WO1989001030A1

WO1989001030A1 - Production of lysin-free phage inoculum and production of lysin

Info

Publication number: WO1989001030A1
Application number: PCT/US1988/002433
Authority: WO
Inventors: Huei-Hsiung Yang; Stephen F. Hiu; John L. Harris
Original assignee: Igene Biotechnology, Inc.
Priority date: 1987-07-27
Filing date: 1988-07-22
Publication date: 1989-02-09
Also published as: US4859597A; CA1298221C; EP0324018A4; JPH02500166A; EP0324018A1

Abstract

A method for producing a lysin-free phage inoculum, which comprises: a) inoculating a growing Streptococcal culture with phage; b) incubating the culture for plurality of lytic cycles of phage until the cells are completely lysed to obtain a lysate; and c) removing cell debris and free lysin from the lysate to form a lysin-free phage suitable for use as an inoculum. Also disclosed is a method of producing lysin.

Description

Production of Lysin-free Phage Inoculum and Production of Lysin

DESCRIPTION OF THE INVENTION

1. Technical Field of the Invention

This invention relates to a process for the production of high titers of phage-associated lysin by growing Streptococcus sp. cells to high density and infecting the Streptococcal culture with an appropriate amount of phage suspension containing no free lysin. In the process, the phages are separated from cell debris and active lysin and are concentrated. The concentrated phage solution is used to infect growing Streptococcal cells at high cell density. After inoculation with phage, the infected Streptococci are harvested at a precise time and resuspended in buffer to release maximal amounts of phage-associated lysin.

2. Background Art Lysates of Group C Streptococci which are infected with Cl bacteriophage contain the enzyme lysin (N-acetylmuramoyl- L-alanine amidase, E.C. 3.5.1.28) that has the ability to lyse Groups A, C, and E Streptococci and their isolated cell walls. Maxted, . R. (1957) J. Gen. Microbiol. 16: 584. This enzyme was found to be ideal for the isolation of Group A Streptococcal cell wall components, M protein and C carbohydrate. A new application of this enzyme for the detection of Streptococcal throat infections was developed by Vincent A. Fischetti and David Bernstein. To make this new application commercially attractive, it is essential to produce the phage- associated lysin efficiently.

In the research laboratory, a procedure for making small quantities of lysin has been documented. However, the process described has certain inherent disadvantages from the commercial standpoint, in that a large volume of phage lysate is required as inoculum for the preparation of phage, and that long periods of time (4 weeks) are required to inactivate a free lysin present in the supernatant. In addition, the titers obtained are considerably low and inconsistent.

A lysin-free phage inoculum is essential for the preparation of both lysin and phage inoculum. Besides inactivation by aging, physical separation provides a nondestructive means to remove undesirable components from the phage inoculum. Among methods of physical separation, ultrafiltration is a commercially available system which will separate components according to molecular size.

Molecules of lysin are smaller than phage particles. By selecting the proper membrane, the phage can be retained and concentrated while lysin passes through. Concentrated phage is then washed with buffer to remove free lysin, and the phage concentrate is ready to be used or stored for future use.

According to the prior art, the aged phage solutio containing inactivated lysin is used to inoculate Streptococcal culture grown to 0.16 OD at 650 nm. The mixtur is allowed to remain at 37°C for 20 minutes, at which time th infected cells are harvested and resuspended in buffer. Th cells are allowed to lyse, releasing phage and the lyti enzyme. In general, the total yield of intracellular enzyme is a function of cell mass. The titer of lysin should increase with increasing concentrations of infected Streptococci. I the prior art, the phage concentration was constant so that

* 5 any increase in the Streptococcal cell density could not increase the number of infected Streptococci or the enzyme

⁸ titer. According to the present invention, the phage can be concentrated. Thus, the Streptococci can be grown to higher density and can be infected by concentrated phage. As a 10 result, the titer of lysin can be increased.

Disclosure of the Invention

Accordingly, it is a general object of the present

15 invention to provide an improved process of preparing phage suspension containing no free lysin and of producing higher titers of lysin which can be used in diagnostic tests for the identification of Group A Streptococci from infected tissue.

20 The advantages of the new method for phage production are three-fold. The amount of phage required to generate a new batch of phage inoculum is greatly reduced. The number of phage produced per initial phage is improved IO⁴ fold. In addition, the phage lysate used as inoculum for phage

25 production may contain active lysin since lysis "from without" does not occur because of the high dilution factor.

