蓝藻抗病毒蛋白-N基因的克隆、表达、纯化及活性鉴定

蓝藻抗病毒蛋白-N(Cyanovirin-N,CVN)具有强效抗HIV及其他包膜病毒活性,该蛋白序列特殊,难以重组制备,在大肠杆菌细胞质中形成包涵体。本研究根据大肠杆菌密码子偏好性对CVN原始核苷酸序列进行优化,通过多次PCR合成SUMO-CVN的全长DNA序列,构建pET3c-SUMO-CVN重组表达质粒,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。通过对诱导剂浓度和诱导时间的优化,发现以0.5mmol/LIPTG在20℃诱导24h可获得最高表达,SDS-PAGE结果显示,SUMO-CVN为可溶性表达,表达量占菌体总蛋白的28%;经特异性的SUMO蛋白酶对融合蛋白进行酶切及两步Ni-NTA凝胶亲和层析可以得到纯度较高的重组CVN蛋白。ELISA结果表明,重组蛋白CVN与gp120蛋白有较高的亲和力。体外抗病毒活性实验表明,重组蛋白CVN在纳摩尔浓度具有很好的抗HSV-1和HIV-1/ⅢB活性;这为开发基于CVN的新型、高效抗病毒药物打下了基础。     

Semisynthetic analogues of PSC-RANTES, a potent anti-HIV protein

New HIV prevention methods are needed, and among those currently being explored are "microbicides", substances applied topically to prevent HIV acquisition during sexual intercourse. The chemokine analogue PSC-RANTES (N(alpha)(n-nonanoyl)-des-Ser(1)-[ L-thioprolyl(2), L-cyclohexylglycyl(3)]-RANTES(4-68)) is a highly potent HIV entry inhibitor which has shown promising efficacy in its initial evaluation as a candidate microbicide. However, a way must be found to produce the molecule by cheaper means than total chemical synthesis. Since the only noncoded structures are located at the N-terminus, a possible solution would be to produce a protein fragment representing all but the N-terminal region using low-cost recombinant production methods and then to attach, site specifically, a short synthetic fragment containing the noncoded N-terminal structures. Here, we describe the evaluation of a range of different conjugation chemistries in order to identify those with potential for development as economical routes to production of a PSC-RANTES analogue with antiviral activity as close as possible to that of the parent protein. The strategies tested involved linkage through oxime, hydrazone/hydrazide, and Psi[CH2-NH] bonds, as well as through a peptide bond obtained either by a thiazolidine rearrangement or by direct alpha-amino acylation of a protein fragment in which 4 of the 5 lysine residues of the native sequence were replaced by arginine (the fifth lysine is essential for activity). Where conjugation involved replacement of one or more residues with a linker moiety, the point in the main chain at which the linker was introduced was varied. The resulting panel of 22 PSC-RANTES analogues was evaluated for anti-HIV activity in an entry inhibition assay. The [Arg (25,45,56,57)] PSC-RANTES analogue has comparable potency to PSC-RANTES, and one of the oxime linked analogues, 4L-57, has potency only 5-fold lower, with scope for improvement. Both represent promising leads for development as microbicide compounds that could be produced at low cost via semisynthesis.     

Expression, purification, and characterization of recombinant cyanovirin-N for vaginal anti-HIV microbicide development

Cyanovirin-N (CV-N) is a prokaryotic protein under development as a topical anti-HIV microbicide, an urgent and necessary approach to prevent HIV transmission in at-risk populations worldwide. We have expressed recombinant CV-N as inclusion bodies in the cytoplasm of Escherichia coli. A purification scheme has been developed that exploits the physicochemical properties of this protein, in particular its stability in a harsh inclusion body purification scheme. Under the conditions developed, this system yields 140 mg of highly purified CV-N per liter of high-density cell culture, which represents a 14-fold increase over the best recombinant CV-N yield reported to date. This purification scheme results in monomeric CV-N as analyzed by SDS-PAGE, isoelectric focusing, and reverse phase- and size exclusion-HPLC. This recombinantly expressed and refolded CV-N binds to gp120 with nanomolar affinity and retains its potent anti-HIV activities in cell-based assays. The expression and purification system described herein provides a better means for the mass production of CV-N for further microbicide development.     

