Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-05-01
2003-10-07
Wang, Andrew (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S091500, C435S091500, C435S091500
Reexamination Certificate
active
06630317
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for obtaining nucleic acid sequences encoding (poly)peptides which increase the expression yields of periplasmic proteins in functional form upon co-expression of said (poly)peptides and said periplasmic proteins. The invention also provides a method for the identification of said (poly)peptides. Furthermore, the present invention relates to a method for increasing the expression yields of periplasmic proteins in functional form by co-expressing (poly)peptides, for example Skp, FkpA, or a homolog of Skp or FkpA, in bacteria.
Expression in the bacterial periplasm is the most convenient route to express foreign recombinant proteins, especially proteins containing disulphides, since the bacterial disulphide forming and isomerization machinery (Bardwell, 1994) can be utilised. Nevertheless, not all proteins can be produced with high functional yield in the
E. coli
periplasm, and no general method for optimizing the expression in functional form of poorly folding proteins secreted into the periplasm exists.
BACKGROUND OF THE INVENTION
Another field where the correct folding of proteins in the periplasm is of crucial importance is in phage display. This method has been used over the last decade to screen libraries not only of peptides but also of a large variety of proteins (Dunn, 1996; McGregor, 1996). These displayed proteins are fused to a phage coat protein, e.g. to the N-terminus of the whole gene-3-protein (g3p) or to its C-terminal domain. These proteins therefore fold in the periplasm, while remaining anchored to the inner membrane by the C-terminal hydrophobic extension of g3p, before being incorporated into the phage coat. Therefore, the g3p fusion-proteins will almost certainly fold in the same environment and use the same machinery as periplasmically expressed proteins. Poorly folding proteins will most likely be lost over multiple screening rounds irrespective of their binding properties.
Co-expression of the cytoplasmic chaperonins GroEL and GroES during M13 phage assembly for Fab display were reported to lead to a 200fold increase in phage titer (Söderlind, 1993). However, the relative amount of functional antibody fragments being displayed by the phage particles was not affected. It was speculated that GroEL/GroES assist in phage packing and assembly, although these steps take place in the periplasm. A general method for increasing the functional display of proteins on phage is not yet available.
Consequently, there has been great interest in the question of the existence of periplasmic chaperones. However, unlike the well-characterized cytoplasmic machinery of
E. coli
, DnaK/DnaJ/GrpE and GroEL/GroES and possibly others (Makrides, 1996 , Martin & Hartl, 1997; Buchner, 1996 ; EP 0 774 512 A3), the chaperone composition of the periplasm has remained poorly understood (Wall & Plückthun, 1995; Missiakas et al., 1996). While progress in elucidating the signal transduction of periplasmic stress has been made (Missiakas & Raina, 1997), the ultimate effector molecules controlling periplasmic folding have remained obscure, although some proteins, such as FkpA or SurA, were believed to act as general periplasmic folding catalysts (Missiakas et al., 1996). FkpA has first been described as very similar to the eukaryotic FK506 binding proteins (FKBPs) (Horne and Young, 1995), a class of well-characterized peptidyl-prolyl cis-trans isomerases (PPIs), which have been shown to be inhibited by the macrolipide FK506. Missiakas and co-workers showed, that the mature FkpA is located in the periplasm and assayed its activity (Missiakas et al., 1996). The estimated Kcat/Km of the cis-trans isomeration of the Ala-Pro peptidyl-prolyl bond using succinyl-Ala-Ala-Pro-Phe-4-nitroanilide (SEQ ID NO: 1) as substrate was 90mM-1s-1. FkpA is directly regulated by &sgr;
E
, which binds in its promoter region (Danese and Silhavy, 1997). The &sgr;
E
pathway is induced by heat stress and conditions, that lead to misfolding or misassembly of outer membrane proteins (OMPs), such as over-expression of OMPs or inactivation of the surA gene.
Another protein which has been discussed in the context of periplasmic folding and protein transport is Skp. Skp is a very basic protein, which at first led to its misassignment as a DNA-binding protein (Holck et al., 1987), later as an outer membrane associated protein (Hirvas et al., 1990; Koski et al., 1990; Koski et al., 1989), and a variety of synonyms (OmpH, HlpA) witness its unclear function. Homologs have been found in
Salmonella typhimurium
(Koski et al., 1990; Koski et al., 1989),
Yersinia enterocolitica
(Hirvas et al., 1991),
Yersinia pseudotuberculosis
(Vuorio et al., 1991),
Haemophilus influenzae
(Fleischmann et al., 1995) and
Pasteurella multocida
(Delamarche et al., 1995). Müller and co-workers (Thome et al., 1990) showed that this protein stimulates the in vitro import of
E. coli
proteins into membrane vesicles and subsequently established its periplasmic location (Thome & Müller, 1991), consistent with its soluble nature and the presence of a signal sequence. More recently, it was proposed to be involved in the transport of outer membrane proteins (Chen & Henning, 1996), and when its promoter region was interrupted by a Tn10 transposon, the extreme heat shock factor &sgr;
E
(&sgr;
24
) dependent response was induced (Missiakas et al., 1996). However, it remained unclear whether this is an effect of the absence of Skp or a polar effect on other proteins located downstream of skp. The heat shock response was probably induced indirectly via a change in the concentration of outer membrane proteins, which is known (Missiakas et al., 1996) to induce a &sgr;
E
(&sgr;
24
).
However, attempts to increase the expression of antibody fragments in functional form by over-expressing
E. coli
disulphide isomerase DsbA and/or proline cis-trans isomerase PPlase A did not significantly change the folding limit (Knappik et al., 1993). It was concluded that aggregation steps in the periplasm compete with periplasmic folding, and that they may occur before disulphide formation and/or proline cis-trans isomerization take place and be independent of their extent.
In summary, no protein has up to now been identified, which unambiguously acts as a periplasmic chaperone and which could be used to optimize the expression yield of a periplasmic protein in functional form.
SUMMARY OF THE INVENTION
Thus, the technical problem underlying the present invention is to identify factors which increase the expression yield of periplasmic proteins in functional form in bacteria and to apply these factors to the optimization of expression of periplasmic proteins. The solution to the above technical problem is achieved by providing the embodiments characterized in the claims. Accordingly, the present invention allows to identify and to apply nucleic acid sequences encoding (poly)peptides which increase the expression yield of periplasmic proteins in functional form, and/or to identify and apply the (poly)peptides. The technical approach of the present invention, i.e. the co-expression of a collection of (poly)peptides with said periplasmic protein in a collection of host cells to screen or select for such nucleic acid sequences and/or (poly)peptides is neither provided nor suggested by the prior art.
Thus, the present invention relates to a method for obtaining a nucleic acid sequence comprising a (poly)peptide coding sequence, which increases the expression yield of a periplasmic protein in functional form in bacteria upon co-expression of said periplasmic protein and said (poly)peptide, comprising the steps of:
(a) providing a collection of host cells wherein each cell contains
(i) a first nucleic acid sequence out of a collection of nucleic acid sequences, and
(ii) a second nucleic acid sequence encoding said periplasmic protein;
(b) causing or allowing expression of
(i) (poly)peptides expressible from said collection of nucleic acid sequences, and
(ii) said periplasmic protein expressible from said secon
Bothmann Hendrick
Plückthun Andreas
Friend Tomas
Heller Ehrman White and McAuliffe
University of Zurich
Wang Andrew
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