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No / Bacterial lipoproteins are a diverse and functionally important group of proteins that are amenable to bioinformatic analyses because of their unique signal peptide features. Here we have used a dataset of sequences of experimentally verified lipoproteins of Gram-positive bacteria to refine our previously described lipoprotein recognition pattern (G+LPP). Sequenced bacterial genomes can be screened for putative lipoproteins using the G+LPP pattern. The sequences identified can then be validated using online tools for lipoprotein sequence identification. We have used our protein sequence datasets to evaluate six online tools for efficacy of lipoprotein sequence identification. Our analyses demonstrate that LipoP (http://www.cbs.dtu.dk/services/LipoP/) performs best individually but that a consensus approach, incorporating outputs from predictors of general signal peptide properties, is most informative.

Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes mammary tumors in mice. MMTV is the only known complex murine retrovirus and encodes Rem, an HIV-1 Rev-like protein. Rem is a 301-amino-acid (33 kDa) protein that is cotranslationally targeted to the ER, where the first 98 amino acids constitute the signal peptide (SP). The SP is cleaved and retrotranslocated to the cytoplasm prior to nuclear entry. In this thesis, the results show that the presence of a leucine at position 71 allows more efficient cleavage of SP and increases Rem activity. Further, in Rem-transfected cells, the majority of SP appears in the nuclear fraction, consistent with fluorescent microscopy data. The C-terminal fragment of Rem (RemCT) is glycosylated in the ER and, although glycosylation sites are present outside the SP, mutations of both these sites abolish SP activity in a reporter assay. Indirect evidence suggests that unglycosylated RemCT is degraded by the proteasome, whereas glycosylated RemCT is likely secreted out of the cell. A variant of MMTV (TBLV) that lacks functional Sag and RemCT has been prepared and will be studied in mice to elucidate the role of RemCT in vivo. Development of an antibody to RemCT will allow tracking of the protein in virus-producing cells. Although there are many other similarities between complex retroviruses like HIV-1 and MMTV, current evidence suggests that Rem lacks an HIV Tat-like transactivator function. / text

This thesis describes experiments investigating the translational recoding activities and the novel dual signalling properties of eukaryotic ribosome skipping 2A sequences. Over twenty years ago, the 19 amino acid 2A region of a Picornavirus; namely, Foot-and-Mouth Disease Virus (FMDV) polypeptide was shown to possess apparent “self-cleaving” abilities, cutting at its own C-terminus during translation (Ryan et al., 1991). Active FMDV 2A-like sequences were subsequently found in a number of related viruses (Luke et al., 2008), with several now utilised as essential biotechnology multi-gene transfer tools (Luke et al., 2010b). Then, in 2006, eukaryotic 2A-like sequences were identified from trypanosome non-LTR sequences. These were found to be functional in vitro (Heras et al., 2006). I have been able to identify over 400 putative eukaryotic 2A-like sequences through searching the freely available online proteomic and genomic databases. Data is presented to show that these 2As were encoded in frame with non-LTRs, or metabolic, or immune function genes, from a wide range of eukaryotic organisms; but I could not discern any obvious phylogenetic distribution for 2A. I have discovered that the majority of eukaryotic 2A sequences tested can mediate ribosome skipping in vitro. Modelling in silico indicated that active 2A-like sequences possessed the propensity to form a central alpha-helical region, whereas the models suggested that inactive 2A-like sequences would be essentially unstructured. I also report that some of these eukaryotic 2A peptides constitute a novel form of dual protein targeting as they play a dual role as exocytic pathway signal peptides mediating extracellular protein trafficking. I have shown that this protein trafficking ability is evolutionarily conserved, with an echinoderm sequence able to direct protein targeting in both plant and mammalian cells. I therefore propose that these novel eukaryotic 2A sequences could potentially become extremely valuable in biotechnological engineering.

