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Microbial degradation of aromatic hydrocarbons is imperative for maintaining the global carbon cycle and removing potentially toxic aromatic xenobiotics. This thesis focuses on the characterization of a pyruvate-specific class II aldolase (BphI) and acetaldehyde dehydrogenase (BphJ), the final two enzymes of the bph meta-cleavage pathway in Burkholderia xenovorans LB400. This pathway is responsible for the degradation of the industrial pollutant polychlorinated biphenyls (PCB) and therefore mechanistic characterization of these enzymes can be applied to improve pollutant degradation. BphI catalyzes the aldol cleavage of 4-hydroxy-2-oxoacids to pyruvate and an aldehyde while BphJ transforms aldehydes to acyl-CoA, using NAD+ and CoASH as cofactors. Size-exclusion chromatography was used to determine that the oligomeric unit of the BphI-BphJ complex is a heterotetramer. The aldolase BphI was shown to exhibit a compulsory order mechanism and utilize 4-hydroxy-2-oxoacids with an S configuration at C4. The generation of BphI active site variants allowed for the proposal of a catalytic mechanism and a greater understanding as to how stereospecificity occurs. Using steady-state kinetic assays, Arg-16 was demonstrated to be essential for catalysis. Molecular modeling of the substrate and pH dependency (wild-type pKa of ~7, lost in H20A and H20S variants) were used to identify His-20 as the catalytic base. Tyr-290 was originally proposed to be the catalytic acid. However, this was refuted as a Tyr-290 (Y290F) variant did not affect the catalytic efficiency of the enzyme. Instead, the variant was observed to exhibit a loss of stereochemical control. From the crystal structure of an orthologous aldolase-dehydrogenase complex, solvent isotope effect studies, and a proton inventory, a water molecule was implicated as the catalytic acid. Based on their position within the crystal structure, Leu-87 and Leu-89 were implicated in substrate specificity. Replacement of Leu-89 with alanine effectively increased the length of the active site, allowing for the accommodation of longer aldehyde substrates. In contrast, Leu-87 was responsible for hydrophobic stabilization of the C4-methyl of the substrate. Double variants L87N;Y290F and L87W;Y290F were constructed to enable the binding of 4(R)-hydroxy-2-oxoacids. Polarimetric analysis confirmed that the double variants were able to synthesize 4-hydroxy-2-oxoacids of up to 8 carbons in lengths, which were of the opposite stereoisomer to those produced by the wild-type enzyme. Cys-131 was identified as the catalytic thiol that forms an acyl-enzyme intermediate in the dehydrogenase, BphJ. This enzyme was shown to exhibit similar specificity constants for acetaldehyde and propionaldehyde and utilize aliphatic aldehydes from two to five carbons in length as substrates. The enzyme was able to use either NAD+ or NADP+ as the cofactor. Finally, we demonstrated that aldehydes produced in the aldolase reaction are not released into the bulk solvent but are channeled directly to the dehydrogenase, providing the first biochemical determination of substrate channeling in any aldolase-dehydrogenase complex. / Chapter 3 - Reprinted (adapted) with permission from Baker, P., Carere, J., and Seah, S. Y. (2011) Probing the Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis in the Class II Pyruvate Aldolase, BphI, Biochemistry 50: 3559-3569. Copyright (2011) American Chemical Society. Chapter 4 - Reprinted (adapted) with permission from Baker, P., and Seah, S. Y. (2012) Rational design of stereoselectivity in the class II pyruvate aldolase BphI, J Am Chem Soc 134: 507-513. Copyright (2012) American Chemical Society. Chapter 6 - Reprinted (adapted) with permission from Baker, P., Hillis, C., Carere, J., and Seah, S. Y. (2012) Protein-protein interactions and substrate channeling in orthologous and chimeric aldolase-dehydrogenase complexes, Biochemistry 51: 1942-1952. Copyright (2012) American Chemical Society. / National Science and Engineering Research Council of Canada (NSERC), Ontario Graduate Scholarship in Science and Technology