Site-specific discrimination by cyanovirin-N for alpha-linked trisaccharides comprising the three arms of Man(8) and Man(9)

Cyanovirin-N (CVN) is a novel cyanobacterial protein that selectively binds with nanomolar affinities the mammalian oligosaccharides Man(8) and Man(9). Consequently, CVN potently blocks HIV entry through highly avid carbohydrate-mediated interactions with the HIV-envelope glycoprotein gp120, and is under preclinical investigation as an anti-HIV microbicide. CVN contains two non-overlapping carbohydrate-binding sites that bind the disaccharide Manalpha(1-2)Manalpha (which represents the terminal disaccharide of all three arms of Man(9)) with low to sub-micromolar affinities. The solution structure of a 1:2 CVN:Manalpha(1-2)Manalpha complex revealed that CVN recognizes the stacked conformation of Manalpha(1-2)Manalpha through a deep hydrophilic-binding pocket on one side of the protein (site 2) and a semi-circular cleft on the other (site 1). With the prominent exception of the C1 hydroxyl group of the reducing mannopyranose ring, the bound disaccharide is positioned so that each hydroxyl group is involved in a direct or water-mediated hydrogen bond to the polar or charged side-chains comprising the binding pocket. Thus, to determine whether the next-most reducing mannopyranose ring will augment CVN affinity and selectivity, we have characterized by NMR and ITC the binding of CVN to three synthetic trisaccharides representing the full-length D1, D2 and D3 arms of mammalian oligomannosides. Our findings demonstrate that site 1 is able to discriminate between the three related trisaccharides methyl Manalpha(1-2)Manalpha(1-2)Man, methyl Manalpha(1-2)Manalpha(1-3)Man and methyl Manalpha(1-2)Manalpha(1-6)Man with remarkable selectivity, and binds these trisaccharides with K(A) values ranging from 8.1x10(3)M(-1) to 6.6x10(6)M(-1). Site 2 is less selective in that it binds all three trisaccharides with similar K(A) values ranging from 1.7 to 3.7(+/-0.3)x10(5)M(-1), but overall binds these trimannosides with higher affinities than site 1. The diversity of pathogenic organisms that display alpha(1-2)-linked mannosides on their cell surfaces suggests a broad defensive role for CVN in its cyanobacterial source.     

Solution structure of a cyanovirin-N:Man alpha 1-2Man alpha complex: structural basis for high-affinity carbohydrate-mediated binding to gp120

BACKGROUND: Cyanovirin-N (CVN) is a novel, 11 kDa cyanobacterial protein that potently inhibits viral entry by diverse strains of HIV through high-affinity carbohydrate-mediated interactions with the viral envelope glycoprotein gp120. CVN contains two symmetry-related carbohydrate binding sites of differing affinities that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinities, the carbohydrates that also mediate CVN:gp120 binding. High-resolution structural studies of CVN in complex with a representative oligosaccharide are desirable for understanding the structural basis for this unprecedented specificity. RESULTS: We have determined by multidimensional heteronuclear NMR spectroscopy the three-dimensional solution structure of CVN in complex with two equivalents of the disaccharide Manalpha1-2Manalpha, a high-affinity ligand which represents the terminal-accessible disaccharide present in Man(8) D1D3 and Man(9). The structure reveals that the bound disaccharide adopts the stacked conformation, thereby explaining the selectivity for Man(8) D1D3 and Man(9) over other oligomannose structures, and presents two novel carbohydrate binding sites that account for the differing affinities of the two sites. The high-affinity site comprises a deep pocket that nearly envelops the disaccharide, while the lower-affinity site comprises a semicircular cleft that partially surrounds the disaccharide. The approximately 40 A spacing of the two binding sites provides a simple model for CVN:gp120 binding. CONCLUSIONS: The CVN:Manalpha1-2Manalpha complex provides the first high-resolution structure of a mannose-specific protein-carbohydrate complex with nanomolar affinity and presents a new carbohydrate binding motif, as well as a new class of carbohydrate binding protein, that facilitates divalent binding via a monomeric protein.     