The phage lysate of the present invention is substantially free of lysin, i.e. a 1 : 4 dilution with

30 Streptococcus cells will not lyse the cells when incubated at

37°C for 15 minutes. Because the lysin content is kept low, there will be no extracellularly induced lysis prior to the

■*^■ first phage replication cycle. Since the phage replication cycle releases about 100 phage particles every 20 minutes in

' 35 comparison with the Streptococcus replication of two cells every 20 - 30 minutes, this "multicyclic" process can readily produce phage concentrations of 10¹⁰ - 10¹¹ PFU/ml. Initial cell infection with phage is preferably made from IO⁹ PFU/ml stock added to a streptococcal culture of O.D. of about 0.2 at 650 nm, representing an initial inoculum of about IO⁴ PFU/ml. While some lysin builds up in the course of the multicyclic culture process and not every phage particle will infect a new cell, there is a maximum concentration of phage at which all cells will be lysed. Nonetheless, this process can generate 10¹⁰ phage particles/ml from an inoculum of only 10/ml in only four hours.

Upon study of the specification and appended claims, further objects, features and advantages of the present invention will become more fully apparent to those skilled in the art to which this invention pertains.

BEST MODE FOR CARRYING OUT THE INVENTION

Briefly, the above and other objects, features, and advantages of the present invention are attained in one aspect thereof by providing a method for producing a lysin-free phage inoculum, which comprises: a) inoculating a growing Streptococcal culture with phage, b) incubating the culture for a plurality of lytic cycles of phage until the cells are completely lysed to obtain a lysate, and c) removing cell debris and free lysin from the lysat to form a lysin-free phage suitable for use as an inoculum.

DETAIIiED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides an improve process of preparing phage suspension containing no free lysi and of producing higher titers of lysin which can be used i diagnostic tests for the identification of Group

Streptococci from infected tissue. A phage inoculum, which contained no free lysin, wa prepared by growing the Streptococcal cells in Todd-Hewit broth at 37°C to a cell density of 0.10 to 1.0 OD, (preferabl 0.1 to 0.4 OD) at 650 nm before the proper amount of phag solution was added. After phage infection, the culture wa incubated to allow for several lytic cycles of phage to occu before the cells were completely lysed. Cell debris wer removed by filtration through a 0.2 micron membrane. Thi phage preparation containing active free lysin was passe through a second ultrafiltration system with a membrane whic had a molecular weight cutoff between 100,000 and 300,000. The phage was separated from free lysin and concentrated. Th final concentrated phage was washed with buffer and used a an inoculum for lysin production or for preparation of th next batch of phage.

For the production of lysin, the above describe procedure was followed with some modification. The phage infected Streptococcal cells were harvested 15-20 minutes after concentrated phage was inoculated into a Streptococcal culture grown to a cell density greater than 0.1 at 650 nm. The harvested cells were resuspended in 0.05 M phosphate buffer, pH 6.1, containing 5 x IO^'4 M dithiothreitol (DTT) and 5 mg bovine pancreatic deoxyribonuclease. The suspension was then incubated at 37°C for 30-60 minutes during which time the cells lysed, releasing the lytic enzyme (lysin) . Ethylene dia ine tetraacetic acid (EDTA) was added to a final concentration of 0.005 M. The cell debris and phage were removed by ultracentrifugation at 30,000 rpm for 4 hours at 4°C in a Sorvall™ A641 rotor (Ivan Sorvall, Inc., Norwich, CT) . The lysin solution was collected and could be used as a diagnostic reagent for detecting Streptococcal infection.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. In the following examples, the temperatures are set forth uncorrected in degrees Celsius; unless otherwise indicated, all parts and percentages are by weight.

EXAMPIiE 1

A phage inoculum was prepared by the new multicyclic process. A 6.5-liter culture of Streptococcal cells was grown to an optical density of 0.20 at 650 nm in Todd-Hewitt broth, inoculated with phage (30 ml, 1.0 x IO⁷ pfu/ml) , and incubated until lysis occurred (90 minutes) . The initial phage titer immediately after inoculation was 4.6 x IO⁴ pfu/ml. After complete cell lysis (90 minutes) the phage titer was 4.5 x 10⁹ pfu/ml, a 10⁵ increase.

As a control, a phage inoculum was prepared according to published procedure. A 12-liter culture of Streptococcal cells was grown to an optical density of 0.15 at 650 nm i Todd-Hewitt broth, inoculated with phage (3 liters, 2.8 x 10 pfu/ml) and incubated until lysis occurred (45 minutes) . Th initial phage titer immediately after inoculation was 5.6 10^s pfu/ml. After complete cell lysis (45 minutes) , the phag titer was 8.6 x IO⁹ pfu/ml, a 15-fold increase over th starting titer.

