WO1994029435A1

WO1994029435A1 - Enhanced expression of protein by using culture medium containing membrane lipids

Info

Publication number: WO1994029435A1
Application number: PCT/SE1994/000530
Authority: WO
Inventors: Carl-Fredrik Mandenius; Åke SPÅNNING; Sven ENGSTRÖM
Original assignee: Pharmacia Ab
Priority date: 1993-06-07
Filing date: 1994-06-02
Publication date: 1994-12-22
Also published as: IL109911A0; AU6986894A; SE9301937D0; IL109902A0

Abstract

The invention relates to microorganism culture medium for the expression of protein which comprises membrane interactive lipids or membrane lipids belonging to class II or III polar lipids or precursors thereof, such as monoolein or ethoxylated vitamin E or inositol, in an amount of more than 50 mg/L. The invention also relates to a method for expressing protein in recombinant microorganisms which is characterised by the addition of the membrane interactive lipid or membrane lipids or precursors. The microorganism is preferably yeast and the protein is preferably IGF-I. The invention also relates to the use of the membrane interactive lipids or membrane lipids belonging to class II or III polar lipids or precursors thereof in an amount of more than 50 mg/L as enhancer for the expression of protein in a microorganism culture medium.

Description

ENHANCED EXPRESSION OF PROTEIN BY USING CULTURE MEDIUM CONTAINING MEMBRANE LIPIDS

The present invention relates to an enhanced expression of protein in recombinant micro-organisms by the addition of special membrane interactive lipids or membrane lipids or precursors thereof to the culture medium.

INTRODUCTION

It is previously known that it is possible to enhance secretion of a primary metabolites from micro-organisms, such as ethanol, by the addition of surface active molecules. It has e.g. been shown that surfactants such as Tween and Triton X 100 result in higher productivity in bakers' yeast cultures (F. Larue et al, Appl. Environ. Microbiol. 39, 808-11, 1980). It has been suggested that the effects is due to a facilitated transport of the ethanol molecules or other smaller biomolecules through the cell membrane caused by penetration or fluidisation of the cell membrane.

From the theoretical knowledge of the action of amphiphilic molecules in lipidic environments, several hypothetical explanations can be assumed that are related to the membrane interaction of these molecules. The fact that clear membrane effects also are observed for phospholipid molecules, such as phosphatidylethanolamine, support the explanations. Further support of the hypothesis is that the spontaneous interaction of these molecules with membrane double layers will change the properties of the layers including its fluidity.

Media for the cultivation of cells from higher organisms such as mammalian cells are complex and sensitive and are different from cultivation of prokaryotic and eukaryotic micro-organism such as bacteria and yeast for recombinant purposes. Our new finding is that the cultivation of prokaryotic(e.g. E. coli ) and eukaryotic micro-organism such as yeast (e.g. Saccharomyces cerevisiae) for recombinant protein (polypeptide) production results in higher expression yields when a high amount of membrane interactive lipids or membrane lipids, compounds belonging to class π or in polar lipids or precursors thereof, are added to a culture.

We have also surprisingly found that the addition of phospholipid precursors in a high amount to a culture of micro-organisms as host for an expression vector, give an unexpected enhanced expression of the desired protein.

In the method according to our invention we have seen an enhanced growth of the micro-organism and higher production of the wanted proteins.

The definition of the membrane interactive lipids or membrane lipids to be used in the claimed culture media is in accordance to an acknowledged definition of lipids, found in Handbook of Lipid

Research, by Donald M Small. 1986, Plenum Press. New York, pages

89-96.

Lipids are classified in two main classes, non polar and polar lipids.

Polar Hpids are then classified in three different classes I, π and III, defied as follows:

Class I: Insoluble, non swelling amphiphiles, such as triglycerides, long chain fatty acids.

Class II: Insoluble swelling amphiphilic lipids, such as phosphatidyl choline, monoglycerides. Class III: Soluble amphiphiles such as salts of long-chain fatty acids.

It is possible tofind the right classification by e.g. x-ray cristallography, polarization microscopy or nuclear magnetic resonance (NMR).

Penetration of pharmaceuticals through epidermal cell layers facilitated by changes of membrane structures as observed for the pharmaceutical laurocapram and oleic acid is known (JP 63196524, WPI Ace No 88-268233/38).

Different designs for media can be found in the literature. In media cultivation of cells from higher organisms, lipids and/or phospholipid precursors are sometimes suggested to be included, but always in very small amounts. Such media are e.g. disclosed in:

EP 112 174 in which a medium comprising fatty acids in an amount of 0.07 mg/1 and phospatidylcholin in an amount of less than 18 mg/L is disclosed. WO 90/12083 discloses a medium comprising retinoid, vitamin D2, fatty acid in an amount of 0.01-1 mg/L and calcium. In EP 389 788 a well defined medium is disclosed, comprising among other components also inositol. The amount of inositol is 21 to 41 mg/L. In WO 90/03430 a cell culture medium for enhanced cell growth, culture longevity and product expression is disclosed. The medium should comprise glutamine or glutamate, amino acids and phospholipid precursors, such as inositol, serine, ethanol amine. It is clearly stated that the supplementation of standard media with phospholipid precursors alone is not sufficient to achieve the desired maximum extension of culture longevity and product formation. The phospholipid precursors are added in a small amount and said to be in synergy with the other components. In WO 84/03710 an animal cell culture medium is disclosed which comprises a mixture of several fatty acids such as stearic acid, cis- oleic acid and cis-linoleic acid. The amount of fatty acids is 7.6 μeq/L and inositol is added in an amount of 0.5 mg/L or less. Some of the added components to these media for the cultivation of animal cells have been shown to affect the proliferation, i.e. a higher amount of cells.

