Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-04-24
2003-03-04
Achutamurtry, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S320100, C435S252800, C536S023200, C536S024300, C536S024100
Reexamination Certificate
active
06528297
ABSTRACT:
This application is a National Stage application under 35 U.S.C. §371 of International Application Serial No. PCT/CN99/00130 filed Aug. 30, 1999 and published in Chinese as WO 00/12721 on Mar. 9, 2000, which claims priority to Chinese Application Serial No. 98111042.8, filed Aug. 31, 1998.
The invention relates to a new polynucletide ,the polypeptide encoded by said polynucleotide, the uses of said polynucleotide and polypeptide, and the methods for preparing same. In particular, the invention relates to a new member of the lysozyme family.
Lysozyme exists ubiquitously in all parts of organisms, including various tissues, organs, and sera; it is especially abundant in egg white. Lysozyme is mainly secreted by the epithelial cell of certain glands and some kinds of leukocyte.
Lysozyme was first reported by Fleming, et al. in 1922. Afterward, lysozyme has been widely studied. A lot of papers concerning its crystal structure, protein catalytic domains, catalytic dynamics, immunology, molecular evolutionary, and so on, have been published. Lysozyme is one of the proteins that are studied most extensively and intensively. However, the study on lysozyme gene is not yet sufficient. Nowadays, only a few lysozyme genes from different species, such as
E.coli
T4, salmonella P22 phage, bacillus &phgr; phage and chicken, etc., have been cloned. (1983 J. Mol. Biol. 165. 229-248; 1985 Virology 143, 280-289; 1987 Proc. Natl. Acad. Sci. USA, 77, 5759-5763). The cloning about human lysozyme gene was also reported (1988, Gene 66,223-234).
The main function of lysozyme is to hydrolyze the beta(1-4) glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylgluconic acid (NAG) of the bacterial cell wall. In the organism, lysozyme can act as a nonspecific immune molecule against bacterial infections, and as a digestive enzyme in enteron and some mollusks which live on bacteria. Further, lysozyme has the function of inhibiting tumor growth. Therefore, lysozyme has important applications in both industry and medicine.
One purpose of the invention is to provide a new polynucleotide which encodes a new member of lysozyme gene family. The new human lysozyme is named LYC1.
Another purpose of the invention is to provide a new member of lysozyme protein family, which is named LYC1.
Still another purpose of the invention is to provide a new method for preparing said new human lysozyme by recombinant techniques.
The invention also relates to the uses of said human lysozyme and its coding sequence.
In one aspect, the invention provides an isolated DNA molecule, which comprises a nucleotide sequence encoding a polypeptide having human LYC1 protein activity, wherein said nucleotide sequence shares at least 70% homology to the nucleotide sequence of nucleotides 84-530 in SEQ ID NO: 4, or said nucleotide sequence can hybridize to the nucleotide sequence of nucleotides 84-530 in SEQ ID NO: 4 under moderate stringency. Preferably, said nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 5. More preferably, the sequence comprises the nucleotide sequence of nucleotides 84-530 in SEQ ID NO: 4.
Further, the invention provides an isolated LYC1 polypeptide, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: 5, its active fragments, and its active derivatives. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 5.
The invention also provides a vector comprising said isolated DNA.
The invention further provides a host cell transformed with said vector.
In another aspect, the invention provides a method for producing a polypeptide with the activity of LYC1 protein, which comprises:
(a) forming an expression vector of LYC1 protein comprising the nucleotide sequence encoding the polypeptide having the activity of LYC1 protein, wherein said nucleotide sequence is operably linked with an expression regulatory sequences, and said nucleotide sequence shares at least 70% homology to the nucleotide sequence of positions 84-530 in SEQ ID NO: 4;
(b) introducing the vector of step (a) into a host cell, thereby forming a recombinant cell of LYC1 protein;
(c) culturing the recombinant cell of step (b) under the conditions suitable for the expression of LYC1 polypeptides;
(d) isolating the polypeptides having the activity of LYC1 protein.
In one embodiment of the present invention, the isolated polynucleotide has a full length of 583 nucleotides, whose detailed sequence is shown in SEQ ID NO: 4. The open reading frame (ORF) locates at nucleotides 84-530.
In the present invention, the term “isolated” or “purified” or “substantially pure” DNA refers to a DNA or fragment which has been isolated from the sequences which frank it in a naturally occurring state. The term also applied to DNA or DNA fragment which has been isolated from other components naturally accompanying the nucleic acid and from proteins naturally accompanying it in the cell.
In the present invention, the term “LYC1 protein encoding sequence” or “LYC1 polypeptide encoding sequence” refers to a nucleotide sequence encoding a polypeptide having the activity of LYC1 protein, such as the nucleotide sequence of positions 84-530 in SEQ ID NO: 4 or its degenerate sequence. The degenerate sequences refer to the sequences formed by replacingone or more codons in the ORF of 84-530 in SEQ ID NO: 4 with degenerate codes which encode the same amino acid. Because of the degeneracy of codon, the sequence having a homology as low as about 70% to the sequence of nucleotides 84-530 in SEQ ID NO: 4 can also encode the sequence shown in SEQ ID NO: 5. The term also refers to the nucleotide sequences that hybridize with the nucleotide sequence of nucleotides 84-530 in SEQ ID NO: 4 under moderate stringency or preferably under high stringency. In addition, the term also refers to the sequences having a homology at least 70%, preferably 80%, more preferably 90% to the nucleotide sequence of nucleotides 84-530 in SEQ ID NO: 4.
The term also refers to variants of the sequence in SEQ ID NO: 4, which are capable of coding for a protein having the same function as human LYC1 protein. These variants includes, but are not limited to: deletions, insertions and/or substitutions of several nucleotides (typically 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) and additions of several nucleotides (typically less than 60, preferably 30, more preferably 10, most preferably 5) at 5′ end and/or 3′ end.
In the present invention, “substantially pure” proteins or polypeptides refers to those which occupy at least 20%, preferably at least 50%, more preferably at least 80%, most preferably at least 90% of the total sample material (by wet weight or dry weight). Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, PAGE or HPLC analysis. A substantially purified polypeptides is essentially free of naturally associated components.
In the present invention, the term “LYC1 polypeptide” or “LYC1 protein” refers to a polypeptide having the activity of LYC1 protein comprising the amino acid sequence of SEQ ID NO: 5. The term also comprises the variants of said amino acid sequence which have the same function of human lysozyme. These variants include, but are not limited to, deletions, insertions and/or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) at C-terminal and/or N-terminal. For example, the protein function are usually unchanged when an amino residue is substituted by a similar or analogous one. Further, the addition of one or several amino acids at C-terminal and/or N-terminal will not change the function of protein. The term also includes the active fragments and derivatives of LYC1 protein.
The variants of polypeptide include homologous sequences, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybri
Bi Anding
Fu Qiang
Yu Long
Zhang Honglai
Zhao Yong
Achutamurtry Ponnathapu
Baker & Botts LLP
Institute of Genetics Fudan University
Pak Yong
LandOfFree
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