We have developed a novel, sensitive and less time-consuming method to detect activity of the SecA-dependent protein-conducting channels. Nanogram levels of E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. The observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein, proOmpA, or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5’-(β, γ-methylene)-diphosphonate, a non-hydrolyzable ATP analog, both of which are known to inhibit SecA protein activity. Channel activity was also stimulated by oocyte endogenous precursor proteins, which could be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides, but not defective signal peptides, stimulated the ionic currents. We also measured SecA-dependent currents with membranes depleted of SecYEG. Wild-type LamB signal peptides, or precursor proteins stimulated ionic currents following a co-injection of SecYEG¯ membranes with puromycin. Excess exogenous SecA stimulated ionic currents through SecYEG¯ membranes. Similar activities of added SecA were observed with reconstituted membranes depleted of SecYEG. Currents through such SecYEG-depleted membranes were also stimulated by addition of defective LamB signal peptides and unfolded mature PhoA protein. In contrast, currents produced by the membranes containing wild-type SecYEG were not so stimulated, but ionic currents were stimulated through mutant strains, similar to PrlA (SecY) suppressors, e.g. PrlA4, or PrlA665 membranes, suggesting that the proofreading function of SecY was bypassed in these membranes. We have observed that azide can inhibit ionic currents when E. coli wild-type MC4100 membranes were injected with proOmpA or LamB signal peptides into Xenopus oocytes. However, such inhibition was lost when observed with oocyte-endogenous signal peptides in the absence of bacterial signal peptides. Moreover, azide did not show complete inhibition upon using SecYEG¯ membranes or SecYEG¯ reconstituted membranes plus excess SecA in the presence or absence of LamB signal peptides. Such conformational alterations reflect different sensitivity in response to azide during the opening of protein-conducting channels.

Chapter 1gives a general introduction to the CXXC motif found in natural proteins. It then reviews the studies where disulphides were engineered in various proteins. The various strategies developed to engineer metal binding activity and redox activity are described. The objectives behind engineering the CXXC motif into a protein, such as imparting it novel metal-binding and redox activities, are discussed next. Alternative strategies which achieve the same objectives are described as well. This chapter then introduces the model proteins used in the course of this thesis: maltose-binding protein (MBP) and E. coli. Thioredoxin (Trx). This chapter also briefly discusses the role of signal peptide in protein export. Chapter 2describes the experimental studies and their results in which we introduced the widely occurring cysteine motif CXXC into the maltose binding protein (one-at-a-time, in five alpha-helices, at the N-termini) to test three hypotheses: 1) Does a disulphide bond form at the N-terminus? 2) Does the protein acquire any oxido-reductase activity? 3) Does it acquire new metal-binding properties? The results confirmed: 1) Each cysteine pair forms a stable intrahelical disulphide bond under non-reducing conditions. 2) The five mutant proteins acquire considerable oxidoreductase activity, tested by the insulin aggregation assay. 3) The mutants acquire novel metal-binding properties for Ni2+, Cd2+, and Zn2+ upon reduction. Further, introducing the CXXC motif neither destabilizes the protein nor affects its global structure. Our results demonstrated that introduction of CXXC motifs can be used to probe alpha-helix start sites and to introduce oxidoreductase and metal binding functionality into proteins. Chapter 3describes further experimentson a few of the metal ion binding mutants discussed in the previous chapter. We explore the effect and usefulness of reducing agents (DTT and TCEP) on the binding of metal salts to the CXXC mutants. We also studied the explore of metal salts on the thermal stability of the mutants and show that metal ions bind to the CXXC motif even when the protein is in the unfolded state. The chapter describes the use of an immobilized metal affinity chromatography (IMAC) based method for the purification of MBP mutants.Yields ranging from 60-85% were obtained for thethree MBP mutants. The cysteines were located at different positions in thesethree MBP mutants (MBP 42-45 Cys, MBP 128-131 Cys, and MBP 359-359 Cys mutants). The yields for wild-type MBP, a single cysteine mutant (MBP S211C), a double cysteine mutant (MBP 230, 30) were all below 15%. Chapter 3 also reports a new crystal structure of the MBP356-359 mutant in ligand bound form:it crystallizes as an intermolecular dimer, bonded by two disulfides formed by the cysteines of the CXXC motif. Chapter 4describes the effects of inserting signal peptide sequences on protein folding and expression. We fused the malE and pelB signal sequences at the N-terminus of the model protein thioredoxin and observed that the wild-type and pelB fusion constructs are soluble when expressed, but the malE construct was targeted to inclusion bodies. Nonetheless, it could be refolded in vitro to yield a monomeric product with a secondary structure identical to the wild-type thioredoxin. This chapter also details the thermodynamic stability, aggregation propensity and activity of the purified recombinant proteins in comparison with the wild-type thioredoxin. The presence of the signal sequences reduces the thermodynamic stability and activity of the recombinants and increases their aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, different signal sequences affect protein stability and aggregation differently. Chapter 5describes three different strategies to label a protein at different sites with cysteine-specific fluorophores using MBP as the model. The first strategy exploits the differential accessibility of residues within MBP in its maltose-bound and maltose-free states. The second strategy involves insertion of a 14-amino-acid loop called V3 from the HIV gp120 protein into MBP; anti-V3 antibodies shield the cysteine residue present inside the inserted loop, while we label another cysteine present outside the loop. In the third strategy, we introduce a third cysteine residue onto the background of the MBP mutant already containing a disulphide bridge at the N-terminus of one of its helices (discussed in Chapter 2). We label the third, free cysteine while the cysteines involved in the disulphide bridge remain protected. We observed successful differential labelling using the first strategy and also observed FRET between the fluorophore labels. Similarly, after trying the second strategy we could individually label all the mutants except one. The third strategy based on the triple-cysteine mutant was not successful because the fluorophore we chose (DBM) did not show site specificity and instead labelled all three cysteines. In addition, the triple-cysteine mutant did not even show disulphide-bridge formation.We showed that indeed the V3 loop inserted in MBP binds anti-V3 antibodies and we could individually label all the mutants expect D41C. The third strategy was not successful because unfortunately in the triple cysteine mutant, the fluorophore we chose (DBM) did not show site specificity and labeled all three cysteines. In addition, the disulfide bridge was not found to be present in the triple cysteine mutant. Chapter 6discusses the synthesis, characterization and binding of various maltolipids, (and their corresponding maltose-free controls) to MBP. The maltolipids were synthesised with varying linker lengths and anchor- & head-groups and then used to prepare liposomes and micelles. Although both liposomal and micellar forms could bind to MBP, only the micelles were screened subsequently for their ability to bind to MBP. The binding was assessed using various techniques such as fluorescence spectroscopy, gel filtration and thermal stability assay. We screened the maltolipids and determined how their anchor group, linker length and charge on the head group influences the binding of MBP to micelles formed by these maltolipids.