Soluble cytoplasmic expression, rapid purification, and characterization of cyanovirin-N as a His-SUMO fusion

Cyanovirin-N (CVN) is a promising antiviral candidate that has an extremely low sequence homology with any other known proteins. The efficient and soluble expression of biologically functional recombinant CVN (rCVN) is still an obstacle due to insufficient yield, aggregation, and abnormal modification. Here, we describe an improved approach to preparing native rCVN from Escherichia coli more efficiently. A fusion gene consisting of cvn and sumo (small ubiquitin-related modifier) and a hexahistidine tag was constructed according to the codon bias of the host cell. This small ubiquitin-related modifier (SUMO)-fused CVN is expressed in the cytoplasm of E. coli in a folded and soluble form (>30% of the total soluble protein), yielding 3 to 4 mg of native rCVN from 1 g of wet cells to a purity up to 97.6%. Matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry and reverse-phase high-performance liquid chromatographic analysis showed that the purified rCVN was an intact and homogeneous protein with a molecular weight of 11,016.68 Da. Potent antiviral activity of rCVN against herpes simplex virus type 1 and human immunodeficiency virus type 1/IIIB was confirmed in a dose-dependent manner at nanomolar concentrations. Thus, the His-SUMO double-fused CVN provides an efficient approach for the soluble expression of rCVN in the cytoplasm of E. coli, allowing an alternative system to develop bioprocess for the large-scale production of this antiviral candidate.     

Overexpression and purification of scytovirin, a potent, novel anti-HIV protein from the cultured cyanobacterium Scytonema varium

Scytovirin (SVN) is a novel anti-human immunodeficiency virus (HIV) protein isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. The protein consists of a single 95-amino acid chain with significant internal sequence duplication and 10 cysteines forming five intrachain disulfide bonds. A synthetic gene that encodes scytovirin was constructed, and expressed in Escherichia coli, with thioredoxin (TRX) fused to its N-terminus (TRX-SVN). Most of the expressed protein was in soluble form, which was purified by a polyhistidine tag affinity purification step. SVN was then cleaved from TRX with enterokinase and separated from the TRX partner by C18 reversed-phase HPLC. This production method has proven superior to earlier synthetic attempts and recombinant procedures using a standard expression system. The current system resulted in yields of 5–10 mg/L of purified SVN for structural studies and for preclinical development of SVN as a topical microbicide for HIV prophylaxis.     

Solution and crystal structures of a sugar binding site mutant of cyanovirin-N: no evidence of domain swapping

The cyanobacterial lectin Cyanovirin-N (CV-N) exhibits antiviral activity against HIV at a low nanomolar concentration by interacting with high-mannose oligosaccharides on the virus surface envelope glycoprotein gp120. Atomic structures of wild-type CV-N revealed a monomer in solution and a domain-swapped dimer in the crystal, with the monomer comprising two independent carbohydrate binding sites that individually bind with micromolar affinity to di- and trimannoses. In the mutant CVN(mutDB), the binding site on domain B was abolished and the protein was found to be completely inactive against HIV. We determined the solution NMR and crystal structures of this variant and characterized its sugar binding properties. In solution and the crystal, CVN(mutDB) is a monomer and no domain-swapping was observed. The protein binds to Man-3 and Man-9 with similar dissociation constants ( approximately 4 muM). This confirms that the nanomolar activity of wild-type CV-N is related to the multisite nature of the protein carbohydrate interaction.     

Functional homologs of cyanovirin-N amenable to mass production in prokaryotic and eukaryotic hosts

Cyanovirin-N (CV-N) is under development as a topical (vaginal or rectal) microbicide to prevent sexual transmission of human immunodeficiency virus (HIV), and an economically feasible means for very large-scale production of the protein is an urgent priority. We observed that N-glycosylation of CV-N in yeast eliminated the anti-HIV activity, and that dimeric forms and aggregates of CV-N occurred under certain conditions, potentially complicating the efficient, large-scale manufacture of pure monomeric CV-N. We therefore expressed and tested CV-N homologs in which the glycosylation-susceptible Asn residue at position 30 was replaced with Ala, Gln, or Val, and/or the Pro at position 51 was replaced by Gly to eliminate potential conformational heterogeneity. All homologs exhibited anti-HIV activity comparable to wild-type CV-N, and the Pro51Gly homologs were significantly more stable proteins. These glycosylation-resistant, functional cyanovirins should be amenable to large-scale production either in bacteria or in eukaryotic hosts.     

