# US 5,707,822 A · Gene serum opacity factor

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- Number: US 5,707,822 A
- Document ID: US5707822A
- Status: Granted
- Kind: A
- Application number: 08/294,189
- Filed: 1994-08-22
- Granted: 1998-01-13
- Earliest claimed: 1993-09-01
- Assignees: The Rockefeller University
- Inventors: Vincent A. Fischetti; Jasna Rakonjac; John Robbins
- Web page: https://patentgrep.com/patent/US5707822A

## CPC

- C12N 9/52: derived from bacteria {or Archaea}
- C12Q 1/37: involving peptidase or proteinase
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Methods and compositions are provided for cloning and expression of serum opacity factor of Streptococcus pyogenes genes. The portion produced by the recombinant DNA techniques described herein may be employed in qualitative and quantitative testing for high density lipoprotein, as a fibronectin binding factor and for the regulation of high density lipoprotein in a mammal. The gene may further be employed as a molecular probe for accurate identification of opacity factors from various strains of Streptococcus pyogenes.

## Claims

### Claim 1

A process for producing a polypeptide having apolipoproteinase activity which comprises culturing a prokaryotic unicellular organism containing a recombinant plasmid comprising a DNA sequence coding for said polypeptide and capable of being replicated, transcribed and translated in the unicellular organism, and isolating said polypeptide from the culture, said polypeptide being encoded by a sequence selected from the group consisting of:
(1) the DNA sequences of FIG. 4 (SEQ ID NO. 2);
(2) DNA sequences that hybridize to the DNA sequence of (1) under high stringency hybridization conditions; and
(3) DNA sequences that encode an amino acid sequence encoded by the DNA sequences of (1) or (2), and enzyme active segments thereof.

### Claim 2 (depends on 1)

A process of claim 1 wherein the DNA sequence is sof22 and codes for SOF22.

### Claim 3 (depends on 1)

A process of claim 1 wherein the DNA sequence is SOF22.1 and codes for the fragment of SOF 22 which enzymatically hydrolyzes apoprotein A1 from high density lipoprotein.

### Claim 4

A process for preparing a prokaryotic unicellular organism having a DNA sequence coding for a polypeptide having apolipoproteinase activity which comprises introducing a recombinant plasmid comprising a DNA sequence coding for said polypeptide and capable of being replicated, transcribed and translated in the unicellular organism, said sequence selected from the group consisting of:
(1) the DNA sequence of FIG. 4 (SEQ ID NO. 2);
(2) DNA sequence that hybridize to the DNA sequence of (1) under high stringency hybridization conditions; and
(3) DNA sequences that encode an amino acid sequence encoded by the DNA sequences of (1) or (2), and enzyme active segments thereof.

### Claim 5 (depends on 4)

A process of claim 4 wherein the DNA sequence is sof22 and codes for SOF22.

### Claim 6 (depends on 4)

A process of claim 4 wherein the DNA sequence is sof22.1 and codes for the fragment of SOF22 which enzymatically hydrolyzes apoprotein A1 from high density lipoprotein.

### Claim 7

A purified DNA sequence coding for a polypeptide having apolipoproteinase activity, said sequence being selected from the group consisting of:
(1) the DNA sequence of FIG. 4 (SEQ ID NO. 2);
(2) DNA sequence that hybridize to the DNA sequence of (1) under high stringency hybridization conditions; and
(3) DNA sequences that encode an amino acid sequence encoded by the DNA sequences of (1) or (2), and enzyme active segments thereof.

### Claim 8 (depends on 7)

A purified DNA sequence of claim 7 which codes for SOF22.

### Claim 9 (depends on 7)

A purified DNA sequence of claim 7 which codes for the fragment of SOF22 which enzymatically hydrolyzes apoprotein A1 from high density lipoprotein.

### Claim 10 (depends on 7)

A cloning vector comprising the DNA sequence of claim 7.

### Claim 11 (depends on 7)

A cloning vector of claim 7 which is LSOF22.4 and codes for the production of SOF22.

### Claim 12 (depends on 7)

A cloning vector of claim 7 which is pSOF22.1 and codes for the production of the fragment of SOF22 which enzymatically hydrolyzes apoprotein A1 from high density lipoprotein.

### Claim 13 (depends on 10)

A unicellular organism containing the vector of claim 10.

### Claim 14 (depends on 11)

A unicellular organism containing the cloning vector of claim 11.

### Claim 15 (depends on 12)

A unicellular organism containing the cloning vector of claim 12.

### Claim 16 (depends on 12)

An Escherichia coli bacterium containing the cloning vector of claim 12.

### Claim 17

A purified DNA probe capable of binding to a DNA sequence coding for a polypeptide having apolipoproteinase activity or any portion of said sequence having such activity, said sequence being selected from the group consisting of:
(1) the DNA sequence of FIG. 4 (SEQ ID NO. 2);
(2) DNA sequence that hybridize to the DNA sequence of (1) under high stringency hybridization conditions; and
(3) DNA sequences that encode an amino acid sequence encoded by the DNA sequences of (1) or (2), and enzyme active segments thereof.

### Claim 18

A diagnostic test for high density lipoprotein in a body fluid which comprises: (a) contacting said body fluid with a polypeptide having apolipoproteinase activity, said polypeptide being encoded by one of the following sequences:
1. the DNA sequence of FIG. 4 (SEQ ID NO:2);
2. DNA sequences that hybridize to the DNA sequences of (1) under high stringency hybridization conditions; and
3. DNA sequences that encode an amino acid sequence encoded by the DNA sequences of (1) or (2);
(b) detecting cleavage of apoprotein A1; and
(c) correlating said cleavage with the presence of high density lipoprotein.

### Claim 19 (depends on 18)

The test of claim 18 wherein the polypeptide is the enzymatic fragment of SOF 22 which enzymatically hydrolyzes apoprotein A1 from high density lipoprotein.