Other known media are known from US 4564594 in which fatty acids or esters or triglycerides are added to a growth medium for the production of carboxylic acids. It was shown that in the production of fumaric acid, the addition of a fatty ester e.g. polyoxyethylene sorbitan mono-laurate (or -palmitate, stearate, oleate)= Tween or the addition of mixed triglycerides e.g. corn oil, the rate of formation of fumaric acid was increased. The amount in the examples is 300- 1000 μg/mL.

A medium used for the overproduction of a carboxylic acids (primary metabolites of the microorganism) should however be different from a medium in which a recombinant protein is produced by a microorganism. No conclusion can be drawn from the information given in US 4564594, that a high amount of membrane interactive lipids or membrane lipids belonging to class II or III polar lipids or precursors thereof could give a surprisingly high expression level of a protein.

US 3 816 261 disloses a culture medium comprising water-soluble lipid source selected from Na-oleate, Na-stearate and polysorbates and basic anion-exchange resin. Polysorbate and Tween are mentioned as lipid source. The medium is suitable for growing Leptospiraorganisms and nothing is mentioned regarding the recombinant expression of a protein. According to the examples the amount is about 10-15 mg/L. The used amount is lower than the amount used according to the present invention.

Regarding the expression of heterologeous proteins in recombinant micro-organisms it may be assumed that the transport process of the proteins through the membranes in the various cellular entities of the cell, such as endoplasmatic reticulum, Golgi and secretory vesicles will be effected. By affecting the membrane structure, the membrane will be more easily penetrable for the protein and the transport time across the cell could be reduced.

THE INVENTION

The present invention relates thus to a micro-organism culture medium comprising membrane interactive lipids or membrane lipids belonging to class π or III polar lipids or precursors thereof in an amount of more than 50 mg/L for the expression of protein. Inositol is an example of a membrane lipid precursor and monoolein or ethoxylated vitamin E are examples of membrane interactive lipid.

The micro-organism culture medium normally comprises a standard medium based on glucose, sodium and potassium phosphate, nitrogen base and ammonium sulphate supplemented with vitamins and trace amounts of mineral salts. The invention also relates to a method for expressing protein, preferably a heterologous protein, in recombinant micro- organisms, which is characterised by the addition of membrane interactive lipid or membrane lipids or precursors thereof. The invention also relates to the use of membrane interactive lipids or membrane hpids belonging to class II or El polar hpids or precursors thereof in an amount of more than 50 mg/L as enhancer for the expression of protein in a micro-organism culture medium.

Examples of suitable Hpids according to the invention are: monoglycerides, such as monoolein and monolinolein and mixtures thereof; phosphoHpids such as diacylphosphatidylcholine (lecithin) diacylphosphatidylethanolamine, diacylphosphatidylinositol,, diacylphosphatidylglycerol and mixtures thereof; galactoHpids such as monogalactosyldiglyceride and digalactosyldiglyceride and mixtures thereof; soaps such as sodium oleate and potassium oleate; ethoxylated vitamin E.

The membrane Upid precursor is here exemplified by inositol.

The micro-organism could be yeast, such as Saccharomyces cerevisiae or a bacteria, such as E. coli.

The expressed protein is exemplified by Insuline-Like Growth

Factor-I, IGF-I, and the micro-organism by Saccharomyces cerevisiae.

Also growth hormone could be expressed, preferably in E. coli.

The membrane interactive lipids or membrane hpids or precursors thereof should be added in an amount which corresponds to the total cell membrane area in the culture.

Inositol is the precursor to phosphatidylinositol, known as a component in the cell membrane of yeast and belonging to class π. Monoolein is a common glyceride with well characterised surface properties that could be expected to form structures in the cell membrane, belonging to class II.

Laurocapram (N-dodecylcaprolactam, l-dodecylhexahydro-2H- azepin-2-on or Azone®) and oleic acid are known for its action on epithelian membrane and belong to class I of polar hpids.

The investigation shows that addition of inositol and monoolein enhance the expression of IGF-I significantly. Laurocapram and oleic acid do not, in spite of its properties, act in a way resulting in enhanced expression.

The experiments described in the following examples are carried out in fed batch cultures with recombinant IGF-I yeast cells.

Addition of molecules that could be expected to act as assumed above are investigated.