Development of an HIV-1 Specific Microbicide Using Caulobacter crescentus S-Layer Mediated Display of CD4 and MIP1α

The development of alternative strategies to prevent HIV infection is a global public health priority. Initial efforts in anti-HIV microbicide development have met with poor success as the strategies have relied on a non-specific mechanism of action. Here, we report the development of a microbicide aimed at specifically blocking HIV entry by displaying molecular components of the HIV/host cell attachment complex on the surface of Caulobacter crescentus, a harmless aquatic bacterium. This bacterium can be readily manipulated to present heterologous proteins at high density on its surface by genetic insertion into its crystalline surface layer protein [1], [2]. In separate constructions, we generated bacteria displaying domain 1 of CD4 and MIP1α. Each moiety reacted with specific antibodies by Western immunoblot and immuno-fluorescence microscopy. Microbicide functionality was assessed using an HIV pseudotype virus assay system representing Clade B subtypes. Bacteria displaying MIP1α reduced infectivity by 35–78% depending on the specific subtype while CD4 display reduced infection by as much as 56%. Combinations of both constructs reduced infectivity by nearly 98%. We demonstrated that HIV infection could be inhibited using a strategy aimed at HIV-specific molecular interactions with Caulobacter surface protein display, and that sufficient protein folding and conformation could be mimicked to bind and block entry. Further, this is the first demonstration that Caulobacter surface protein display may be a useful approach to preventing HIV infection or other viruses as a microbicide. We propose that this harmless bacterium, which is inexpensive to produce and formulate, might be suitable for topical applications as a viable alternative in the search for effective microbicides to counteract the world wide incidence of HIV infection.     

Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

Background

Previous studies have shown that 3-hydroxyphthalic anhydride (HP)-modified bovine milk protein, β-lactoglobulin (β-LG), is a promising microbicide candidate. However, concerns regarding the potential risk of prion contamination in bovine products and carcinogenic potential of phthalate derivatives were raised. Here we sought to replace bovine protein with an animal protein of non-bovine origin and substitute HP with another anhydride for the development of anti-HIV microbicide for preventing HIV sexual transmission.

Results

Maleic anhydride (ML), succinic anhydride (SU) and HP at different conditions and variable pH values were used for modification of proteins. All the anhydrate-modified globulin-like proteins showed potent anti-HIV activity, which is correlated with the percentage of modified lysine and arginine residues in the modified protein. We selected maleic anhydride-modified ovalbumin (ML-OVA) for further study because OVA is easier to obtain than β-LG, and ML is safer than HP. Furthermore, ML-OVA exhibited broad antiviral activities against HIV-1, HIV-2, SHIV and SIV. This modified protein has no or low in vitro cytotoxicity to human T cells and vaginal epithelial cells. It is resistant to trypsin hydrolysis, possibly because the lysine and arginine residues in OVA are modified by ML. Mechanism studies suggest that ML-OVA inhibits HIV-1 entry by targeting gp120 on HIV-1 virions and also the CD4 receptor on the host cells.

Conclusion

ML-OVA is a potent HIV fusion/entry inhibitor with the potential to be developed as an effective, safe and inexpensive anti-HIV microbicide.     

Rhizosecretion improves the production of Cyanovirin‐N in Nicotiana tabacum through simplified downstream processing

Rhizosecretion has many advantages for the production of recombinant pharmaceuticals, notably facile downstream processing from hydroponic medium. The aim of this study was to increase yields of the HIV microbicide candidate, Cyanovirin‐N (CV‐N), obtained using this production platform and to develop a simplified methodology for its downstream processing from hydroponic medium. Placing hydroponic cultures on an orbital shaker more than doubled the concentration of CV‐N in the hydroponic medium compared to plants which remained stationary, reaching a maximum of approximately 20μg/ml in one week, which is more than 3 times higher than previously reported yields. The protein composition of the hydroponic medium, the rhizosecretome, was characterised in plants cultured with or without the plant growth regulator alpha‐napthaleneacetic acid by LC‐ESI‐MS/MS, and CV‐N was the most abundant protein. The issue of large volumes in the rhizosecretion system was addressed by using ion exchange chromatography to concentrate CV‐N and partially remove impurities. The semi‐purified CV‐N was demonstrated to bind to HIV gp120 in an ELISA and to neutralise HIV_Ba‐L with an IC₅₀ of 6nM in a cell‐based assay. Rhizosecretion is therefore a practicable and inexpensive method for the production of functional CV‐N.     