Escherichia coli O157:H7 is an enteric pathogen that can cause severe gastrointestinal disease, sometimes leading to hospitalization and death. These bacteria have a variety of virulence factors that can be encoded for on pathogenicity islands (PAIs). The goal of this study was to characterize specific E. coli O157:H7 PAI deletion mutants using three methods: Phentotype Microarrays (PM), growth curves and survival curves were used to elucidate possible roles for the PAIs. Results from the PM study suggest that PAIs have a role in carbon substrate utilization; i.e., four of the O-island (OI) deletion mutants (OI-87, 98, 102 and 172) and an S-Loop (SL-72) deletion mutant exhibited differences in substrate utilization (gains and losses in utilization) compared to parental O157:H7 strains EDL933 (OI) and Sakai (SL), respectively. All of the mutants with the exception of the OI-135 mutant exhibited differences in level of substrate utilization for substrates shown to have important roles in the bacterium. Cell growth results showed that three OI deletion mutants (OI-55, 87 and 102) and the SL (SL-72) mutant exhibited a difference in rate of growth compared to the parental strains. Cell viability results showed that seven of the OI deletion mutants (OI-51, 55, 98, 108, 135, 172 and 176) exhibited different rates of decline in cell number when transferred to sterile water compared to the parental strain. The results show that removal of PAIs from E. coli O157:H7 can affect carbon utilization, growth and survival demonstrating the importance of PAIs in the ecology of these bacteria. / xx, 208 leaves : ill. (some col.) ; 29 cm

DNA damage is important to understand since it has the potential to lead to disease if unrepaired. In particular, bulky C8 guanine adducts (addition products) are known to induce a variety of mutations due to their conformational flexibility. C-linked C8-phenoxyl-deoxyguanosine adducts (PhOH-dG) have been poorly understood despite their potential for genotoxicity. This thesis systematically develops a computational model to predict the conformational and base-pairing preferences of PhOH-dG by gradually increasing the size of the system. The structure of PhOH-dG in DNA is determined, where the bulky C8 group induces a syn conformation of the base similar to other C8-adducts. A stabilized guanine mismatch is identified for the syn adducts, which implies that the primary mechanism of genotoxicity may be base-substitution mutations resulting in G→C transversions. This thesis has contributed to a growing body of literature dedicated to understanding the role of conformational heterogeneity in the mutagenicity of bulky C8-adducts. / xix, 192 leaves : ill. (some col.) ; 29 cm

Here we developed multiple genetic systems through which genetic modifications driven by DNA breaks caused by the I-SceI nuclease can be assayed in the yeast Saccharomyces cerevisiae and in human cells. Using the delitto perfetto approach for site-directed mutagenesis in yeast, we generated isogenic strains in which we could directly compare the recombination potential of different I-SceI variants. By genetic engineering procedures, we generated constructs in human cells for testing the recombination activity of the same I-SceI variants. Both in yeast and human cells we performed gene correction experiments using oligonucleotides (oligos) following modification and/or optimization of existing gene targeting protocols and development of new ones. We demonstrated that an I-SceI nicking enzyme can stimulate recombination on the chromosome in S. cerevisiae at multiple genomic loci. We also demonstrated in yeast that an I-SceI-driven nick can activate recombination 10 kb distant from the initial site of the chromosomal lesion. Moreover we demonstrated that an I-SceI nick can stimulate recombination at the site of the nick at episomal and chromosomal loci in human cells. We showed that an I-SceI double-strand break (DSB) could trigger recombination up to 2 kb distant from the break at an episomal target locus in human cells, though the same was not observed for the nick. Overall, we demonstrated the capacity for I-SceI nick-induced recombination in yeast and human cells. Importantly, our findings reveal that the nick stimulates gene correction by oligos differently from a DSB lesion, as determined by genetic and molecular analyses in yeast and human cells. This research illustrates the promise of targeted gene correction following generation of a nick.

