerol and additional methanol (Gelissen & Melber 1996). The common process strategy is described in more details by Weydemann et al. (1995) for the production of hirudin. A process for the production of thrombomodulin with a
Pichia pastoris
strain using also glycerol as a non repressive carbon source is described by Chen et al. (1996).
However, due to the high price of glycerol this process is not cost-effective for the production of low cost proteins like feed or industrial enzymes. Methanol is not a useful alternative due to handling problems caused by its toxicity and volatility. Therefore in the production of low cost proteins, methylotrophic yeast systems are not typically used. Glucose-metabolizing filamentous fungi are the standard expression system. However, these fungi require complex media, form a viscous culture difficult to handle, and produce significant quantities of undesired proteins such as proteases, decreasing yield and making purification of the target protein awkward. Thus the methylotrophic yeast system would be a much preferable production method for low cost proteins if expensive or dangerous substrates were not required.
SUMMARY OF THE INVENTION
Surprisingly, it is now possible to use glucose, glucose-containing compositions or a similar sugar as a carbon source for protein production with a promoter of the methylotrophic yeast methanol metabolic pathway, even though glucose and other monosaccharides are known repressors of such promoters. It has been discovered that this is possible if culture conditions are controlled such that the carbon source is a limiting factor, while oxygen is not.
Therefore it is an object of the present invention to overcome such obstacles and to provide a process for the preparation of an endogenous or heterologous protein by cultivation of a transformed or non-transformed eukaryotic cell which process is characterized therein that a substrate known to be repressive is used as a carbon source, the carbon source is limiting during the feeding phase and there is no continuous oxygen limitation during the whole cultivation; or such a process wherein the protein is an enzyme, especially a feed enzyme, e.g. phytase, cellulase, xylanase or an industrial enzyme, e.g. amylase, protease, invertase, lipase, catalase, cellulase, glucose oxidase, alcohol oxidase, pectinase, naraginase, collagenase, peroxidase or pullalanase; or such a process wherein the cell is methylotrophic and/or is transformed with a DNA sequence comprising a promoter for enzymes involved in the methanol metabolism, e.g. the formate dehydrogenase (FMD or FMDH) promoter, the methanol oxidase (MOX) promoter or the dihydroxyacetone synthase (DAS or DHAS) promoter; or such a process wherein the cell is a methylotrophic yeast, preferably Hansenula, Pichia, Candida or Torulopsis, e.g.
Hansenula polymorpha
or
Pichia pastoris;
or such a process wherein the repressive substrate counts for 1-100%, preferably 40-100% or more preferably 90-100% of the carbon source or is the sole carbon source; or such a process wherein the substrate is a sugar or sugar polymer like mono-, di-, oligo- or poly-saccharide, e.g. glucose, fructose, sucrose, maltose, starch, glycogen, cellulose or dextrose or a sugar containing compound, e.g. molasses, glucose syrups or fructose syrups especially glucose-containing compositions like glucose syrups; or such a process which is effected in form of repeated fed-batches. Furthermore such process is characterized therein that the feeding rate range is limited by the metabolic characteristics of the microorganisms and the mass transfer performance of the bioreactor, in particular for oxygen. Feeding rates are preferentially maintained between the minimum to allow production and the maximum to avoid oxygen limitation in the culture.
Therefore, this invention is directed to a method for producing a protein which comprises culturing a cell capable of expressing said protein and which contains a methylotrophic yeast promoter for an enzyme of the methanol metabolic pathway controlling expression of said protein, in a fermentative batch process comprising a batch phase and a feeding phase, under conditions such that dissolved oxygen is continually present in the culture medium throughout the process, and about 1% to about 100% of the carbon source during the feeding phase is a sugar or sugar polymer, which sugar or sugar polymer is provided in such an amount that the sugar or sugar polymer is continually depleted by the cell and therefore is substantially undetectable in the culture medium; and isolating the protein on completion of the feeding phase.


REFERENCES:
patent: 5667986 (1997-09-01), Goodey et al.
patent: 59543/98 (1998-01-01), None
patent: 173 378 (1986-03-01), None
patent: 183 071 (1986-06-01), None
patent: 299 108 (1989-01-01), None
patent: 0 533 492 A2 (1993-03-01), None
patent: 684 313 (1995-11-01), None
patent: WO 90/03431 (1990-04-01), None
Fieschko, et al., “Controlled Expression and Purification of Human Immune Interferon from High-Cell-Density Fermentations ofSaccharomyces cerevisiae,” Biotechnology and Engineering,vol. 29, pp. 1113-1121 (1987).
Mayer, et al., “An Expression System Matures: A Highly Efficient and Cost-Effective Process for Phytase Production by Recombinant Strains of Hamsenula Polymorpha,”Biotechnology and Bioengineering,vol. 63, No. 3, pp. 373-381 (1999).
J.M. Cregg et al. Developments in Industrial Microbiology 29, pp. 33-41 (1988).
Y. Sakai et al. J. Bacteriol, 179 pp. 4480-4485 (1997).
Gelissen and K. Melber, Drug Res. 46, pp. 943-948 (1996).
G. Gelissen et al. Gene Expression in Recombinant Microorganisms, Dekker, New York, pp. 195-239 (1994).
Weydemann, P. et al. High-level secretion of hirudin by Hansenula polymorpha, Appl. Microbiol. Biotechnol. 44, pp. 377-385 (1995).
Th. Egli, et al. Regulation of the synthesis of catabol

Affiliated with

Also associated with

Rating

Protein production process does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Protein production process, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Protein production process will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2540605