Protein modification method

Details Protein modification method Protein modification method

1995-11-29

2001-11-27

Wortman, Donna C. (Department: 1642)

Chemistry: molecular biology and microbiology

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

Enzymatic production of a protein or polypeptide

C435S128000, C530S402000, C530S409000, C530S411000, C562S563000, C564S463000, C564S511000

Reexamination Certificate

active

06322996

ABSTRACT:

TECHNICAL FIELD
This invention relates to a method of the modification of a Gln residue in the peptide moiety of a fused protein in which a peptide is linked through the amide bonding to the N-terminal or C-terminal or to the amino acid sequence of a physiologically active protein, more particularly a method of the selective modification of a Gln residue in a physiologically active protein with polyethylene glycol, a polysaccharide, a polyamino acid or a branched sugar derivative.
BACKGROUND ART
Recently, a number of physiologically active proteins are used or ready to be used as pharmaceutical drugs. Since these physiologically active proteins are all easily metabolized, decomposed or excreted when administered to the body of animals including human beings, their retention time in blood is short and their target directivity is low, thus posing a problem in that the protein is not accumulated in the affected part in a required amount for a required period of time.
Various attempts have been made with the aim of overcoming this problem. For example, as described in a report by Karte et al (
Proc. Natl. Acad. Sci., USA,
84 (1987), pp.1487-1491), there is a method in which a physiologically active protein is chemically modified with polyethylene glycol.
However, all such chemical modification methods find difficulty in modifying a specified part of the protein or strictly controlling the degree of modification. For example, when a protein is modified with polyethylene glycol, polyethylene glycol is introduced mainly at the &egr;-position amino group of a lysine residue in the protein, but, since a protein molecule generally contains a plurality of lysine residues, polyethylene glycol may be incorporated or not incorporated into a plurality of lysine residues. As the result, the protein loses its inherent physiological activity in some cases or its quality as a pharmaceutical drug cannot be controlled easily.
For these reasons, great concern has been directed toward the development of a method which can modify a specified site of a physiologically active protein or can strictly control degree of the modification when the physiologically active protein is modified with a modification agent such as polyethylene glycol, a polysaccharide or the like.
DISCLOSURE OF THE INVENTION
The inventors of the present invention have found that the physiologically active protein can be modified in a position-selective manner with polyethylene glycol, a polysaccharide, a polyamino acid or a branched sugar derivative, and the modification degree can be controlled strictly, when a physiologically active protein having a molecular weight of from 5×10
3
to 2×10
5
and containing at least one glutamine residue capable of receiving the action of transglutaminase is allowed to react with an amino group donor in the presence of transglutaminase, thereby effecting the formation of amido linkage between the &ggr;-carboxyamido group of said glutamine residue and the primary amino group of said amino group donor, and have accomplished the present invention on the basis of these findings.
Accordingly, the present invention relates to a method of modifying protein which comprises allowing a physiologically active protein having a molecular weight of from 5×10
3
to 2×10
5
and containing at least one glutamine residue to react with an amino group donor represented by any one of the following general formulae (I) to (IV) or an alkylamine-introduced polysaccharide or a modified product thereof in the presence of transglutaminase, thereby effecting the formation of amido linkage between the &ggr;-carboxyamido group of said glutamine residue and the primary amino group of the amino group donor.
NH
2
(CH
2
)
n
T(CH
2
)
m
(OCH
2
CH
2
)
p
OR  (I)
NH
2
(CH
2
)
n
T(CH
2
)
m
(OCH
2
CH
2
)
p
T(CH
2
)
n
NH
2
  (II)
In the above general formulae (I) and (II), n is an integer of 1 to 8, m is an integer of 0 to 2, p is an integer of 1 to 400, T represents a bond selected from —O—, —C(O)O—, —OC(O)—, —NHCO—, —OCNH—, —NHCONH—, —OOCNH— or —HNCOO—, and R represents a hydrogen atom, a lower alkyl group having 1 to 5 carbon atoms or a lower acyl group having 2 to 6 carbon atoms.
In the above general formula (III), n is an integer of 1 to 8, q is an integer of 2 to 6, and R represents a galactose, a glucose or an N-acetylgalactosamine.
H
2
N—(CH
2
)
n
—NH—CO—T′  (IV)
In the above general formula (IV), n is an integer of 1 to 8, and T′ represents the residue of a polyamino acid excluding its terminal carboxyl group. In this connection, component amino acids of the polyamino acid are not particularly limited, but an acidic or basic amino acid may be preferred from the viewpoint of solubility. Also, preferred amino acid polymerization degree is 1 to 400.
The alkylamine-introduced polysaccharide or a modified product thereof is a compound obtained by subjecting the reducing end-group of a polysaccharide to reductive amination in the presence of a compound represented by the following general formula (1) or a salt thereof (hydrochloride or the like) and then eliminating the protecting group V.
V—NH—(CH
2
)
n
—NH
2
  (1)
In the above general formula (1), n is an integer of 1 to 8, and V represents a protecting group generally used for the protection of an amino group such as Fmoc (9H-fluoren-9-ylmethoxycarbonyl).
Examples of such polysaccharide include pullulan, dextran, dextran sulfate, chondroitin sulfate and carboxymethylated products thereof, having a molecular weight of 1×10
3
to 1×10
5
dalton.
The alkylamine-introduced polysaccharide or a modified product thereof can be obtained for example by dissolving a polysaccharide or a carboxymethylated polysaccharide in an acetic acid-water-DMF mixture solution or in water, successively adding to the resultant solution a compound of the above general formula (1) in an amount of about 10 to 1000 equivalents, more preferably about 100 equivalents, per 1 equivalent of the polysaccharide and a reducing agent such as sodium cyanoboron hydride, maintaining the mixture at a temperature of from room temperature to 80° C., more preferably from 50 to 70° C., for 1 to 3 days whereby the reaction is carried out, and then eliminating the amino group-protecting group with an alkali such as diethylamine or 0.1 N NaOH.
The aforementioned physiologically active protein has a molecular weight of 5×10
3
or more, and dansyl cadaverine can be introduced thereinto when 10 &mgr;M of said physiologically active protein and monodansylcadaverine in an amount of 100 equivalents per 1 equivalent of said physiologically active protein are kept at 37° C. for 60 minutes in a 100 mM Tris-HCl buffer solution containing 10 mM CaCl
2
and having a pH value of 7.5, in the presence of the aforementioned transglutaminase.
The present invention will be described in detail, as follows.
Examples of the physiologically active protein to be modified with polyethylene glycol, a polysaccharide, a polyamino acid or a branched sugar derivative by the method of the present invention include human blood plasma components such as albumin, immunoglobulin, blood coagulation factors and the like; enzymes such as superoxide dismutase, urokinase and the like; hormones such as growth hormone, erythropoietin and the like; cell growth regulating factors such as cell growth factors, cell growth inhibitors and the like; immune reaction controlling factors such as cell differentiation, induction, stimulation and the like factors; and biologically active cellular proteins such as monokine, cytokine, lymphokine and the like. Origins of these physiologically active proteins are not particularly limited, which include animals, plants and microorganisms. Also useful are proteins which are produced by integrating genes of the above proteins into
Escherichia coli,
yeast, Chinese hamster ovary and the like and expressing the integrated genes.
Physiologically active proteins if having a molecular weight of smaller than 5×10
3
have a smaller number of

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