Engineering soya bean seeds as a scalable platform to produce cyanovirin‐N,a non‐ARV microbicide against HIV

There is an urgent need to provide effective anti‐HIV microbicides to resource‐poor areas worldwide. Some of the most promising microbicide candidates are biotherapeutics targeting viral entry. To provide biotherapeutics to poorer areas, it is vital to reduce the cost. Here, we report the production of biologically active recombinant cyanovirin‐N (rCV‐N), an antiviral protein, in genetically engineered soya bean seeds. Pure, biologically active rCV‐N was isolated with a yield of 350 μg/g of dry seed weight. The observed amino acid sequence of rCV‐N matched the expected sequence of native CV‐N, as did the mass of rCV‐N (11 009 Da). Purified rCV‐N from soya is active in anti‐HIV assays with an EC₅₀ of 0.82–2.7 nM (compared to 0.45–1.8 nM for E. coli‐produced CV‐N). Standard industrial processing of soya bean seeds to harvest soya bean oil does not diminish the antiviral activity of recovered rCV‐N, allowing the use of industrial soya bean processing to generate both soya bean oil and a recombinant protein for anti‐HIV microbicide development.     

New Candidate Biomarkers in the Female Genital Tract to Evaluate Microbicide Toxicity

Vaginal microbicides hold great promise for the prevention of viral diseases like HIV, but the failure of several microbicide candidates in clinical trials has raised important questions regarding the parameters to be evaluated to determine in vivo efficacy in humans. Clinical trials of the candidate microbicides nonoxynol-9 (N9) and cellulose sulfate revealed an increase in HIV infection, vaginal inflammation, and recruitment of HIV susceptible lymphocytes, highlighting the need to identify biomarkers that can accurately predict microbicide toxicity early in preclinical development and in human trials. We used quantitative proteomics and RT-PCR approaches in mice and rabbits to identify protein changes in vaginal fluid and tissue in response to treatment with N9 or benzalkonium chloride (BZK). We compared changes generated with N9 and BZK treatment to the changes generated in response to tenofovir gel, a candidate microbicide that holds promise as a safe and effective microbicide. Both compounds down regulated mucin 5 subtype B, and peptidoglycan recognition protein 1 in vaginal tissue; however, mucosal brush samples also showed upregulation of plasma proteins fibrinogen, plasminogen, apolipoprotein A-1, and apolipoprotein C-1, which may be a response to the erosive nature of N9 and BZK. Additional proteins down-regulated in vaginal tissue by N9 or BZK treatment include CD166 antigen, olfactomedin-4, and anterior gradient protein 2 homolog. We also observed increases in the expression of C-C chemokines CCL3, CCL5, and CCL7 in response to treatment. There was concordance in expression level changes for several of these proteins using both the mouse and rabbit models. Using a human vaginal epithelial cell line, the expression of mucin 5 subtype B and olfactomedin-4 were down-regulated in response to N9, suggesting these markers could apply to humans. These data identifies new proteins that after further validation could become part of a panel of biomarkers to effectively evaluate microbicide toxicity.     

Design of fusion protein for efficient preparation of cyanovirin-n and rapid enrichment of pseudorabies virus

Biotechnology Letters - Cyanovirin-N (CVN) is a cyanobacterial protein with potent neutralizing activity against enveloped virus. To achieve the economic and functional production of CVN, the CVN...     

High-level expression of the HIV entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves

Key message

The potent anti-HIV microbicide griffithsin was expressed to high levels in tobacco chloroplasts, enabling efficient purification from both fresh and dried biomass, thus providing storable material for inexpensive production and scale-up on demand.

Abstract

The global HIV epidemic continues to grow, with 1.8 million new infections occurring per year. In the absence of a cure and an AIDS vaccine, there is a pressing need to prevent new infections in order to curb the disease. Topical microbicides that block viral entry into human cells can potentially prevent HIV infection. The antiviral lectin griffithsin has been identified as a highly potent inhibitor of HIV entry into human cells. Here we have explored the possibility to use transplastomic plants as an inexpensive production platform for griffithsin. We show that griffithsin accumulates in stably transformed tobacco chloroplasts to up to 5% of the total soluble protein of the plant. Griffithsin can be easily purified from leaf material and shows similarly high virus neutralization activity as griffithsin protein recombinantly expressed in bacteria. We also show that dried tobacco provides a storable source material for griffithsin purification, thus enabling quick scale-up of production on demand.