Background: Cyclodextrin glucanotransferases (CGTases) catalyze the synthesis of cyclodextrins, cyclic oligosaccharides composed of glucose monomers that find applications in the pharmaceutical, food, and cosmetic industries. An economic application of these industrially important enzymes requires their efficient production and recovery. In this study, the effect of Sec-type signal peptides on the recombinant expression of a CGTase derived from Bacillus sp. G825-6 was investigated in Escherichia coli BL21(DE3) using a codon-adapted gene. In addition, a novel purification method for the CGTase using starch adsorption was developed. Results: Expression vectors encoding N-terminal PelB, DacD, and the native Bacillus sp. G825-6 CGTase signal peptides (SP) were constructed for the recombinant CGTase. With the DacD SP derived from E. coli, a 3.9- and 3.1-fold increase in total enzyme activity was obtained compared to using the PelB and the native CGTase SP, respectively. DacD enabled a 7.3-fold increase of activity in the extracellular fraction after induction for 24 h compared to the native CGTase SP. After induction for 48 h, 75% of the total activity was detected in the extracellular fraction. By a batch wise adsorption to starch, the extracellular produced CGTase could be purified to homogeneity with a yield of 46.5% and a specific activity of 1637 U/mg. Conclusions: The signal peptide DacD promoted the high-level heterologous extracellular expression of a recombinant CGTase from Bacillus sp. G825-6 with a pET20b(+) vector in E. coli BL21(DE3). A protocol based on starch adsorption enabled a fast and efficient purification of the recombinant enzyme.