Protein expression system

Details Protein expression system Protein expression system

1993-09-30

2001-10-16

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Vector, per se

C536S023100, C536S024200

Reexamination Certificate

active

06303369

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to processes for expression of proteins and to expression vectors and host cells used therefor.
BACKGROUND OF THE INVENTION
The lck gene product, p56
lck
, is a member of the src family of protein tyrosine kinases. Cooper, J. A. (1990) in
Peptides and Protein Phosphorylation
(Kemps, B. E., ed) pp. 85-113, CRC Press, Boca Raton, Fla. The lck protein is normally expressed in T lymphocytes and natural killer cells, where it likely performs a variety of functions relating to signal transduction through ligand binding to selected surface proteins. Bolen, J. A., and Veillette, A. (1989)
Trends Biochem. Sci
. 14, 404-407; Rudd, C. E. (1990)
Immunol. Today
11, 400-406. In T-cells, p56
lck
forms a non-covalent complex with the CD4 and CD8a. Veillette, A., Bookman, M. A., Horak, E. M., and Bolen, J. A. (1988). For this reason, p56
lck
is believed to aid in mediation of signals emanating from the T-cell antigen receptor through ligation of CD4 or CD8 to non-polymorphic determinants on antigen-bearing major histocompatibility molecules. Shaw, A. S., Chalupny, J., Whitney, J. A., Hammond, C., Amrein, K. E., Kavathas, P., Sefton, B. M., and Rose, J. K., (1990)
Mol. Cell. Biol
. 10, 1853-1862; Doyle, C., and Strominger, J. L. (1987)
Nature
330, 256-259; Norment, A. M., Salter, R. D., Parham, P., Engelhard, V. H., and Littman, D. R. (1988)
Nature
336, 79-81. More recently, p56
lck
has been implicated as a signaling component of the high affinity interleukin-2 receptor. Hatakeyama, M., Kono, T., Kobayashi, N., Kawahara, A., Levin, S. D., Perlmutter, R. M., and Tanaguchi, T. (1991)
Science
252, 1523-1528.
A better understanding of the structure and regulation of p56
lck
and similar proteins would clearly contribute to our knowledge of early signal transduction events and a source of large quantities of purified p56
lck
would be useful. While early analysis of p56
lck
functions have been greatly facilitated by antibodies directed against this protein, immunoaffinity purification has been hampered by lack of an abundant source of enzyme. This difficulty has been addressed in part by baculovirus expression systems. Summers, M. D., and Smith, G. E. (1987).
A Manual for baculovirus vectors and insect cell culture procedures
, Texas A&M bulletin No. 1555, (College Station, Texas Agricultural Experimental Station and Texas A&M University), 10-39. Recent studies using a baculovirus expression system have reported significant purification of p56
lck
using conventional chromatography methodologies. Ramer S. E., Winkler, D. G., Carrera, A., Roberts, T. M., and Walsh, C. T. (1991)
Proc. Natl. Acad. Sci. USA
88, 6254-6258; Watts, J. D., Wilson, G. M., Ettehadieh, E., Clark-Lewis, I., Kubanek, C., Astell, C. R., Marth, J. D., and Aebersold, R, (1991)
J. Biol, Chem
. 267, 901-907. While this approach results in purified enzyme, multiple column enzyme purification is costly, time-consuming, and requires large amounts of starting material.
Glutathione-s-transferase (Gst) is a protein well known to bind to glutathione (Smith, D. B., and Johnson, K. S. (1988)
Gene
67, 31-40). Glutathione resin may be used in column chromatography. The above baculovirus expression systems, however, do not employ Gst.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to processes for expressing isolated forms of proteins and to expression vectors and host cells useful for such processes. In particular, this invention relates to an expression vector, comprising:
(a) a first coding region, which codes for a polypeptide capable of binding to gluthathione, operatively connected to a promoter,
(b) a second coding region in-frame with the first coding region, and
(c) at least one restriction site between the first and second coding regions;
wherein a fusion protein of the first and second coding regions would result from expression of the vector. Vectors derived from baculovirus are preferred.
Further in accordance with this invention is a host cell comprising such a vector. The preferred host cell is a
Spodoptera frugiperda
cell, particularly an Sf9 cell, although other host cells are suitable (see below).
Such vectors and host cells are useful in a process for expressing a protein in isolated form, which comprises:
(a) treating such a host cell under conditions allowing expression of the vector, whereby a fusion protein of the first and second coding regions will be expressed;
(b) exposing proteins from the host cell to glutathione resin, whereby the fusion protein will adhere to the resin; and
(c) cleaving the expression product of the second coding region from the resin-bound fusion protein.
Further in accordance with the present invention is a process for expressing a nucleic acid sequence, which comprises:
(a) inserting the nucleic acid sequence into a baculovirus expression vector in-frame with a first coding region for a polypeptide capable of binding to glutathione,
wherein the coding region is operatively linked to a promoter;
(b) placing the vector into a host cell;
(c) treating the host cell under conditions allowing expression of the vector, resulting in expression of a fusion protein of the first coding region and the sequence inserted in step (a);
(d) exposing proteins from the host cell to glutathione resin, whereby the fusion protein adheres to the resin; and
(e) treating the adhered fusion protein with a protease to release the expression product of the nucleic acid sequence from the resin.
For the first coding region, the inventors prefer a sequence encoding glutathione-s-transferase (nucleotide SEQ. ID. NO.: 1; amino acid SEQ. ID. NO.: 2) or a fragment thereof capable of binding to glutathione. This system combines the high level expression of foreign proteins with baculovirus vectors (e.g., in Sf9 cells) and the ability of Gst fusion proteins to bind to glutathione resin. Treatment of the glutathione-binding fusion protein with a proteolytic substance such as thrombin can thus liberate the desired protein from the glutathione-binding portion of the fusion protein. The glutathione-binding portion remains bound to the resin, thus purifying the desired protein.
This expression system presents advantages over other systems, because it allows the practitioner (1) to produce large quantities of protein, (2) to purify significant amounts of active protein by a single chromatography step, (3) to use a wide range of extraction conditions, including non-denaturing detergents to maintain protein function, (4) to use anti-Gst antibodies, allowing for screening of recombinant baculoviruses that express cloned sequences to which antibodies have not been generated or proteins whose function can not be measured, (5) to use a multiple cloning site with many restriction sites for convenient ligation, and (6) to use and/or study thrombin because it includes a thrombin cleavage site.
DETAILED DESCRIPTION OF THE INVENTION
The following definitions apply to the terms as used throughout this specification, unless otherwise limited in specific instances.
The term “fusion protein” refers to a protein or polypeptide that has an amino acid sequence having portions corresponding to amino acid sequences from two or more proteins. The sequences from two or more proteins may be full or partial (i.e., fragments) of the proteins. Such fusion proteins may also have linking regions of amino acids between the portions corresponding to those of the proteins. Such fusion proteins may be prepared by recombinant methods, wherein the corresponding nucleic acids are joined through treatment with nucleases and ligases and incorporated into an expression vector. Preparation of fusion proteins is generally understood by those having ordinary skill in the art.
The phrase “polypeptide capable of binding to glutathione” refers to proteins, protein fragments, and synthetic polypeptides capable of binding to glutathione. Examples include glutathione-s-transferase and fragments thereof. Suitable fragments may be generated by gene amplification using 5′ and 3′ p

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