Preventing norvaline and norleucine misincorporation in recombinant proteins

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Previous efforts to prevent norleucine misincorporation into recombinant proteins involved supplementing medium with leucine and/or methionine (e.g., U.S. Patent 5,599,690; Apostol et ah, J. Biol. Chem. 272, 28980-88, 1997) or supplementing the medium with combinations of leucine, methionine, and cysteine (e.g., U.S. Patent 5,622,845). There is a need in the art for methods of preventing misincorporation of norvaline and norleucine in recombinant proteins.

The invention provides a method for preventing misincorporation of norvaline in place of leucine in recombinant proteins. The method can be utilized in the production of any recombinant protein, and may be used with both large and small scale production. The method involves feeding the culture with the amino acids leucine, isoleucine, and methionine and is particularly suited for the prevention of norvaline and norleucine misincorporation in recombinant proteins without compromising cell densities and titers.

Accordingly, in one embodiment, the invention is directed to a method for preventing norvaline and norleucine misincorporation into a recombinant protein comprising providing a culture of host cells, which inducibly express the recombinant protein, an amount of amino acids leucine, isoleucine, and methionine sufficient to maintain a constant amino acid concentration of each of the amino acids throughout induction of recombinant protein expression.

In certain embodiments of the method, the leucine, isoleucine, and methionine are present in a glucose feed. In additional embodiments of the method, the glucose feed comprises 5 g/L leucine, 6.3 g/L isoleucine, and 4 g/L methionine.

I*1 still further embodiments, the method further comprises supplying leucine and isoleucine to the culture at glucose exhaustion.

In other embodiments, the recombinant protein is TFPI or ala-TFPI.

In yet an additional embodiment, the culture is in a fermentor.

In certain embodiments of the subject invention, the host cells are E. cailL

These and other embodiments of the subject invention will readily occur to those of skill in the art in view of the disclosure herein.

The invention provides a method for preventing misincorporation of norvaline in place of leucine in recombinant proteins. The method involves feeding the culture with leucine, isoleucine, and methionine. Addition of methionine prevents incorporation of norleucine. Leucine inhibits and represses the enzymes involved in the synthesis of both norvaline and norleucine. However, leucine also affects other cellular functions and results in lower cell densities and titers at the end of fermentation. Addition of isoleucine results in greatly increased amount of recombinant protein production. Thus, the present method prevents norvaline and norleucine incorporation without compromising on cell densities and titers achieved in the absence of leucine.

Methods of the invention can be used in the production of any recombinant protein and can be used with both large and small scale production. It is particularly useful for use with high density fermentation, for example, of E. coli host cells. One embodiment of the invention is illustrated in the examples below for the production of recombinant ala-TFPI (TFPI as shown in SEQ ED NO:1 comprising an N-terminal alanine). These examples are provided for purposes of illustration only and are not intended to limit the scope of the invention.

All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference.