Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2000-04-03
2004-09-21
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
C435S212000
Reexamination Certificate
active
06794159
ABSTRACT:
FIELD OF THE INVENTION
This invention describes a method of removing N-terminal alanine residues from polypeptides, preferably recombinant proteins, using an aminopeptidase derived from the marine bacterium
Aeromonas proteolytica
. Accordingly, Aeromonas aminopeptidase (AAP; E.C. 3.4.11.10) can be used to remove N-terminal alanyl residues from derivatives of human somatotropin (hST, human growth hormone, or hGH), porcine somatotropin (pST), and bovine somatotropin (bST), for example, to yield proteins having their native amino acid sequences. The enzyme reactions can be carried out in free solution, or the AAP can be immobilized on a solid support, for reactions carried out in vitro. An efficient method for converting Ala-hGH to hGH, for example, comprises expression of Ala-hGH in
E. coli
, recovery of inclusion bodies, solubilization and refolding in detergent, detergent removal by ultrafiltration, selective precipitation, enzyme cleavage, followed by two column chromatography steps.
BACKGROUND OF THE INVENTION
Recombinant proteins that mimic or have the same structure as native proteins are highly desired for use in therapeutic applications, as components in vaccines and diagnostic test kits, and as reagents for structure/function studies. Mammalian, bacterial, and insect cells are commonly used to express recombinant proteins for such applications. Bacterial expression systems, however, are often used when large quantities of protein are needed for experimental or clinical studies, and the protein is capable of being refolded to its proper conformation. Bacterial systems, in particular, offer significant cost advantages over other expression vector systems when eukaryotic post-translational modifications (e.g., glycosylation) are not required, or desired, in the final protein product.
Recombinant proteins expressed in bacteria, such as
E. coli
, are often sequestered into insoluble inclusion bodies. Heterologous proteins harvested from inclusion bodies often retain an additional amino acid residue such as methionine at their amino terminus. This methionine residue (encoded by the ATG start codon) is often not present, however, on native or recombinant proteins harvested from eukaryotic host cells. The amino termini of many proteins made in the cytoplasm of
E. coli
, however, are processed by enzymes, such as methionine aminopeptidase (Ben Bassat et al.,
J. Bacteriol
. 169: 751-757, 1987), so that upon expression the methionine is ordinarily cleaved off the N-terminus.
The amino acid composition of protein termini are biased in many different manners (Berezovsky et al.,
Protein Engineering
12(1): 23-30, 1999). Systematic examination of N-exopeptidase activities led to the discovery of the ‘N-terminal’-or ‘N-end rule’: the N-terminal (f)Met is cleaved if the next amino acid is Ala, Cys, Gly, Pro, Ser, Thr, or Val. If this next amino acid is Arg, Asp, Asn, Glu, Gln, Ile, Leu, Lys or Met, the initial (f)Met remains as the first amino acid of the mature protein. The radii of hydration of the amino acid side chains was proposed as physical basis for these observations (Bachmain et al.,
Science
, 234: 179-186, 1986; Varshavsky,
Cell
, 69: 725-735, 1992). The half-life of a protein (from 3 min to 20 hours), is dramatically influenced by the chemical structure of the N-terminal amino acid (Stewart et al.,
J. Biol. Chem
., 270: 25-28, 1995; Griegoryev et al.,
J. Biol., Chem
., 271: 28521-28532, 1996). Site-directed mutagenesis was subsequently used to confirm the ‘N-end rule’ by monitoring the life-span of recombinant proteins containing altered N-terminal amino acid sequences (Varshavsky,
Proc. Natl. Acad. Sci. USA
, 93: 12142-12149, 1996). A statistical analysis of the amino acid sequences at the amino termini of proteins suggested that Met and Ala residues are over-represented at the first position, whereas at positions +2 and +5, Thr is preferred (Berezovsky et al.,
Protein Engineering
12(1): 23-30, 1999). C terminal biases, however, show a preference for charged amino acids and Cys residues (Berezovsky et al.,
Protein Engineering
, 12(1): 23-30, 1999).
