# US 5,700,665 A · Method for the extraction of periplasmic proteins from prokaryotic microorganisms in the presence of arginine

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- Number: US 5,700,665 A
- Document ID: US5700665A
- Status: Granted
- Kind: A
- Application number: 08/594,469
- Filed: 1996-01-31
- Granted: 1997-12-23
- Earliest claimed: 1995-01-31
- Assignees: Sanofi
- Inventors: Richard Legoux; Paul Maldonado; Marc Salome
- Web page: https://patentgrep.com/patent/US5700665A

## CPC

- C07K 14/61: Growth hormone [GH], i.e. somatotropin
- C07K 1/145: {by extraction or solubilisation}
- C07K 14/5437: {IL-13}
- C12N 15/67: General methods for enhancing the expression
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The invention relates to a method for the extraction of recombinant periplasmic proteins wherein arginine is used as the extraction agent. In particular, the invention relates to a method for the extraction of a periplasmic protein of interest, which essentially consists in: (i) suspending the pellet of cells or of cell debris from cells, which cells originate from the culture of a prokaryotic microorganism transformed with an expression vector containing a gene coding for the protein and means for its expression in the periplasm of the microorganism, in a buffer solution containing arginine; and (ii) recovering the protein of interest in the supernatant of the bacterial suspension thereby obtained.

## Claims

### Claim 1

In a method for the extraction of recombinant periplasmic proteins, wherein the improvement comprises carrying out the extraction in the presence of arginine.

### Claim 2

A method for the extraction of a periplasmic protein of interest, which comprises the steps of
(i) culturing a prokaryotic microorganism of the Enterobacteriaceae family transformed with an expression vector containing a gene coding for said protein of interest and means for a periplasmic expression thereof,
(ii) subjecting the resulting culture to centrifugation or microfiltration thereby producing a cell pellet,
(iii) suspending the resulting cell pellet in a buffer solution containing arginine thereby producing a suspension,
(iv) centrifuging the resulting suspension thereby producing a supernatant, and
(v) recovering the protein of interest from the supernatant thereby obtained.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the buffer solution containing arginine is an alkaline aqueous solution having an arginine concentration of at least 0.4M.

### Claim 4 (depends on 2)

The method according to claim 2, wherein the protein of interest is IL-13.

### Claim 5 (depends on 2)

The method according to claim 2, wherein the protein of interest is hGH.

### Claim 6 (depends on 3)

The method according to claim 3, wherein the arginine concentration is between 0.4M and 0.8M.

### Claim 7 (depends on 3)

The method according to claim 3, wherein the protein of interest is IL-13.

### Claim 8 (depends on 3)

The method according to claim 3, wherein the protein of interest is hGH.

### Claim 9

A method for the extraction of a periplasmic protein of interest, which comprises the steps of
(i) culturing a prokaryotic microorganism of the Enterobacteriaceae family transformed with an expression vector containing a gene coding for said protein of interest and means for a periplasmic expression thereof,
(ii) subjecting the resulting culture to centrifugation or microfiltration thereby producing a cell pellet,
(iii) lysing the cells from the cell pellet thereby obtained and centrifuging the resulting lysate thereby producing a pellet of cell debris,
(iv) suspending the pellet of cell debris thereby obtained in a buffer solution containing arginine thereby producing a suspension,
(v) centrifuging the resulting suspension thereby producing a supernatant, and
(iv) recovering the protein of interest from the supernatant thereby obtained.

### Claim 10 (depends on 9)

The method according to claim 9, wherein the buffer solution containing arginine is an alkaline aqueous solution having an arginine concentration of at least 0.4M.

### Claim 11 (depends on 9)

The method according to claim 9, wherein the protein of interest is IL-13.

### Claim 12 (depends on 9)

The method according to claim 9, wherein the protein of interest is hGH.

### Claim 13 (depends on 10)

The method according to claim 10, wherein the arginine concentration is between 0.4M and 2.5M.

### Claim 14 (depends on 10)

The method according to claim 10, wherein the protein of interest is IL-13.

### Claim 15 (depends on 10)

The method according to claim 10, wherein the protein of interest is hGH.

### Claim 16

A method for the culture of a prokaryotic microorganism of the Enterobacteriaceae family transformed with an expression vector containing a gene coding for a protein of interest, which comprises culturing said microorganism in the presence of arginine at a concentration of at least 2 g/l.

### Claim 17 (depends on 16)

The method according to claim 16, wherein said arginine concentration is between 2 g/l and 10 g/l.

### Claim 18 (depends on 16)

The method according to claim 16, wherein the protein of interest is IL-13.