Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-10-26
2004-05-11
Hines, Ja-Na (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C424S134100, C424S145100, C424S185100, C424S192100, C435S069900, C435S070100, C435S070200, C435S158000, C435S173300, C435S173300, C435S332000, C436S086000, C530S300000, C536S023100, C930S300000, C930S310000
Reexamination Certificate
active
06733997
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the use of nucleic acids (termed “SS” for “secretory signal” or “Vtgss” for “vitellogenin secretory signal”) that function as universal secretory signals for rapidly directing recombinant proteins out of a heterologous host cell. The invention is also applicable to improved and more convenient means of detecting reporter proteins by simply sampling culture medium in which cells harboring constructs of the invention are maintained. The invention is also applicable to biosensor technology, to for screening of genetically-engineered clones harboring selectable marker(s) to be expressed as an indicator, and to achieving systemic circulation/distribution of recombinant proteins in a transgenic or chimeric host organism.
DESCRIPTION OF THE RELATED ART
Although protein expression in bacteria is commonly used to obtain recombinant proteins, bacterial hosts present several drawbacks to expression of recombinant proteins. First, eukaryotic proteins expressed in
E. coli
are often not properly folded and thus non-functional (Cleland, J. L. 1993, In Protein Folding In Vivo and In Vitro: pp. 1-21). This is a large problem, since most proteins used for pharmaceutical/diagnostic purposes are eukaryotic in nature. Second, proteins expressed in large amounts in
E. coli
often precipitate into insoluble aggregates called “inclusion bodies”, from which they can only be recovered in an active form by solubilization in denaturing agents followed by careful renaturation (Chang and Swartz, 1993, in “Protein Folding In Vivo and In Vitro”, Cleland, J. L. ed., pp. 178-188). Third, it is difficult to engineer the secretion of large amounts of expressed protein in
E. coli
, although it has often been possible to secrete small amounts into the periplasmic space and to recover them by osmotic shock. Finally, the recovered recombinant protein is usually contaminated with an unacceptable level of endotoxin/pyrogen that makes proteins for pharmaceutical/diagnostic use extremely difficult to validate (Walsh and Headon, 1994, in “Protein Biotechnology”, pp. 118-162).
Although, protein expression in yeast (
Saccharomyces cerevisiae
) can solve some of the problems associated with bacterial expression, the harsh conditions necessary to lyse the yeast often result in poor yield and denaturation of the recombinant proteins. Secretion of recombinant proteins from yeast has largely been a trial and error effort. Furthermore, most secreted proteins are trapped in the yeast periplasmic space and thus, difficult to retrieve (Lemontt et al., DNA 4:419-428 (1985); Martegani et al.,
Appl. Microbiol. Biotechnol
. 37:604-608 (1992)).
The mammalian and insect expression systems have a number of advantages that have contributed to the recent popularity (Straten et al., 1989, in “Invertebrate Cell Systems Applications”, pp. 183-193; Yarranton, G. T.,
Curr. Opinion Biotech
. 1:133-140 (1990)). Recombinant proteins are almost always expressed at high levels and the expressed proteins are usually properly modified and accumulated in the proper cellular compartment. However, there have been occasions where the use of the homologous secretory signal of the gene insert resulted in mistargetting of the expressed proteins, which consequently remained within the yeast. Generally, protein expression in higher eukaryotes such as insect cells and mammalian cells is challenging, time-consuming and expensive although they are suitable hosts for small- and medium-scale work.
An example of a Drosophila expression system utilizes a cell line derived from
Drosophila melanogaster
, Schneider 2 (S2) cells, and a simple plasmid vector for the expression of heterologous proteins (Ivey-Hoyle, M.,
Curr. Biol
. 2:704-707 (1991)). S2 cells and mammalian cells such as CHO cells have been used to express proteins for both biochemical assays and therapeutics. In both the insect and mammalian cell systems, there is a therefore a need to employ powerful secretory signals to drive the secretion of the expressed recombinant proteins out of the heterologous host cells for easy detection, quantification and large-scale purification.
For an efficient expression system, several criteria need to be satisfied. First, the recombinant protein synthesis must be functional. Second, a reasonable yield of the protein is required to justify its usage. Third, the system must enable homogenous synthesis of the recombinant protein. Last, the system should allow easy scaling-up and subsequent downstream processing, for example, purification. Consequently, we have constructed recombinant fusion vectors that direct the secretion of heterologous proteins into the medium used to culture various host cells.
An important aspect of the study of gene regulation is assay of transcription levels observed from wild-type and mutant versions of putative cis-acting control elements in transfected mammalian cells. In most cases, the ability of the cis-acting elements to modulate transcription is not assayed directly. Instead, the cis-acting elements are joined to a reporter gene that codes for a measurable enzyme activity. The level of the enzymatic activity that accumulates during the course of an experiment is taken as a measure of the ability of the cis-acting element to regulate transcription. Although this is an indirect assay of the activity of the cis-acting elements that control transcription, it is often the only one that is practicable. A number of different genes have been used as reporters of the transcriptional activity of eukaryotic promoters. These include chloramphenicol acetyltransferase (CAT), &bgr;-galactosidase and luciferase. However, all these require lysis of the transfected cells and thus are subject to the problems associated with set-up and variability of multiple cultures.
To date, the Great EscAPE SEAP Genetic Reporter System™ (Clontech) is one available system that utilizes a secreted form of an enzyme, human placental alkaline phosphatase, as a reporter for the analysis of cis-acting DNA sequences and trans-acting factors. However, a drawback of the SEAP assay is that it has to be measured using either a fluorescent or chemiluminescent assay, requiring a fluorimeter or a luminometer. Furthermore, it requires the investigator to switch from their established protocols for CAT, &bgr;-galactosidase or luciferase.
SUMMARY OF THE INVENTION
The present invention stems from the finding that the secretory signal sequence (SS or Vtgss) of the vitellogenin gene, especially the vitellogenin gene from
Oreochromis aureus
, can be used to direct the secretion of recombinant proteins out of cells. The cells that are able to utilize the
O. aureus
SS are not particularly limited; the present inventors have found that cells of many kinds, especially eukaryotic cells, will efficiently process exogenous fusion proteins comprising the SS of the invention and secrete them from the cell.
Thus, one aspect of the present invention is isolated nucleic acids that encode a SS that provides the biological activities of directing secretion of a protein linked to the SS from a host cell and cleavage of the SS from the fusion protein at a particular point in the SS sequence. The isolated nucleic acids of the invention are easily incorporated into vectors for expression of recombinant proteins and subsequent secretion of the fusion protein, processed by appropriate cleavage of the SS portion, into the medium used to culture the host cells. An advantage of the present invention is that the secretion is very efficient regardless of the particular protein secreted or the host cell used to express the recombinant protein.
Thus, the present invention resides in part in the development of an efficient and versatile vector for high level expression and immediate secretion of recombinant proteins into serum-free medium used to culture the host cells. The host cells can be immobilized, or else easily removed from the culture, thus providing easy purification of the recombinant proteins. Also, vectors comprising the SS of the invention an
Ding Jeak Ling
Ho Bow
Lam Toong Jin
Tan Nguan Soon
Birch Stewart Kolasch & Birch, LLP.
Hines Ja'Na
National University of Singapore
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