Protein secretion

Details Protein secretion Protein secretion

2001-03-12

2002-09-17

McKelvey, Terry (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...

Reexamination Certificate

active

06451560

ABSTRACT:

The invention relates to a novel prokaryotic expression system and proteins expressed thereby.
The industrial production of proteins has, in many instances, exploited the native expression and secretory systems of microorganisms and specifically bacteria. For example and without limitation the bacterium
Bacillus subtilis
(
B.subtilis
) is known to produce and secrete a number of proteins. One family of these proteins, &agr;-amylases, is of industrial importance and therefore the harvesting of this secreted protein is an activity currently undertaken by industry. However, the yield of some &agr;-amylases is significantly reduced by protein degradation during an/or following passage through the cell membrane.
It therefore follows that there is a need to provide a protein expression system which enhances the production of native and/or heterologous and/or recombinant protein and more specifically effectively enhances the secretion of protein from the cell.
The expression and secretion of heterologous and/or recombinant protein (i.e. proteins that are not native to that particular bacteria) typically involves transformation of a bacterial cell with heterologous DNA with a view to manufacturing or producing heterologous and/or recombinant proteins.
Microorganisms such as
Escherichia coli
(bacteria),
Saccharomyces cerevisiae, Aspergillus nidulans
and
Neurospora crassa
(fungi) have been used in this fashion. The expression of heterologous protein in primitive eukaryotes also allows some desirable eukaryotic post-translational modifications to occur in heterologous and/or recombinant proteins leading to an increase in the stability of the expressed proteins and subsequent improvement in yield. More recently the use of mammalian and insect cells have been developed to facilitate the expression of eukaryotic proteins that for various reasons cannot be expressed in a prokaryotic host cell.
However, the cost effectiveness of producing heterologous and/or recombinant protein still remains the major advantage offered by genetically engineered prokaryotic expression systems and indeed significant advances have been made in the development of genetically engineered
E.coli
strains that increase the yield of specific proteins. The development of these bacterial strains has also been married with an ever increasing development of more efficient vectors adapted to optimise the expression of recombinant protein. These vectors contain promoter elements that are genetically engineered to create hybrid promoters that can be switched on or off with ease.
However, there are three major disadvantages when using
E.coli
as a means of expressing heterologous and/or recombinant protein. Firstly, the high levels of expression lead to a precipitation of recombinant protein in the bacterial cytoplasm as “inclusion bodies”. This feature was thought to be advantageous as it can provide a simple means of separating the insoluble recombinant protein from the soluble endogenous
E.coli
protein. However, in reality this advantage is not a general feature of the system as in many cases proteins remain an insoluble precipitate that can only be released into solution by using strong chaotropic agents. This presents a major problem if the protein in question is particularly labile and therefore loses biochemical or biological activity upon denaturation. Secondly, the expression of foreign protein in
E.coli
leads to rapid degradation of these proteins via an efficient proteolytic system. Thirdly, it is known by those skilled in the art that
E.coli
usually does not naturally secrete protein into its surrounding environment. Therefore, the purification of native, heterologous or recombinant protein has the major disadvantage that the desired protein has to be purified from endogenous
E.coli
protein.
E.coli
strains have been engineered to allow the expression of recombinant proteins that would ordinarily be difficult to express in traditional laboratory strains of
E.coli
. However, these engineered
E.coli
strains are invariably not as biologically disabled as traditional laboratory strains of
E.coli
and as a consequence require containment levels that are higher than would normally be required.
The identification of alternative prokaryotic host cells and the development of means that facilitate the production of soluble, intact and biologically active protein is obviously desirable. However, notably the number of potential prokaryotic host cells is huge.
With a view to producing a novel protein and expression system we have chosen to genetically engineer, as our example, Bacillus, ideally
B.subtilis
, in order to provide an expression system that overcomes the problems of yield associated with prior art systems. We have focussed our attention on providing a Bacillus expression system that produces and ideally secretes protein(s) into the culture medium because this system enables an initial purification of the manufactured protein due to the absence of contaminating endogenous bacterial protein(s) and other macromolecules.
The biochemical composition of the
B.subtilis
cell wall is quite different from that of
E.coli
. The cell walls of
E.coli
and
B.subtilis
contain a framework that is composed of peptidoglycan, a complex of polysaccharide chains covalently cross-linked by peptide chains. This forms a semi-rigid structure that confers physical protection to the cell since the bacteria have a high internal osmotic pressure and can be exposed to variations in external osmolarity. In Gram-positive bacteria, such as the members of the genus Bacillus, the peptidoglycan framework may represent as little as 50% of the cell wall complex and these bacteria are characterised by having a cell wall that is rich in accessory polymers such as wall teichoic acids. In addition, teichoic acids may be attached to the outside of the cytoplasmic membrane in the form of lipoteichoic acids or membrane anchored wall teichoic acids.
Teichoic acids are simple polymers of alditol phosphate molecules linked to each other by phosphodiester bridges. The free hydroxyl groups of the alditol phosphate backbone may be occupied by alanine or sugar residues. The alanylation of teichoic acids has a major effect of neutralising the negative charge conferred by adjacent phosphate residues, thereby reducing the overall negative charge of the cell wall.
The cell wall therefore provides, amongst other things, protection to the cell membrane to prevent rupture. The peptidoglycan framework represents upto approximately 50% of the cell wall mass. The remaining wall material consists of components which differ significantly between Gram negative (
E.coli
) and Gram positive (
B.subtilis
) bacteria.
B.subtilis
, and many other Gram positive bacteria, is characterised by having a cell wall that is rich in the accessory molecule teichoic acid.
The alanylation of teichoic acids is controlled by the D-alanyl-lipoteichoic acid (dlt) operon, a cluster of five genes encoding proteins necessary for the alanylation of teichoic acid. The genes are termed dltA, dltB, dltC, dltD and dltE. With the exception of dltE, each of these genes have known functions, Perego et.al 1995, please see FIG.
1
.
The partial or complete deletion of any individual member of the dlt operon, with the exception of the dltE, completely inhibits the alanylation of teichoic acid. However, there is no obvious phenotypic effect of deleting one or more of the dltA-D genes other than the inhibition of alanylation and consequential changes in the overall surface charge. Cell division and growth are apparently unaffected in
B.subtilis.
An unrelated gene, prsA, encodes a cell membrane located chaperone like molecule. The protein is involved in the folding of secreted proteins on the extracytoplasmic side of the cytoplasmic membrane (Kontinen et.al. 1991; Jacobs et.al 1993). Sequence homology with several peptidyl-prolyl-isomerases suggests that the PrsA protein is involved in the isomerisation of proline residues between cis and trans isomers in secreted proteins. A number of

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