Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1995-07-21
2001-12-18
Whisenant, Ethan (Department: 1655)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S380000
Reexamination Certificate
active
06331611
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of the invention is expression and purification of cloned human alpha-fetoprotein.
Alpha-fetoprotein (AFP) is a serum protein normally found at significant levels only in fetal blood. In adult blood increased alpha-fetoprotein levels are associated with liver regeneration and certain carcinomas.
The specific function of alpha-fetoprotein is not known. Suggested roles for the protein include: fetal albumin; protection from maternal immune attack; and protection from maternal estrogen.
Morinaga et al. (
Proc. Natl. Acad. Sci.
USA 80:4604, 1983) report the cloning of human AFP.
Innis et al. (
Arch. Biochem. Biophys.
195:128, 1979) report the cloning of an approximately 950 base-pair fragment of human AFP into
E. coli
plasmid pBR322.
Nishi et al. (
J. Biochem.
104:968, 1988) report the expression of rat AFP in
E. coli
and
Saccharomyces cerevisiae.
Nishi et al. also report that, in an estradiol-binding assay, yeast-produced rat rAFP is as active a authentic AFP, while bacterial-produced rat rAFP is essentially inactive. Further, when characterized by radioimmunoassay or an Ouchterlony double immunodiffusion assay, yeast-produced rat rAFP bears a closer resemblance to authentic rat AFP than does bacterial-produced rat rAFP. Nishi et al. state that:
“In the Ouchterlony double immunodiffusion test, authentic and yeast rAFP formed a completely fused precipitin line with antibody to rat AFP while
E. coli
rAFP showed a reaction of partial identity in a similar test . . . . It is likely that the functionally active yeast rAFP in this study had the correct pairs of disulfide bridges. On the other hand the
E. coli
rAFP probably failed to form them”.
Yamamoto et al. (
Life Sciences
46:1679, 1990) report the expression of human AFP in yeast and report that the rAFP so produced was “indistinguishable immunologically from authentic AFP.”
Giuliani et al. (
Protein Engineering
2:605, 1989) report the expression of a portion of human AFP (amino acids 38 to 119) in
E. coli.
Japanese Patent Application 88158596 reports a method for preparing recombinant human domain I AFP in
E. coli.
SUMMARY OF THE INVENTION
In general, the invention features substantially pure biologically-active recombinant human alpha-fetoprotein produced using a prokaryotic cell. In preferred embodiments, the pure recombinant human alpha-fetoprotein of includes a sequence substantially identical to amino acids 1 to 590 of
FIG. 1
(SEQ ID NO: 4).
In related aspects, the invention also features substantially pure biologically-active recombinant human alpha-fetoprotein including a sequence that is substantially identical to either amino acids 1 to 389 of
FIG. 1
(SEQ ID NO: 4) or a fragment thereof; amino acids 198 to 590 of
FIG. 1
(SEQ ID NO: 5) or a fragment thereof; amino acids 198 to 389 of
FIG. 1
(SEQ ID NO: 6) or a fragment thereof; amino acids 390 to 590 of
FIG. 1
(SEQ ID NO: 7) or a fragment thereof; and amino acids 267 to 590 of
FIG. 1
(SEQ ID NO: 8) or a fragment thereof.
Such recombinant human alpha-fetoprotein may be used in therapeutic compositions.
In another related aspect, the invention features a method for using an insect cell for producing biologically active recombinant human alpha-fetoprotein or a fragment or analog thereof involving
a) providing a transformed insect cell (e.g.,
Spodoptera frugiperda
) including a recombinant DNA molecule encoding the human alpha-fetoprotein or fragment or analog thereof operably linked to an expression control element which directs the expression of the human alpha-fetoprotein or fragment or analog thereof;
b) culturing the transformed cell; and
c) recovering the biologically active human alpha-fetoprotein or fragment or analog thereof.
The invention also features substantially pure human alpha-fetoprotein or fragment or analog thereof produced using the above-described method, and therapeutic compositions including substantially pure human alpha-fetoprotein or fragment or analog thereof produced using an insect cell.
By “human alpha-fetoprotein” is meant a polypeptide having substantially the same amino acid sequence as the protein encoded by the human alpha-fetoprotein gene. Morinaga et al. (
Proc. Natl. Acad. Sci.
USA 80:4604, 1983) reports the sequence of cDNA complementary to human alpha-fetoprotein.
By “expression control element” is meant a nucleotide sequence which includes recognition sequences for factors that control expression of a protein coding sequence to which it is operably linked. Accordingly, an expression control element generally includes sequences for controlling both transcription and translation, for example, promoters, ribosome binding sites, repressor binding sites, and activator binding sites.
By “substantially the same amino acid sequence” is meant a polypeptide that exhibits at least 80% homology with naturally occurring amino acid sequence of human alpha-fetoprotein, typically at least about 85% homology with the natural human alpha-fetoprotein sequence, more typically at least about 90% homology, usually at least about 95% homology, and more usually at least about 97% homology with the natural human alpha-fetoprotein sequence. The length of comparison sequences will generally be at least 16 amino acids, usually at least 20 amino acids, more usually at least 25 amino acids, typically at least 30 amino acids, and preferably more than 35 amino acids.
Homology, for polypeptides, is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisoonsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Protein analysis software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
As used herein, the term “substantially pure” describes a protein or polypeptide which has been separated from components which naturally accompany it. Typically, a protein of interest is substantially pure when at least 60% to 75% of the total protein in a sample is the protein of interest. Minor variants or chemical modifications typically share the same polypeptide sequence. A substantially pure protein will typically comprise over about 85 to 90% of the protein in sample, more usually will comprise at least about 95%, and preferably will be over about 99% pure. Normally, purity is measured on a chromatography column, polyacrylamide gel, or by HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from the native contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Thus the term can be used to describe polypeptides and nucleic acids derived from eukaryotic organisms which have been synthesized in
E. coli
and other prokaryotes.
The present invention provides for substantially pure human alpha-fetoprotein. Various methods for the isolation of human AFP from biological material may be devised, based in part upon the structural and functional properties of human alpha-fetoprotein. Alternatively, anti-AFP antibodies may immobilized on a solid substrate to generate a highly specific affinity column for purification of human AFP.
Besides substantially full-length polypeptides, the present invention provides for biologically active recombinant fragments of alpha-fetoprotein. For example, fragments active in ligand binding or immunosuppression.
The natural or synthetic DNA fragments coding for human alpha-fetoprotein or a desired fragment thereof will be incorporated into DNA constructs capable of introduction to and expression in cel
Clark & Elbing LLP
McGill University
Whisenant Ethan
LandOfFree
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