The primary aim of the work presented in this thesis was to change the inducer specificity of the DntR protein in order to improve the response to DNT. The long-term goal is to use this protein in a biosensor for DNT, a signature compound for detection of the explosive TNT. Another aspect of this work was to understand the mechanisms of inducer binding and how the binding of an inducer molecule changes the DntR structure into a state that triggers transcriptional activation. In the papers included in this thesis the inducer specificity of wt DntR has been investigated under different conditions. The functional effects of specific mutations have also been investigated, in some cases in combination with structure determination using X-ray crystallography. In addition, structural data offering insights into the details of inducer binding and conformational changes upon inducer binding are presented and discussed in terms of mechanisms for transcriptional activation by DntR. Furthermore, a directed evolution strategy was employed in order to find variants of DntR with improved response to DNT. A variant with a large improvement in the DNT response was isolated and characterized. In optimized growth conditions, this DntR variant had a nearly 10-fold increase in fluorescence in response to DNT compared to wt DntR. Specific substitutions found in this DntR variant are suggested to be important for changing the inducer response. / Syftet med denna avhandling har varit att förbättra förmågan hos proteinet DntR att upptäcka DNT. Det långsiktiga målet har varit att använda DntR i en biosensor för att upptäcka sprängämnet TNT, som avger DNT som en ”signaturmolekyl”. En annan aspekt har varit att bättre förstå den detaljerade mekanismen för hur DntR fungerar. DntR är ett protein som binder till en viss DNA sekvens (promotor) och reglerar hur gener intill denna promotorsekvens läses av. När en inducerande molekyl som t.ex. DNT binder till DntR förändras proteinets struktur på ett sådant sätt att DntR kan aktivera transkription av de gener som finns intill promotor-sekvensen. För att mäta hur DntR reagerar på olika inducerande molekyler har DntR uttryckts i bakterien Escherichia coli, som också innehållit promotorn som DntR binder till. Intill promotorn sitter en gen som kodar för proteinet GFP. När en inducerande molekyl binder till DntR, slås avläses gfp-genen, och det fluorescerande proteinet GFP produceras. Ju mer GFP som produceras i cellerna, desto högre fluorescens kan uppmätas när cellerna analyseras.   I de artiklar som presenteras i avhandlingen har vi undersökt hur olika substitutioner i DntR proteinet påverkar specificiten och sensitiviteten och hur dessa egenskaper kan påverkas av olika experimentella faktorer. Effekten av substitutioner har relaterats till strukturdata, där bilder av hur proteinet ser ut på molekylär nivå har tagits fram. Dessutom presenteras även en bild av hur DntR förändras beroende på om inducerande molekyler är bundna eller inte. En sådan strukturbild ökar förståelsen för de mekanismer som gör att bindning av en inducerande molekyl orsakar en förändring av formen hos DntR på så sätt att avläsning av gener kan aktiveras. Vi har också använt en metod där evolutionära processer härmats för att få fram varianter av DntR med förbättrad respons till DNT. En variant med en drastisk ökning av DNT-responsen har isolerats, och dess egenskaper har karaktäriserats. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript

Dengue virus (DENV) is the causative agent of dengue fever (DF), the most prevalent arthropod-borne viral disease in the world and therefore is considered an emerging global health threat. The four DENV serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) that infect humans are distinguished from one another by unique antigenic determinants (epitopes) on the DENV envelope (E) protein. The E protein is the primary antigenic site of the DENV and is responsible for inducing neutralising antibody (Ab) and cell mediated immune response in DENV infected hosts. The DENV E protein also mediates attachment of virions to host cell receptors and entry of virions into host cells by membrane fusion. The study of epitopes on DENV E protein is necessary for understanding viral function and for the design of unique polyvalent vaccines capable of inducing a neutralising antibody response against each DENV serotype. Reverse genetics using infectious cDNA clones has enabled the construction of functional intertypic DENV, where the E protein of one DENV serotype is put in the genetic background of a different DENV serotype. In addition, observations from our laboratory indicate that chimeric E proteins, consisting of E protein structural domains from different DENV serotypes can fold into functional proteins. This suggests that there is potential to engineer viruses with intertypic DENV E proteins as potential DENV vaccine candidates, which is the long term goal of studies within our research group. However, if a chimeric E protein was to be constructed containing epitopes involved in antibody mediated neutralisation of each DENV serotype, then knowledge of the location of these epitopes on the E protein of each DENV serotype would be essential. Prior to this study, monoclonal antibodies (MAbs) had been used to identify epitopes involved in antibody mediated