Process for bacterial production of polypeptides

Details Process for bacterial production of polypeptides Process for bacterial production of polypeptides

1999-10-21

2001-01-30

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069700, C435S252100, C435S252300

Reexamination Certificate

active

06180367

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a process for producing and recovering heterologous polypeptides from bacterial cells. More particularly, this invention relates to a process wherein recovery of soluble or aggregated recombinant heterologous polypeptides from bacterial cytoplasm and periplasm is facilitated or increased.
2. Description of Related Disclosures
Escherichia coli
has been widely used for the production of heterologous proteins in the laboratory and industry.
E. coli
does not generally excrete proteins to the extracellular medium apart from colicins and hemolysin (Pugsley and Schwartz, FEMS (Federation of European Microbiological Societies) Microbiology Reviews, 32: 3-38 (1985)). Heterologous proteins expressed by
E. coil
may accumulate as soluble product or insoluble aggregates. See
FIG. 1
herein. They may be found intracellularly in the cytoplasm or be secreted into the periplasm if preceded by a signal sequence. How one proceeds initially in the recovery of the products greatly depends upon how and where the product accumulates. Generally, to isolate the proteins, the cells may be subjected to treatments for periplasmic extraction or be disintegrated to release trapped products that are otherwise inaccessible.
The conventional isolation of heterologous polypeptide from gram-negative bacteria poses problems owing to the tough, rigid cell walls that surround these cells. The bacterial cell wall maintains the shape of the cell and protects the cytoplasm from osmotic pressures that may cause cell lysis; it performs these functions as a result of a highly cross-linked peptidoglycan (also known as murein) backbone that gives the wall its characteristic rigidity. A recent model described the space between the cytoplasmic and outer membranes as a continuous phase filled with an inner periplasmic polysaccharide gel that extends into an outer highly cross-linked peptidoglycan gel (Hobot et al.,
J. Bact
., 160: 143 (1984)). This peptidoglycan sacculus constitutes a barrier to the recovery of any heterologous polypeptide not excreted by the bacterium into the medium.
To release recombinant proteins from the
E. coli
periplasm, treatments involving chemicals such as chloroform (Ames et al.,
J. Bacteriol
., 160: 1181-1183 (1984)), guanidine-HCl, and Triton X-100 (Naglak and Wang,
Enzyme Microb. Technol
., 12: 603-611 (1990)) have been used. However, these chemicals are not inert and may have detrimental effects on many recombinant protein products or subsequent purification procedures. Glycine treatment of
E. coli
cells, causing permeabilization of the outer membrane, has also been reported to release the periplasmic contents (Ariga et al.,
J. Ferm. Bioeng
., 68: 243-246 (1989)). These small-scale per plasmic release methods have been designed for specific systems. They do not translate easily and efficiently and are generally unsuitable as large-scale methods.
The most widely used methods of periplasmic release of recombinant protein are osmotic shock (Nossal and Heppel,
J. Biol. Chem
., 241: 3055-3062 (1966); Neu and Heppel,
J. Biol. Chem
., 240: 3685-3692 (1965)), hen eggwhite (HEW)-lysozyme/ethylenediamine tetraacetic acid (EDTA) treatment (Neu and Heppel,
J. Biol. Chem
., 239: 3893-3900 (1964); Witholt et al.,
Biochim. Biophys. Acta
, 443: 534-544 (1976); Pierce et al.,
ICheme Research Event
, 2: 995-997 (1995)), and combined HEW-lysozyme/osmotic shock treatment (French et al.,
Enzyme and Microb. Tech
., 19: 332-338 (1996)). Typically, these procedures include an initial disruption in osmotically-stabilizing medium followed by selective release in non-stabilizing medium. The composition of these media (pH, protective agent) and the disruption methods used (chloroform, HEW-lysozyme, EDTA, sonication) vary among specific procedures reported. A variation on the HEW-lysozyme/EDTA treatment using a dipolar ionic detergent in place of EDTA is discussed by Stabel et al.,
