Vectors for differential expression

Details Vectors for differential expression Vectors for differential expression

1998-01-20

1999-08-03

Ketter, James

Chemistry: molecular biology and microbiology

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

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

4353201, C12P 2102

Patent

active

059324414

DESCRIPTION:

BRIEF SUMMARY
This invention relates to nucleic acid constructs capable of being differentially expressed, and to vectors comprising such constructs. The invention further relates to a method for differential expression of a protein.
Various expression systems are available for synthesising proteins. For expression in eukaryotic cells such as mammalian cells or yeast, a protein coding DNA sequence may be placed downstream of a promoter and introduced into the appropriate host cell. RNA transcribed under control of the promoter will then be translated by the host cell translation machinery including ribosomes.
Proteins may also be expressed in vitro. In this case, the coding sequence is most usually placed downstream from a bacteriophage promoter which directs the synthesis of RNA in the presence of a suitable polymerase. The RNA is then used to programme a cell-free extract, derived eg. from rabbit reticulocytes, which contains ribosomes and other components of the translation machinery.
A further alternative is to express proteins in bacteria. This can be achieved by placing the coding sequence downstream of a promoter DNA sequence which acts as a recognition sequence for either a bacterial or bacteriophage RNA polymerase. A commonly used system involves introducing a ribosome binding site 5' to the ATG initiation codon for the protein sequence, where the ribosome begins translation of the RNA. The DNA sequence thus produced is introduced into bacteria which constitutively express a compatible RNA polymerase. The bacteriophage promoter drives the production of RNA which will then be translated by ribosomes recognising the ribosome binding site and the ATG initiation codon.
As a result of the differences between mammalian, yeast, bacterial and bacteriophage promoters, in order to achieve transcription and subsequent translation of a particular protein coding sequence in the different systems, it is necessary to transfer the coding sequence from one expression vector to another. The transfer is a time-consuming step.
In some cases it is also desirable to express different but related forms of the same protein in the different systems. For example, expression of a protein in yeast may be "tagged" with an epitope for identification with an antibody, or with part of a heterologous protein, such as a transcriptional activation domain.
In the two-hybrid assay technique of Fields and Song described in U.S. Pat. No. 5,283,173, two chimeric genes which encode fusion proteins are used to test the interaction between a known protein and a protein of interest. The first chimeric gene codes for a known protein, fused to a protein domain which binds to any specific DNA sequence such as the DNA binding domain of a transcriptional activator. The second chimeric gene codes for the protein of interest fused to a transcriptional activation domain. Alternatively, the chimeric genes may be reversed, with the protein of interest fused to the DNA-binding domain and the known protein fused to the transcriptional activation domain. Additionally, the protein of interest may not be known and could be derived for example from a cDNA library. In a suitable host cell, if the protein of interest and the known protein do interact, they bring into proximity the DNA-binding and transcriptional activation domains. This proximity is sufficient to cause transcription of a marker gene placed under the control of a promoter containing a binding site for the DNA-binding domain. Once an interaction has been demonstrated, it is often desirable to further investigate the test protein eg. by expressing it in isolated form. The interactions detected ex vivo (that is, in the cell expressing the hybrids) or in vivo eg. yeast or plants or animals require confirmation in vitro and the test proteins may need to be over-expressed in bacteria for the production of antibodies. Transfer to suitable vectors is therefore required.
The present invention aims to avoid the need for transfer between vectors, for example in the situations described above. investigating a protein,

REFERENCES:
S. Fields et al. "A novel genetic system to detect protein-protein interactions", Letters to Nature, vol. 340, pp. 245-246, Jul. 20, 1989.
S. Green et al., "A versatile in vivo and in vitvo eucaryotic expression vector for protein engineering", Nucleic Acids Research, vol. 16, No. 1, pp. 268-269, Jan. 11, 1988.

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