EXAMPLES

Example 1: Expression of IGF-I in a Saccharomyces cerevisiae fed batch culture

Recombinant yeast cells of Saccharomyces cerevisiae was modified with a vector construction containing the gene sequence for human insulin-like growth factor I. The cultivation was carried out in a standard medium based on glucose, sodium and potassium phosphate, nitrogen base and ammonium sulphate supplemented with vitamins (pantotenate, inositol (in an amount of 7 mg/L), nicotinamide, pyridoxin, riboflavine, thiamin, pholic acid, biotin) and trace amounts of mineral salts (magnesium chloride, ferric chloride, zink sulphate, cobolt chloride, sodium molybdate, cupper sulphate, potassium iodine, manganese sulphate). Glucose was added according to a predetermined profile. After 65 h of fed batch cultivation the cells were harvested and separated and the concentration of IGF-I in the medium measured with an enzyme linked immunosorbent assay specific to IGF-I. The final IGF-I concentration is according to ELISA was 14 mg/L. Example 2: Expression of IGF-I in a fed batch culture with a medium supplemented with inositol

The cultivation conditions as described in example 1 were used. Initially, additional inositol is supplemented to the primary medium to a concentration of 1OO mg/L. After 65 h cultivation time the yeast cells were harvested and separated and the concentration of IGF-I measured. According to ELISA the IGF-I concentration was 43 mg/L.

Example 3: Expression of IGF-I in a fed batch culture supplemented with a medium containing monoolein The cultivation was carried out as described in Example 1. At a cultivation time of 40 h the medium was supplemented with 482 mg/L of monoolein. After 65 h cultivaltion time the cells yeast were harvested and the IGF-I concentration determined. According to ELISA the IGF-I concentration was 39 mg/L.

Example 4: Expression of IGF-I in a fed batch culture with a medium supplemented with laurocapram

The cultivation was carried out as described in Example 1. At a cultivation time of 40 h the medium was supplemented with 160 mg of N-dodecylcaprolactam (l-dodecylhexahydro-2H-azepin-2-on, Azone). After 65 h cultivaltion time the yeast cells were separated and harvested and the IGF-I concentration determined. According to ELISA the IGF-I concentration was 17 mg/L.

Example 5: Expression of IGF-I in a fed batch cultivation supplemented with a medium with oleic acid

The cultivation was carried out as described in Example 1. At a cultivation time of 40 h the medium was supplemented with 340 mg of oleic acid (9-cis-octadecenoic acid). After 65 h cultivaltion time the yeast cells were harvested and separated and the IGF-I concentration determined. According to ELISA the IGF-I concentration was 17 mg/L. Conclusion

When IGF-I was expressed in a fed batch cultivation supplemented with a medium with laurocapram or oleic acid the amount of IGF-I was the same as when IGF-I was expressed in a medium without supplement., as in Example 1.

When, however, inositol or monoolein were added in a large amount in comparison to what has been added in to media for cultivation of mammalian cells, the amount of harvested IGF-I was three times the expected amount in a medium without this supplement.

This finding could not have been foreseen by a person skilled in the art and gives an important contribution to the production of heterologeous protein in recombinant micro-organisms.

Claims

CLAIMS 5 1. Micro-organism culture medium comprising membrane interactive hpids or membrane hpids belonging to class II or III polar hpids or precursors thereof in an amount of more than 50 mg/L, for the expression of protein.

10 2. Micro-organism culture medium according to claim 1 in which the membrane lipid precursor is inositol.

3. Micro-organism culture medium according to claim 1 in which the membrane interactive lipid is monoolein.

15

4. Micro-organism culture medium according to claim 1 in which the membrane interactive lipid is ethoxylated vitamin E.

5. Micro-organism culture medium according to any of claims 1 to 20 4, which comprises a standard medium based on glucose, sodium and potassium phosphate, nitrogen base and ammonium sulphate supplemented with vitamins and trace amounts of mineral salts.

6. Method for expressing protein in recombinant micro-organisms, 25 characterised by the addition of membrane interactive lipid or membrane hpids or precursors thereof according to any of claims 1 to 5 to the micro-organism culture medium.

7. Method according to claim 6, in that the membrane lipid 30 precursor is inositol.

_μ 8. Method according to claim 6, in that the membrane interactive molecules is monoolein. i

35 9. Method according to claim 6 in which the micro-organism is yeast.

10. Method according to claim 9 in which the micro-organism is Saccharomyces cerevisiae.

11. Method according to claim 6 in which the micro-organism is E Coli.

12. Method according to claim 6 in which the expressed protein is Insuline-like Growth factor -I, (IGF-I).

13. Method according to claim 6 in which the expressed protein is IGF-I and the used micro-organism is Saccharomyces cerevisiae.

14. Method according to claim 6 in which the expressed protein is growth hormone.

15. Method according to claim 6 in which the expressed protein is IGF-I and the used micro-organism is E. coli.

16. Use of membrane interactive hpids or membrane hpids belonging to class π or in polar hpids or precursors thereof in an amount of more than 50 mg/L as enhancer for the expression of protein in a micro-organism culture medium.