Veterinary Microbiol
., 38: 307-314 (1994). For a general review of use of intracellular lytic enzyme systems to disrupt
E. coli
, see Dabora and Cooney in
Advances in Biochemical Engineering/Biotechnology
, Vol. 43, A. Fiechter, ed. (Springer-Verlag: Berlin, 1990), pp. 11-30.
Conventional methods for the recovery of recombinant protein from the cytoplasm, as soluble protein or retractile particles, involved disintegration of the bacterial cell by mechanical breakage. Mechanical disruption typically involves the generation of local cavitation in a liquid suspension, rapid agitation with rigid beads, sonication, or grinding of cell suspension (
Bacterial Cell Surface Techniques
, Hancock and Poxton (John Wiley & Sons Ltd, 1988), Chapter 3, p. 55). These processes require significant capital investment and constitute long processing time.
HEW-lysozyme acts biochemically to hydrolyze the peptidoglycan backbone of the cell wall. The method was first developed by Zinder and Arndt,
Proc. Natl. Acad. Sci. USA
, 42: 586-590 (1956), who treated
E. coli
with egg albumin (which contains HEW-lysozyme) to produce rounded cellular spheres later known as spheroplasts. These structures retained some cell-wall components but had large surface areas in which the cytoplasmic membrane was exposed.
U.S. Pat. No. 5,169,772 discloses a method for purifying heparinase from bacteria comprising disrupting the envelope of the bacteria in an osmotically-stabilized medium, e.g., 20% sucrose solution using, e.g., EDTA, lysozyme, or an organic compound, releasing the non-heparinase-like proteins from the periplasmic space of the disrupted bacteria by exposing the bacteria to a low-ionic-strength buffer, and releasing the heparinase-like proteins by exposing the low-ionic-strength-washed bacteria to a buffered salt solution.
There are several disadvantages to the use of the HEW-lysozyme addition for isolating periplasmic proteins. The cells must be treated with EDTA, detergent, or high pH, all of which aid in weakening the cells. Also, the method is not suitable for lysis of large amounts of cells because the lysozyme addition is inefficient and there is difficulty in dispersing the enzyme throughout a large pellet of cells.
Many different modifications of these methods have been used on a wide range of expression systems with varying degrees of success (Joseph-Liauzun et al.,
Gene
, 86: 291-295 (1990); Carter et al.,
Bio/Technology
, 10: 163-167 (1992)). Although these methods have worked on a laboratory scale, they involve too many steps for an efficient large-scale recovery process.
Efforts to induce recombinant cell culture to produce lysozyme have been reported. EP 155,189 discloses a means for inducing a recombinant cell culture to produce lysozymes, which would ordinarily be expected to kill such host cells by means of destroying or lysing the cell wall structure. Russian Pat. Nos. 2043415, 2071503, and 2071501 disclose plasmids and corresponding strains for producing recombinant proteins and purifying water-insoluble protein agglomerates involving the lysozyme gene. Specifically, the use of an operon consisting of the lysozyme gene and a gene that codes for recombinant protein enables concurrent synthesis of the recombinant protein and a lysozyme that breaks the polysaccharide membrane of
E. coli.
U.S. Pat. No. 4,595,658 discloses a method for facilitating externalization of proteins transported to the periplasmic space of
E. coli
. This method allows selective isolation of proteins that locate in the periplasm without the need for lysozyme treatment, mechanical grinding, or osmotic shock treatment of cells. U.S. Pat. No. 4,637,980 discloses producing a bacterial product by transforming a temperature-sensitive lysogen with a DNA molecule that codes, directly or indirectly, for the product, culturing the transformant under permissive conditions to express the gene product intracellularly, and externalizing the product by raising the temperature to induce phage-encoded functions. JP 61-257931 published Nov. 15, 1986 discloses a method for recovering IL-2 using

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