Formylglycine-generating Enzyme Catalyzed Site-specific Aldehyde-modification Of Nanobody And Its Applications

Nanobodies,also called single-domain antibodies or VHHs,are a unique type of antibody derived from Camelidae animals which only have the 1/10 size of conventional antibody.Nanobodies are regarded as the smallest antibody molecules which can bind antigen.Because of small sizes and robust structures,nanobodies performed better in stability,solubility,recombinant expression and penetrating than conventional IgG-like antibodies.In the past two decades,nanobodies have been extensively applied for both fundamental researches and various applications like therapeutics and diagnosis.However,the function of individual nanobody is limited by its lacking of Fc fragments or other effectors.Therefore,modification and engineering of nanobody is necessary to expand the scope of its applications.Despite the advantages due to its small size,that also brings the challenges to further modification and engineering.Classical chemical modification methods that employ the native lysine or cysteine residues on the protein surface are considered to destroy the structure and affect the antigen-binding of a nanobody.However,the available site-specific modification strategies are neither efficiency nor convenient to manipulate.Therefore,to develop efficient and convenient site-specific modification technology of nanobody is the key point to extend its application.Due to the small size of aldehyde-tag and the facility to manipulate,formylglycine-generating enzyme(FGE)catalyzed aldehyde-modification has attracted considerable attention in site-specific modification of protein.Since the N terminus is near the antigen-binding region of nanobody,this dissertation proposed to use FGE to achieve the site-specific aldehyde-modification at C terminus of nanobody.In further,based on the site-specific modification,this dissertation developed the application of one-step site-direct immobilization and the construction of C-C linked bivalent and biparatopic nanobody.Also,this paper explored the in situ polymerization of nanobody for one-pot preparing macroporous cryogel-monolith for the capture of target proteins.This work has been carried out in four fractions:(1)Firstly,we tried to co-express nanobody and FGE in the cytoplasm of Escherichia coli(E.coli).However,nanobody was neither expressed at a acceptable solubility nor efficiently modified by FGE.In a further study,we found protein A which is much more soluble than nanobody could achieve site-specific aldehyde-modification in vivo by co-expressing FGE in E.coli.Then the co-expression E.coli cells were cultured,collected,and split.By using hydrazide agarose beads,protein A could be one-step immobilized from the crude cell lysate with specificity of about 80%.Due to the elimination of the prior purification of the affinity ligand,the preparation process of affinity chromatography media is simplified to a large extent.However,because of the poor versatility of in vivo aldehyde-modification,which depend on the solubility or individual property of the target protein,we would focus on the in vitro aldehyde-modification in the further study.(2)We further explored the in vitro FGE catalyzed aldehyde-modification of nanobody.With the anti-human ?-2 microglobulin(h?2M)nanobody sdAb-55 as the model protein,the Box-Behnken design was used to optimize the reported catalytic condition of FGE.The optimized catalytic temperature was 18?37? pH value was 9.2,with the addition of 2 mM?-mercaptoethanol,3 ?M copper sulfate,with only 0.1 eq.mole of sdAb-55,about 85%aldehyde conversion could be achieved within 4 hours.The time yield of converted aldehyde within nanobody by unit FGE is 2.13 mol·h-1·mol-1 at the optimized catalytic condition,it was 40 times of the reported time yield.Besides,the auto-aldehyde modification of FGE was found and explored.Peptide mapping of FGE(trypsin digested)was analyzed on LC-MS/MS,which proved the auto-aldehyde modification occurred at the C23 8 or C23 9 site.Furthermore,through the site-directed mutation at either site,the mutant FGE-C239A eliminated the aldehyde-modification.Not only the catalytic efficiency of FGE-C239A was maintained,but also the thermostability was enhanced.More importantly,the FGE-C239A mutant would provide a strong support for the subsequent application of aldehyde-modified nanobody.(3)Base on the in vitro FGE catalyzed aldehyde-modification of nanobody,we explored the preparation of C-C linked bivalent and bispecific nanobody.To our knowledge,this is the first time to achieve the preparation through the concentration-effect within the ice crystals.The C-terminal aldehyde was coupled with flexible bi-hydrazide or bi-aminooxy linkers via the hydrazone or oxime formation which accelerated by freezing.This freezing method figured out the difficulty of ligation between two protein molecules and one linker.The results showed that the yield of bivalent nanobody was significantly increased at the temperature from-10? to-20? especially at-10? which could achieve up to 40%yield of bivalent nanobody within 24 hours.Furthermore,with the anti-h?2M nanobody sdAb-55 and sdAb-83 as the building blocks,the bivalent and bispecific nanobody with linkers of different length were synthesized by freezing.Affinity of these C-C or C-N linked nanobody constructs were also measured and compared with each other.The C-C linked bivalent and bispecific nanobody constructs were proved to maintain almost all of its binding activity and exhibited an increase by two orders of magnitudes in affinity kinetics,demonstrating the superiority of C-C over the C-N linking approach.This technology can also be used as a platform technology for high throughput and rapid synthesis of C-C linked bivalent and bispecific nanobodies.(4)Base on the in vitro FGE catalyzed aldehyde-modification of nanobody,the last part of this paper developed the preparation of nanobody functionalized biomaterials.4-Vinylphenylamine(4-VA)was efficiently modified to the aldehyde group at C-terminus of anti-h?2M nanobody NB1.Then took 4-VA labeled nanobody NB1 as the monomer,we achieved in situ polymerization of nanobody for preparing the macroporous cryogel in one-pot.By frozen the mixture of the appropriate concentration of acrylamide(AAm),poly(ethylene glycol)diacrylate(PEGDA)and 4-VA modified nanobody NB1 at-18? for 18 hours,the AAm-NB1-PEGDA cryogel could be obtained.The prepared AAm-NB1-PEGDA cryogel contains large pores range from 10 to 100 ?m and the high porosity which was above 80%,which indicated excellent mechanical properties.Moreover,the h?2m adsorption capacity of AAm-NB1-PEGDA can reach up to 3.42 mg/mL gel.The in situ polymerization method of nanobody is convenient and simple to prepare nanobody or other proteins coupled cryogel in different containers.The processes are mild,neither introduce any chemical groups nor affect the activity of proteins,which are promising to be applied in preparing the biomaterials for precision blood purification and the resins for affinity chromatography.In summary,the FGE-catalyzed site-specific aldehyde modification of nanobody is a convenient and efficient method that can be extensively applied in site-direct immobilization and synthesis of C-C linked nanobody constructions.Besides,this method provides a novel strategy to furtherly modify nanobody with the alkene group for the preparation of nanobody functionalized biomaterials.