neutralisation on the E protein of all DENV serotypes, except DENV-4. The primary objective of this study was to identify epitopes on the DENV-4 E protein involved in neutralisation by antibodies. In order to achieve this objective, a panel of 14 MAbs was generated against DENV-4 in BALB/c mice and characterised using various serological and functional assays. The identification of DENV-4 specific neutralising MAbs in the panel was essential for subsequent experiments aimed at determining antigenic domains, structural domains or specific epitopes (peptides or amino acids) involved in the neutralisation of DENV-4. The majority of MAbs (11/14) generated against DENV-4 recognised the E protein. The remaining three MAbs reacted with the non-structural (NS) 1 protein. The majority of MAbs against the E protein were DENV or Flavivirus group reactive, but four MAbs were DENV-4 specific. All MAbs against the E protein recognised conformationally dependent epitopes and were able to capture DENV-4 in an enzyme linked immuno-adsorbent assay (ELISA). Eighty percent (9/11) of the anti-E MAbs produced for this study neutralised infection of cells by DENV-4 in vitro. Three of the neutralising MAbs (F1G2, 18F5 and 13H8) were DENV-4 specific and also demonstrated the strongest neutralisation activity of the panel, reducing DENV-4 infectivity by 100-1000 fold. The amount of virus neutralised by the MAbs was not related to the avidity of the MAbs. The DENV-4 specific MAbs F1G2, 18F5 and 13H8 were used to identify epitopes involved in neutralisation of DENV-4. The MAbs that effectively captured DENV-4 were used in competitive binding assays (CBAs) to determine spatial relationships between epitopes and therefore define antigenic domains on the DENV-4 E protein. The CBAs indicated that the epitopes recognised by the panel of MAbs segregated into two distinct domains (D4E1 and D4E2) and both contained epitopes involved in neutralisation. CBAs incorporating human serum from DENV-4 infected patients suggested that the MAbs recognised the same, or spatially related, epitopes in domain D4E2 as antibodies from humans who had experienced natural dengue infections, indicating the clinical relevance of such epitopes for the development of DENV vaccines. The reactivity of the capture MAbs with low pH treated DENV-4 was also evaluated in an attempt to identify epitopes that might be more accessible during low pH-mediated virus fusion. Only one of the MAbs (13H8) recognised an acid resistant epitope. Initial attempts to identify epitopes on the DENV-4 E protein involved in neutralisation followed the traditional epitope mapping approach of selecting subpopulations of DENV-4 which escaped neutralisation by MAbs. These attempts were unsuccessful so a variety of strategies for mapping epitopes were used including DENV-4 variant analysis and site directed mutagenesis of the DENV-4 E protein, MAb screening of chimeric DENV-3/4 E proteins and MAb screening of a bacterial peptide display library. DENV-4 variants including DENV-4 isolates from different geographical locations or chemically mutagenised DENV-4 were screened with neutralising MAbs to identify neutralisation escape mutant (n.e.m.) viruses. Site directed mutagenesis of the DENV-4 E protein confirmed whether amino acid changes identified in DENV-4 n.e.m.s were essential for the binding of neutralising MAbs to an epitope. The MAb screening of DENV-4 variants identified n.e.m.s with amino acid changes at residues E95, E96, E156, E157, E203, E329 and E402 of the DENV-4 E protein. Site directed mutagenesis of the DENV-4 E protein identified two epitopes recognised by the DENV-4 specific neutralising MAbs F1G2 and 18F5 at specific amino acid residues within domains II and III of the DENV-4 E protein. No specific epitopes were identified for the MAb 13H8; however this MAb did recognise domain I and II of the DENV-4 E protein, when screened against DENV-3/4 chimeric DENV E proteins. The first epitope, which was recognised by the MAb F1G2, contained residue E95 which was located in domain II of the DENV-4 E protein. The aspartate (Asp) to alanine (Ala) change at E95 prevented the binding of F1G2 to the DENV-4 E protein. The binding of F1G2 to the E95 residue was confirmed using the pFlitrX bacterial peptide display library, which demonstrated binding of F1G2 to a peptide homologous with residues E99-E104. No peptides recognised by 13H8 and 18F5 were identified by this method. The MAb F1G2 also bound to the domain III region (E300-E495) of the DENV-4 E protein when screened against DENV-3/4 chimeric DENV E proteins. This implied that F1G2 may be recognising a discontinuous epitope consisting of domains II and III. The second epitope, which was recognised by MAb 18F5, contained residue E329 which was located in domain III of the DENV-4 E protein. The alanine (Ala) to threonine (Thr) change at E329 prevented the binding of 18F5 to the DENV-4 E protein. MAb 18F5 also bound to the domain III region (E300-E495) of the DENV-4 E protein when screened against DENV-3/4 chimeric E proteins, thus confirming the E329 epitope. The potential mechanisms by which the DENV-4 specific MAbs neutralise virus infection were evaluated by the virus overlay protein binding assay (VOPBA). The binding of MAb 18F5 to a domain III (E329) epitope of the DENV-4 E protein and the