Recombinant proteins that retain the N-terminal methionine, in some cases, have biological characteristics that differ from the native species lacking the N-terminal methionine. Human growth hormone that retains its N-terminal methionine (Met-hHG), for example, can promote the induction of undesireable antibodies, compared to hGH purified from natural sources or recombinant hGH that is prepared in such a way that has the same primary sequence as native hGH (lacking an N-terminal methionine). Low-cost methods of generating recombinant proteins that mimic the structure of native proteins are often highly desired for therapeutic applications (Sandman et al.,
Bio/Technology
13:504-6 (1995)).
One method of preparing native proteins in bacteria is to express the desired protein as part of a larger fusion protein containing a recognition site for an endoprotease that specifically cleaves upstream from the start of the native amino acid sequences. The recognition and cleavage sites can be those recognized by native signal peptidases, which specifically clip the signal peptide of the N-terminal end of a protein targeted for delivery to a membrane or for secretion from the cell. In other cases, recognition and cleavage sites can be engineered into the gene encoding a fusion protein so that recombinant protein is susceptible to other non-native endoproteases in vitro or in vivo. The blood clotting factor Xa, collagenase, and the enzyme enterokinase, for example, can be used to release different fusion tags from a variety of proteins. Economic considerations, however, generally preclude use of endoproteases on a large scale for pharmaceutical use.
Another method of preparing native proteins in bacteria is to use the enzyme methionine aminopeptidase (MAP) to process the N-terminal methionine from
E. coli
-derived recombinant proteins. Met-hGH, for example, can be treated with MAP to generate hGH. U.S. Pat. Nos. 4,870,017 and 5,013,662 describe the cloning, expression, and use of
E. coli
methionine aminopeptidase to remove Met from a variety of peptides and Met-IL-2. The ability to release amino acids from a variety of peptide substrates was analyzed, revealing that MAP cleaves only N-terminal methionine on peptides that are at least three amino acids long. The nature of amino acids in the second and third positions also appear to be significant. Methionine was released, for example, from Met-Ala-Met; Met-Gly-Met, Met-Ala-Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Leu (SEQ ID NO: 1), and Met-Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Gln-Cys (SEQ ID NO: 2), but not Met-Phe-Gly, Met-Leu-Phe, Met-Met-Met, among others. No amino acids were released from Leu-Ala-Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Leu (SEQ ID NO: 3), Ala-Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Leu (SEQ ID NO: 4), or Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Gln-Cys (SEQ ID NO: 5).
WO 84/02351 discloses a process for preparing ripe (native) proteins such as human growth hormone or human proinsulin from fusion proteins using leucine aminopeptidase. A fusion protein having the amino acid sequence (Y
m
. . . Y
2
Y
1
)-(Pro)
p
-(X
1
X
2
. . . X
n
) in which the (Y
m
. . . Y
2
Y
1
)-(Pro)
p
is the pro-sequence and the rest is the ripe protein, m is an integer greater than 2, Y is an arbitrary amino acid, p is 0, if X
1
or X
2
is Pro, and 1 if X, or X
1
is different from Pro, X is an arbitrary amino acid, and n is an integer greater than or equal to 4, is converted by stepwise cleavage with aminopeptidase removing amino acids Y
m
. . . Y
2
if p=1 or X
1
=Pro, or the groups Y
m
. . . Y
2
-Y
1
if X
2
=Pro and then the two amino acids Y
1
-Pro if p=1 are cleaved off enzymatically in one or two steps in a manner known per se, and similarly Y
1
alone is cleaved off, if X
1
=Pro.
European Patent Application EP 0 204 527 A1 discloses a method of removing the N terminal methionine from proteins of the formula H-Met-X-Pro-Y-OH to yield a pr
Taylor Douglas W.
Tou Jacob S.
Pharmacia Corporation
Weber Jon P.
LandOfFree
Method of removing n-terminal alanine residues from... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of removing n-terminal alanine residues from..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of removing n-terminal alanine residues from... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3266772