Clostripain catalyzed amidation of peptides

Details Clostripain catalyzed amidation of peptides Clostripain catalyzed amidation of peptides

1998-12-16

2002-10-08

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

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

Enzymatic production of a protein or polypeptide

C435S220000

Reexamination Certificate

active

06461834

ABSTRACT:

BACKGROUND OF THE INVENTION
In vitro DNA manipulation allows the transfer of foreign genetic information into a host cell to affect efficient expression of endogenous and foreign proteins in a wide variety of host cells, such as microbial hosts. Recombinant DNA techniques have made possible the selection, amplification and manipulation of expression of proteins and peptides.
Some modifications to a recombinantly produced protein or peptide, however, cannot be accomplished by altering the DNA sequence. Many naturally occurring proteins and peptides contain a C-terminal amino acid residue that has an &agr;-carboxamide group but the amide group is not produced directly through expression. Instead, a precursor protein is produced by genetic expression and the amide is introduced in vivo by enzymatic modification of the precursor protein. In vitro, a variety of methods exist for converting a C-terminal &agr;-carboxylic acid group into an &agr;-carboxamide group, however, the available methods generally have limitations in terms of a number of factors, such as the reaction conditions, selectivity, type of reagent(s) employed and/or types of substrates which may be used.
Moreover, many small foreign proteins and oligopeptides often cannot be successfully overproduced in most cellular hosts, since the host may reassimilate the peptide after expression. For example, where the size of the desired peptide is no more than about 60 to 80 amino acid units in length, degradation rather than end product accumulation usually occurs.
In response to this problem, small peptides have typically been expressed either as part of fusion proteins which include a second larger peptide (e.g., &bgr;-galactosidase or chloramphenicol acetyl transferase) or as a recombinant construct which includes multiple copies of the desired peptide (a multicopy construct). In either instance, the initially expressed construct generally needs to be cleaved to produce the desired peptide(s). Very often, the recombinant construct is cleaved to produce a precursor peptide(s) which may then be subjected to posttranslational modification to produce the desired peptide(s). It would be extremely advantageous to have additional method(s) which would allow cleavage of a peptide precursor to be carried out simultaneously with the introduction of an &agr;-carboxamide group into the C-terminal amino acid residue of the cleavage product.
SUMMARY OF THE INVENTION
The invention relates to a method of producing a polypeptide having a C-terminal &agr;-carboxamide group. It particularly concerns an enzymatic modification of selected arginine-containing substrate polypeptides which result in cleavage of the substrate polypeptide to form a product polypeptide having a C-terminal &agr;-carboxamide group. The method includes contacting a substantially aqueous solution which includes (a) the substrate polypeptide (“first polypeptide”) and (b) ammonia reagent with (c) clostripain. The substrate polypeptide includes at least one copy of a core amino acid sequence and typically includes more than one copy of the core amino acid sequence (i.e., a multicopy construct). The C-terminal residue of the core amino acid sequence is an arginine residue which is bonded to the adjacent amino acid residue through an &agr;-carboxyl peptide bond (i.e., an “Arg-Xaa” peptide linkage). Since clostripain is an endopeptidase, the Xaa amino acid residue represents an amino acid residue which has its &agr;-carboxylic group bonded to either another amino acid residue through a peptide bond (“Arg-Xaa-Xaa′”) or to a carboxyl blocking group (“Arg-Xaa-R”). Carboxyl blocking groups are organic functional groups which replace the acid functionality of the carboxylic acid (the “—OH” portion of the —C(O)OH group) and are capable of being cleaved or hydrolyzed to regenerate a carboxylic acid group (“—C(O)OH group”). Examples of suitable carboxyl blocking groups include groups include the alkoxy portion of an ester group (e.g., the ethoxy or benzyloxy portion of a —C(O)OR group) and the —NRR′ portion of a non-peptide amide linkage (e.g., the NRR′ portion of a —C(O)NRR′ group). The —NRR′ portion may be unsubstituted (i.e., NH
2
) or may be substituted with one or two substituents (e.g., NHEt or NMe
2
). When such a substrate polypeptide in an aqueous-based solution is contacted with the ammonia reagent in the presence of clostripain, the substrate polypeptide is cleaved at the &agr;-carboxyl peptide bond of the arginine residue and a second polypeptide (“product polypeptide”) having a C-terminal arginine residue containing an &agr;-carboxamide group (“Arg-NH
2
” residue) is produced.
As employed herein, the term “ammonia reagent” refers to a reagent which includes “dissolved free ammonia” (i.e., NH
3
dissolved in the aqueous solution) and/or is capable of releasing free dissolved ammonia in an aqueous solution under conditions where clostripain will amidatively cleave an arginine-containing peptide. For example, the ammonia reagent may include one or more salts of ammonia in equilibrium with dissolved free ammonia. The relative amounts of free ammonia and the various salts will generally be a function of various parameters well known to those skilled in the art, such as the pH of the solution, the relative concentrations of different anions present in the solution and/or the solubility of particular individual salts of ammonia. Since the pK
a
of ammonia (“NH
3
”) is about 9.2 in aqueous solution, a substantial portion of the ammonia reagent will generally be present as free ammonia at pHs of about 9 or above. In solutions with a pH above the pK
a
of ammonia, more than half of the ammonia will generally be present either as dissolved free ammonia or as ammonium hydroxide (“NH
4
OH”). It also will be understood that the anion portion of a salt of ammonia generally undergoes a very rapid exchange with other anions present in a given solution. Thus, if a pH 10.0 aqueous solution includes chloride salt(s) (“Cl
−
”), acetate salt(s) (“OAc
−
”) and sulfate salt(s) (“SO
4
=
”), ammonia reagent in this solution will likely include ammonium chloride (“NH
4
Cl”), ammonium acetate (“NH
4
OAc”) and ammonium sulfate (“(NH
4
)
2
SO
4
”), as well as dissolved free ammonia and ammonium hydroxide (“NH
4
OH”). The present method typically employs the aqueous-based reaction medium which includes at least about 0.5 M ammonia reagent. It appears that a concentration of ammonia reagent of about 0.75 M to about 1.5 M strikes a balance between optimizing the rate and yield of amidated product formation while avoiding substantial inhibition of the enzyme activity. As employed herein, the concentration of ammonia reagent is based on the equivalents of free dissolved NH
3
that are present in the medium. One embodiment of the present method includes forming a solution of the substrate polypeptide in a first aqueous-based medium having a pH of no more than about 8.5 and, preferably having a substantially neutral pH. The substrate polypeptide may be cleaved at the &agr;-carboxyl peptide bond to produce the product polypeptide having a C-terminal Arg-NH
2
residue by adjusting the pH of the solution to at least about 9.0 and, typically between about 9.0 to about 11.0, and contacting the substrate polypeptide with an immobilized form of clostripain (“immobilized clostripain”) in the presence of ammonia reagent. The substrate and ammonia reagent are preferably contacted with the immobilized clostripain for no more than about 20 minutes and, more preferably, for no more than about 5 minutes.
Typically, the first aqueous-based medium is mixed with a basic aqueous solution (“alkaline medium”) to raise the pH shortly before the substrate polypeptide and ammonia reagent are brought into contact with the immobilized clostripain. One manner of practicing this embodiment of the invention is to pack resin containing immobilized clostripain in a chromatography column. The substrate stock solution and basic solutions are mixed just prior to introduction to the column,

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