binding of MAb F1G2 to domain II (E95, E99-E104) and domain III epitopes (chimeric E protein) of the DENV-4 E protein, prevented the attachment of DENV-4 to a 40 kDa C6/36 cell protein. In contrast the binding of MAb 13H8 to domains I and II of the DENV-4 E protein did not prevent attachment of DENV-4 to the same protein. This was preliminary evidence that the binding of domain III epitopes by the MAbs F1G2 and 18F5 may be important in preventing virus attachment. The binding of MAb 13H8 to domains I and II, and the ability of this MAb to recognise DENV-4 treated at low pH, suggested that MAb 13H8 may block epitopes exposed at low pH that are required for low pH mediated virus fusion to host cell membranes. Overall, the different methods used in this study identified epitopes involved in the neutralisation of DENV-4. The distribution of epitopes involved in neutralisation throughout the DENV-4 E protein were similar to the distribution of epitopes involved in neutralisation on the DENV-1, 2 and 3 E proteins. This suggested that it might be possible to elicit neutralising antibodies against multiple DENV serotypes using chimeric E-proteins derived from two or more DENV serotypes and therefore, facilitate the design of novel tetravalent DENV vaccines.

Bovine herpesvirus-1 (BoHV-1) is an important pathogen of cattle associated with respiratory and reproductive disease and is the most common viral agent implicated in the bovine respiratory disease complex (BRDC). BRDC is an economically significant multifactorial disease of feedlot cattle estimated to cost Australian feedlot producers $AU60 million/year in lost production, therapeutics and disease management. Worldwide BRDC is attributed to cost $US2 billion to cattle industries. In an effort to limit the associated economic costs and enhance animal health and welfare of feedlot cattle, the concerted use of vaccination and diseased animal management are practiced. Numerous vaccines are available in North America and Canada however, in Australia, feedlot producers are reliant on three vaccines. These vaccines target either the bacterial or viral agents of the BRDC and encompass antibody, subunit and attenuated live BoHV-1 preparations. Live attenuated vaccines are developed by numerous methods including, deletion or disruption of certain genes. The development of an attenuated live virus vaccine was traditionally a laborious task requiring numerous rounds of in vitro purification. Contrastingly, technological advances introduced this decade, allowing the stable maintenance of the complete herpesvirus genome in bacteria as a bacterial artificial chromosome (BAC), has advanced herpes virology exponentially in that investigation and manipulation of the herpesvirus genome can be conducted independent of a cell culture system. With respect to BRDC and the generation of vaccines to combat the disease, the tools to fully utilise the potential of BoHV-1 as a live vaccine vector are now routine. It is now possible to vii construct BoHV-1 as a delivery vector by inserting appropriate antigens of those bacterial and viral pathogens implicated in the BRDC into a BAC maintained BoHV-1 genome. However, there is a significant lack of genetic information regarding BoHV-1 and inserting several antigenic sequences would expand the genome of BoHV-1 inducing non-viability. Therefore, to further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and nonessential genes required for the in vitro viability of BoHV-1. Identifying the essential and nonessential genes will establish which genes may be preferentially deleted or replaced with exogenous antigenic sequences in a BoHV-1 derived vaccine vector. To define the requirement of genes encoded by BoHV-1, random-insertion mutagenesis utilising a Tn5 transposition system and targeted gene deletion catalysed by GET recombination was employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion was determined by direct sequencing. with the essential or nonessential requirement of either transposed or deleted open reading frames (ORFs) assessed by transfection of respective BoHV- 1 BAC DNA into host cells. Of the 73 recognised ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be nonessential for virus viability in cell culture with the requirement of the two dual copy ORFs inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by Human herpesvirus 1. However, ORFs encoding for glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to Human herpesvirus-1 encoded homologues. Further analysis of clones encompassing restriction digestion profiling, one-step growth and replication kinetic analysis defined the genetic constitution and replicative capacity of the mutant clones. Thirty-three individual ORFs of the 36 defined nonessential ORF were identified as being amenable to deletion without causing significant replicative detriment to a potential BoHV-1 vaccine vector. This study has provided the foundational information required for the future development of BoHV-1 as a multivalent vaccine vector for the protection of feedlot cattle from BRDC. Furthermore, the genetic information generated in this study contributes to the general knowledge of the prototype ruminant herpesvirus, BoHV-1, and contributes to the comparative study of gene function between the large and diverse family that is Herpesviridae.