     

Solution structure of the monovalent lectin microvirin in complex with Man(alpha)(1-2)Man provides a basis for anti-HIV activity with low toxicity

Lectins that bind surface envelope glycoprotein gp120 of HIV with high avidity can potently inhibit viral entry. Yet properties such as multivalency that facilitate strong interactions can also cause nonspecific binding and toxicity. The cyanobacterial lectin microvirin (MVN) is unusual as it potently inhibits HIV-1 with negligible toxicity compared with cyanovirin-N (CVN), its well studied antiviral homolog. To understand the structural and mechanistic basis for these differences, we solved the solution structure of MVN free and in complex with its ligand Manα(1-2)Man, and we compared specificity and time windows of inhibition with CVN and Manα(1-2)Man-specific mAb 2G12. We show by NMR and analytical ultracentrifugation that MVN is monomeric in solution, and we demonstrate by NMR that Manα(1-2)Man-terminating carbohydrates interact with a single carbohydrate-binding site. Synchronized infectivity assays show that 2G12, MVN, and CVN inhibit entry with distinct kinetics. Despite shared specificity for Manα(1-2)Man termini, combinations of the inhibitors are synergistic suggesting they recognize discrete glycans and/or dynamic glycan conformations on gp120. Entry assays employing amphotropic viruses show that MVN is inactive, whereas CVN potently inhibits both. In addition to demonstrating that HIV-1 can be inhibited through monovalent interactions, given the similarity of the carbohydrate-binding site common to MVN and CVN, these data suggest that gp120 behaves as a clustered glycan epitope and that multivalent-protein interactions achievable with CVN but not MVN are required for inhibition of some viruses.     

A topical microbicide gel formulation of CCR5 antagonist maraviroc prevents HIV-1 vaginal transmission in humanized RAG-hu mice

For prevention of HIV infection many currently licensed anti-HIV drugs and new ones in the pipeline show potential as topically applied microbicides. While macaque models have been the gold standard for in vivo microbicide testing, they are expensive and sufficient numbers are not available. Therefore, a small animal model that facilitates rapid evaluation of potential candidates for their preliminary efficacy is urgently needed in the microbicide field. We previously demonstrated that RAG-hu humanized mouse model permits HIV-1 mucosal transmission via both vaginal and rectal routes and that oral pre-exposure chemo-prophylactic strategies could be tested in this system. Here in these proof-of-concept studies, we extended this system for topical microbicide testing using HIV-1 as the challenge virus. Maraviroc, a clinically approved CCR5 inhibitor drug for HIV treatment, was formulated as a microbicide gel at 5 mM concentration in 2.2% hydroxyl ethyl cellulose. Female RAG-hu mice were challenged vaginally with HIV-1 an hour after intravaginal application of the maraviroc gel. Our results showed that maraviroc gel treated mice were fully protected against vaginal HIV-1 challenge in contrast to placebo gel treated mice which all became infected. These findings highlight the utility of the humanized mouse models for microbicide testing and, together with the recent data from macaque studies, suggest that maraviroc is a promising candidate for future microbicide clinical trials in the field.     

Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands

Cyanovirin-N (CVN) is an 11 kDa pseudosymmetric cyanobacterial lectin that has been shown to inhibit infection by the human immunodeficiency virus by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work, we describe rationally designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson-Boltzmann calculations and protein repacking algorithms were used to select stabilizing mutations in the protein core. By substituting the buried polar side chains of Ser11, Ser20, and Thr61 with aliphatic groups, we stabilized CVN by nearly 12 °C against thermal denaturation, and by 1 M GuaHCl against chemical denaturation, relative to a previously characterized stabilized mutant. Glycan microarray binding experiments confirmed that the specificity profile of carbohydrate binding is unperturbed by the mutations and is identical for all variants. In particular, the variants selectively bound glycans containing the Manα(1→2)Man linkage, which is the known minimal binding unit of CVN. We also report the slow denaturation kinetics of CVN and show that they can complicate thermodynamic analysis; in particular, the unfolding of CVN cannot be described as a fixed two-state transition. Accurate thermodynamic parameters are needed to describe the complicated free energy landscape of CVN, and we provide updated values for CVN unfolding.