EXAMPLE 2

Phage inoculum can be concentrated and free lysin remove by the new process. In prior art, this phage preparation wa stored at 4°C for 4 weeks to allow the residual lysin t become inactive, thus preventing lysis by the enzyme whe phage lysate was added to the Group C cells for enzym preparation (Fishchetti, V.A. , Gotschlich, E.C. an Bernheimer, A.W. (1971) J. Exp. Med. , 133: 1105-1117). According to the present invention, the lysate can b processed immediately after lysis has occurred. The lysat was passed through a Millipore GVLP 0.2 micron membran cassette to remove uninfected cells and cell debris. Th filtrate then went through a Millipore PTMK 300K membran cassette which concentrated the phage and removed the fre lysin. The resulting phage retentate was washed with a buffe containing 2 g/1 sodium chloride, 0.4 g/1 dibasic sodiu phosphate and 2.5 g/1 sodium carbonate, pH 7.5. This washe phage retentate, containing no active free lysin, was read to be used as an inoculum for lysin production. The result of phage concentration and lysin removal through the 300 membrane are shown in Tables 1 and 2, respectively.

EXAMPLE 3

The improved process of producing lysin is described as follows. Group C Streptococcal strain 26RP66 (ATCC #21,597) was grown in 12-liters of Todd-Hewitt broth at 37°C to an O.D. of 0.41 at 650 nm. Three liters of Group C bacteriophage (Cl) (ATCC #21,597-B1) containing 2.4 x 10¹⁰ pfu/ml was added to the Streptococcal culture. The mixture was allowed to remain at 37°C for 16 minutes at which time the infected culture was poured over ice cubes to reduce the temperature of the broth to below 15°C. The infected cells were then harvested in a refrigerated centrifuge at 3700 x g and resuspended in 0.05 M phosphate buffer, pH 6.1 containing 5 x IO^"3 M dithiothreitol and 5 mg of bovine pancreatic deoxyribonuclease I (Boehringer, Grade II) . The cells lysed, releasing the lytic enzyme. Cell debris and phage were removed by centrifugation at 30,000 rp for 4 hours m a Sorvall A641. rotor. The enzyme solution was aliquoted, tested for its ability to lyse Group A Streptococci, and stored at -70°C.

EXAMPLE 4

The number of units per ml in a batch of enzyme is determined to be the reciprocal of the highest dilution of enzyme required to reduce the OD₆₅₀ of a suspension of Group A Streptococci from 0.3 to 0.15 in 15 minutes at 37°C. The yield of lysin in a 12 liter batch obtained by infecting different cell densities of Streptococci with increasing phage inoculum is shown in Table 3. TABLE 3

Cell Density Phage Inoculum Lysin Titer (°D₆₅₀> (pfu) (units/batch)

The preceding examples can be repeated with simila success by substituting the generically or specificall described reactants and/or operating conditions of thi invention for those specifically used in the examples. Fro the foregoing description, one skilled in the art to whic this invention pertains can easily ascertain the essentia characteristics thereof and, without departing from the spiri and scope of the present invention, can make various changes and modifications to adapt it to various usages an conditions.

Industrial Applicability

As can be seen from the preceding discussion, the present invention is industrially useful in providing a commercially attractive method for the production of phage-associated lysin which is useful, e.g. in the detection of Streptococcal throat infections.

Claims

WHAT IS CLAIMED IS:

1. A method for producing a lysin-free phage inoculum, which comprises: a) inoculating a growing Streptococcal culture with phage, b) incubating the culture for a plurality of lytic cycles of phage until the cells are completely lysed to obtain a lysate, and c) removing cell debris and free lysin from the lysate to form a lysin-free phage suitable for use as an inoculum.

2. A method according to claim 1, wherein the free- lysin is removed by ultrafiltration of lysate through a membrane having a molecular weight cutoff of about 100,000 - 300,000.

3. A method according to claim 1 wherein the cells are Group- C Streptococcus cells.

4. A Streptococcal phage inoculum substantially free of extracellular lysin.

5. An inoculum of claim 4, wherein the phage is Cl.

6. A method of producing lysin, which comprises: a) inoculating growing Streptococcal cells with a Streptococcal lysin-free phage inoculum; b) culturing the cells under conditions suitable for the production of lysin; c) lysing the cells to release the lysin; and d) recovering the lysin from the lysate.

7. A process according to claim 7 wherein the phage is Cl.

8. The method of claim 6 wherein the cells are group Streptococcus.

9. A method of claim 6 wherein the lysate contains a least 70,000 units of lysin per liter of fermentation broth.