Protein aggregation is commonly encountered during the manufacture of protein-based bioproducts in processing such as protein expression, purification, refolding, shipping and storage (Volkin and Middaugh, 1992; Brange, 2000). Aggregation may shorten the shelf-life of pharmaceutical proteins (Frokjaer and Otzen, 2005) and induce severe hypersensitivity (Rosenberg, 2006). In addition, several diseases ranging from Alzheimer’s disease to cystic fibrosis are associated with protein aggregation in the form of amyloid fibrils and plaques (Dobson, 1999; Luheshi et al., 2008). Hence, studies on protein aggregation, especially those dealing with high concentrations of proteins, are highly demanded in both academic and industrial laboratories. To address the aforementioned issues, physicochemical factors affecting protein aggregation were investigated systematically in this project. Strategies were developed to inhibit protein aggregation during renaturation and to enhance protein stability against aggregation during and after production, especially when dealing with high protein concentrations. ∆5-3-Ketosteroid isomerase (KSI) was used as a model for aggregation studies during protein renaturation due to its intrinsic aggregation properties. KSI was overexpressed as inclusion bodies (IBs) in Escherichia coli (E. coli). Cost- and time-efficient combination of chemical extraction and one-step affinity purification ensured the production of denatured KSI with high purity at high yield. Several key factors, including protein concentration and ionic strength, were determined to greatly influence KSI aggregation during renaturation. Polymer addition (PEG 3000 and Eudragit S-100) was found to alter KSI aggregation behaviour in a polymer-specific manner, as quantified using reversed phase-high performance liquid chromatography (RP-HPLC) analysis. Light scattering for second virial coefficient (SVC) measurement, surface plasmon resonance (SPR), and microfluidics were applied to study the fundamental mechanism of protein aggregation. Lysozyme was further introduced as a control protein for comparison with KSI. A rapid lumped method was established to measure specific refractive index (∂n/∂c) and SVC values for KSI and lysozyme, which provided quantitative and qualitative information on thermodynamic interactions of molecules in solution. SPR and microfluidics were also used to explore protein aggregation properties. To our best knowledge, it is the first time SPR and microfluidics have been used to investigate protein aggregation behaviour. Both SPR and microfluidics present significant potential for assessing protein aggregation and diagnosis or drug screening of protein aggregation related diseases. The chemical and physical stability of proteins needs to be maintained after successful refolding to ensure an acceptably long shelf life, especially at high protein concentration (Chang and Hermsdorf, 2002). The pharmaceutical effects of lectins on cell growth provided incentive for studies to improve their stability. Human galectin-2 (hGal-2, a homodimeric lectin) was used as a study model in this project. Mutations were introduced at one of the two Cys residues (C57A, C57M, and C57S). Only the C57M variant was highly expressed in bacteria in soluble form. No aggregate of this mutant was detected during 3 weeks of storage. hGal-2 C57M also facilitated site-directed introduction of poly(ethylene glycol) (PEG) into the remaining sulfhydryl group (Cys75). Product analysis revealed rather complete conjugation with one PEG chain per protein subunit in homodimer. Neither secondary structure alteration nor the absence of binding ability to a glycoprotein (asialofetuin) was observed. The results document the feasibility of tailoring a human galectin for enhanced stability against aggregation as well as monoPEGylation, which enables further testing of biological properties including functionality as a growth regulator and the serum clearance rate of hGal-2.