Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-12-02
2000-06-06
Railey, II, Johnny F.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 696, 435 698, 435 711, 435 712, 435476, 435488, C12N 121, C12N 1566, C12N 1570, C12P 2102
Patent
active
060717254
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to surface anchoring vectors containing gene segments of ice nucleation protein (INP), which can express foreign proteins onto a cell surface.
The present invention also relates to a method for preparing foreign proteins onto a cell surface by using INP and use of foreign proteins prepared by the surface expression system.
Particularly, the present invention relates to surface anchoring vectors, a method for preparing foreign proteins onto a cell surface and use thereof, which uses outer cell membrane proteins ice nucleation protein (INP) derived from Pseudomonas syringae, a gram-negative bacterium.
BACKGROUND ARTS
Presently, surface expression of proteins has been investigated in unicellular organisms such as bacteriophage, bacteria, yeast and the like to produce new vaccines, to perform screening of various antigens and antibodies and to localize useful enzymes onto a cell surface.
Surface expression of proteins has been tried at first to express antigenic peptides onto a cell surface for stable production of vaccines. Hitherto, in order to produce vaccines pathogenic bacteria were mutated arbitrarily and screened for selecting safe bacteria which can induce immunization consistently. However, the screening method has a disadvantage of losing the antigenic activities when the vaccines are administered orally into human body and animal body. Thus, many investigations have been performed to overcome the disadvantage.
First, surface expression has been performed by the process, wherein gene segments of cell surface protein were exploited in gram-negative bacteria, ligated to genes of antigenic polypeptides and used to transform proper bacteria hosts for effective production of fusion proteins. The fusion proteins prepared by the process can work as effective antigens since they are extruded onto a cell surface stably. Especially in gram-negative bacteria, outer member lipopolysaccharides (LPS) increase antigenicity of proteins expressed onto a cell surface.
To express proteins onto a cell surface, a secretion signal is necessary within primary sequences of proteins, which helps foreign proteins produced intracellularly to pass through a cell membrane. Especially in gram-negative bacteria, proteins should pass through the inner cell membrane and periplasmic space, localize onto the outer cell membrane stably and be extruded. For example, surface proteins, specific enzymes and toxin proteins have a secretion signal and a targeting signal localizing the proteins onto the cell surface. Practically, foreign proteins can be expressed on the cell surface successfully by using such a secretion signal, a targeting signal and the like combined with proper promoters.
Up to now, surface proteins present in gram-negative bacteria have been utilized mainly to produce foreign polypeptides which are necessary onto the cell surface. There are 4 kinds of proteins used for the cell surface expression, such as outer cell membrane protein, lipoprotein, secretion protein and cell surface structure protein. As outer cell membrane proteins, Lam B, Pho E, Omp A and the like have been utilized for the surface expression. In these cases, however, sizes of proteins expressed onto the cell surface are limited since the proteins should be inserted into loops extruded from the cell surface. In addition, the C- and N-termini of the foreign protein should be close to each other 3- dimensionally. Thus, when the distance between the two termini is long the C- and N-termini should be joined.
Practically, if Lam B or Pho E is utilized to insert foreign polypeptides comprising more than 50-60 amino acids, membrane proteins cannot be produced stably due to structural imitation [Charbit, et al., J. Immuol., 139: 1658-1664 (1987); Agterberg, et al., Vaccines, 8: 85-91 (1990)]. To overcome the structural limitation, a part of the Omp A protein which contains a miniumum targeting sequence localizing foreign proteins onto the outer cell membrane is used, although the whole Omp A protein was also used
REFERENCES:
Green et al., Mol. Gen. Genet. 215:165-172 (1988).
Microbiology, Bernard D. Davis et al., eds. Hagerstown: Harper & Row, Publishers, 1980. pp. 73, 82 and 83.
Han Moon Hi
Jung Heung Chae
Pan Jae Gu
Park Seung Hwan
Park Young Hoon
Korea Institute of Science and Technology
Railey II Johnny F.
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