Recombinant Expression,Purification And Application Of Microbial Transglutaminase

Transglutaminases(TGases;EC 2.3.2.13)catalyze the acyl transfer reaction from the γ-carboxamide of glutamine residue(acyl donor)to the ε-amine of lysine residue(acyl acceptor)to form protein polymers cross-linked by isopeptide bonds.TGases are widely found in mammals,plants,and microorganisms.Among them,TGases from microorganisms(MTG)has the advantages of being easy to obtain and store,independence on Ca2+,excellent specific activity,good substrate adaptation,and being stable in wide range of temperature and pH conditions.The commercial MTG was produced by Streptomyces mobaraensis and expressed in the form of zymogen(ProMTG)with a propeptide sequence(Pro)at the N-terminal,which was hydrolyzed by endogenous proteases to generate active mature MTG.In addition to being used in the food and textile industries,MTG has been exploited as a tool enzyme to modify pharmaceutical proteins including the generation of antibody-drug conjugates(ADCs)and protein PEGylation with polyethylene glycol(PEG)polymers.In the process of expression and secretion of native MTG,the Pro domain plays a crucial role.Not only can Pro assist the correct folding of MTG but also mask the active center of MTG to inhibit its activity,so as to avoid MTG-catalyzed protein cross-linking in the host cell to produce toxic effects on the cell.Recombinant expression of MTG through expression systems such as E.coli can obtain tool enzymes with controllable quality and easy isolation after modification,which has been intensively studied by previous studies.There were mainly four strategies for producing MTG in E.coli:1)expressing mature MTG directly in E.coli,but the expression level was low or inclusion bodies would be formed;2)expressing full-length Pro-MTG in E.coli and then cleaving Pro by exogenous proteases(dispase or trypsin)in vitro;3)constructing fusion protein with artificial linker between Pro and MTG,the linker was a recognition sequence for an exogenous specific protease or an intein domain which can be cleaved by specific protease or pH-induced self-cleaved in the case of intein;4)polycistronic gene strategy,cloning of a polycistronic gene with the pro-domain required for proper folding and the mature enzyme after cleavage expressed by the same promoter and expression of these genes into periplasm to obtain mature MTG but with low expression level.In most of these strategies,the additional downstream processing of inclusion bodies or zymogen complicated the production and increased the cost.More importantly,the cleaved Pro peptide is difficult to be decontaminated owing to its strong affinity with mature MTG.Although the contamination of Pro has negligible impact on MTG applications in food industry,it affects the product purity and enzyme efficiency during MTG-catalyzed modification on pharmaceutical proteins.The decontamination of Pro from MTG has been rarely studied except a report by Pfizer Inc.,in which site-directed mutagenesis has been performed to screen Pro-domain mutants maintaining chaperone function but being easy to strip.In this thesis,strategies were developed to produce MTG in E.coli cells and to strip Pro domain efficiently.The obtained MTG has the advantages of high activity and purity,as well as easy separation after modification.The properties and application on protein modification of produced MTG were further studied.In chapter 2,we first attempted to directly produce mature MTG in E.coli.The results confirmed that the expression of mature MTG was low in E.coli and affected the growth of host cells.Therefore,we designed a "molecular chaperone" expression strategy.The Pro domain,formed a fusion protein with a tag protein,was co-expressed with mature MTG as a "molecular chaperone" during expression.We found that using the Trx-Pro fusion protein with thioredoxin(Trx)tag at the N-terminal and Pro domain at the C-terminal as the chaperone protein,high-level and soluble expression of MTG was achieved.After purification via nickel affinity chromatograph facilitated by fused His6 tag at the C-terminal of mature MTG,the target protein yield of up to 300 mg/L can be reached with shake flask fermentations.Although the obtained protein contained Trx-Pro fusion proteins,the specific activity of the recombinant enzyme was determine to be 30 U/mg,which was similar to that of commercial MTG.In chapter 3,in an attempt to obtain MTG with high purity,good activity and low contamination for its application in the modification of medicinal proteins,we explored the use of small molecular reagents to strip residual Trx-Pro protein.The study found that several amine-reactive reagents can strip the Pro domain,among which acetic acid N-hydroxysuccinimide ester(AcNHS)was both effective and economical.Thus,we chose AcNHS as the treatment reagent during the secondary purification process.The effects of reagent concentration,solvent,treatment temperature and time on the specific activity and recovery rate of MTG were studied.The results showed that the optimum concentration of AcNHS was 50 mM,the optimum temperature was 37℃,and the optimum time was 3 h.High-purity MTG-His6 can be obtained when using protein concentration of 1-5 mg/mL during treatment and the specific activity of MTG-His6 after stripping Trx-Pro was 1.6 folds of that before treatment.In addition,in order to verify the influence of the Pro on the enzymatic activity of MTG,we constructed an intein expression and purification system to obtain Trx-Pro.The results showed that with the increase of Trx-Pro concentration in the reaction system,the specific activity of MTG decreased with an IC50 of 2.02 μM(concentration of MTG=1.02 μM).In Chapter 4,we compared the enzyme properties of commercial MTG,one-step purified MTG-His6 and two-step purified MTG-His6.The results showed that the optimum reaction pH of the three kinds of MTG was 6.0,stable range of pH was 5-7;the optimum reaction temperature was 55℃,and the stable range of temperature was below 50℃;Na+,K+,Ca2+ possessed the same effects on enzyme activity.Subsequently,we verified the protein cross-linking ability of the commercial MTG,one-step purified MTG-His6 and two-step purified MTG-His6 with bovine serum albumin as the substrate.Finally,after construction,expression and purification of the medicinal protein interferon-α2b in E.coli,site-specific PEGylation of IFN-α2b was investigated with two-step purified MTG-His6,which could be effectively removed by affinity chromatography magnetic beads after usage.