Construction and characterization of a pseudo-immune human antibody library using yeast surface display

Lymphocytes from eight individuals out of 60 healthy donors, whose plasmas showed relatively higher antibody titer for a target antigen of death receptor 5 (DR5), were selected for the source of antibody genes to construct so called an anti-DR5 pseudo-immune human single-chain fragment variable (scFv) library on the yeast cell surface (approximately 2x10(6) diversity). Compared with a large nonimmune human scFv library (approximately 1x10(9) diversity), the repertoire of the pseudo-immune scFv library was significantly biased toward the target antigen, which facilitated rapid enrichments of the target-specific high affinity scFvs during selections by fluorescence activated cell sortings. Isolated scFvs, HW5 and HW6, from the pseudo-immune library showed much higher specificity and affinity for the targeted antigen than those from the nonimmune library. Our results suggest that a pseudo-immune antibody library is very efficient to isolate target-specific high affinity antibody from a relatively small sized library.     

两种主流载体蛋白在大肠杆菌表面展示抗体应用中的系统分析

抗体表面展示技术对于新抗体的筛选和抗体亲和力的成熟是非常重要的工具．目前,较为广泛应用的表面展示技术是噬菌体的表面展示和酵母的表面展示．大肠杆菌,以其培养简单和基因改造便捷,有望成为非常好的一种表面展示的宿主．但是,目前为止,大肠杆菌还没有被广泛地应用于抗体的表面展示技术中．主要的原因之一是在大肠杆菌外膜展示抗体的效率还不够高．作为外膜展示的载体,许多蛋白都被研究过,其中自转运蛋白(autotransporter,AT)和冰核蛋白(ice nucleation protein,INP)是人们研究最多的两种载体蛋白．还有一个原因是大肠杆菌作为宿主,在表达异源基因和展示异源蛋白过程中的存活率问题．在本研究中,系统地研究了Ag43β(一种自转运蛋白Antigen43的β结构域)和INPNC(去掉中间冗余序列的冰核蛋白的N端和C端)两种载体蛋白在强弱不同的三种启动子(T7、araBAD和lac)诱导表达的情况下,表达量、展示率、抗原亲和力以及宿主菌存活率的差异．我们发现,Ag43β展示的抗体在抗原亲和力上优于INPNC展示的抗体．在存活率方面,T7启动子诱导表达的存活率很低：用INPNC作为载体蛋白时只有0.0033%,用Ag43β作为载体蛋白时只有0.02%存活率．lac启动子诱导表达的存活率：用INPNC作为载体蛋白时为2.04%,用Ag43β作为载体蛋白时为13.27%．araBAD启动子诱导表达的存活率很高：用INPNC作为载体蛋白时为37.80%,用Ag43β作为载体蛋白时高达90.23%．但是araBAD诱导表达量和展示率都很低,所以其表现出的宿主高存活率意义有限．综合看来,由lac启动子驱动的、以Ag43?茁为载体蛋白的抗体表面展示系统是最好的选择．     

Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions

Yeast surface display, a well‐established technology for protein analysis and engineering, involves expressing a protein of interest as a genetic fusion to either the N‐ or C‐terminus of the yeast Aga2p mating protein. Historically, yeast‐displayed protein variants are flanked by peptide epitope tags that enable flow cytometric measurement of construct expression using fluorescent primary or secondary antibodies. Here, we built upon this technology to develop a new yeast display strategy that comprises fusion of two different proteins to Aga2p, one to the N‐terminus and one to the C‐terminus. This approach allows an antibody fragment, ligand, or receptor to be directly coupled to expression of a fluorescent protein readout, eliminating the need for antibody‐staining of epitope tags to quantify yeast protein expression levels. We show that this system simplifies quantification of protein‐protein binding interactions measured on the yeast cell surface. Moreover, we show that this system facilitates co‐expression of a bioconjugation enzyme and its corresponding peptide substrate on the same Aga2p construct, enabling enzyme expression and catalytic activity to be measured on the surface of yeast.     

Synthesis and spectroscopic characterization of 4-butoxyethoxy-N-octadecyl-1,8-naphthalimide as a new fluorescent probe for the determination of proteins

A novel 4-butoxyethoxy-N-octadecyl-1,8-naphthalimide (BON) was synthesized as a fluorescent probe for the determination of proteins. The interactions between BON and bovine serum albumin (BSA) were studied by fluorescence spectroscopy and UV-vis absorption spectroscopy. Fluorescence data revealed that the fluorescence quenching of BSA by BON was likely the result of the formation of the BON-BSA complex. According to the modified Stern-Volmer equation, the binding constants of BON with BSA at four different temperatures were obtained. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated to be −23.27 kJ mol⁻¹ and 10.40 J mol⁻¹ K⁻¹ according to van’t Hoff equation, indicating that the hydrogen bonds and hydrophobic interactions played a dominant role in the binding of BON to BSA. Furthermore, displacement experiments using warfarin indicated that BON could bind to site I of BSA. The effect of BON on the conformation of BSA was also analyzed by synchronous fluorescence and three-dimensional fluorescence spectra. A new fluorescence quenching assay of the proteins BSA using BON in the HCl-Tris (pH 7.4) buffer solution was developed with maximum excitation and emission wavelengths of 373 and 489 nm, respectively. The linear range was 0.1-10.0 × 10⁻⁵ mol L⁻¹ with detection limits were determined to be 1.76 × 10⁻⁸ mol L⁻¹. The effect of metal cations on the fluorescence spectra of BON in ethanol was also investigated. Determination of protein in human serum by this method gave results which were very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry.     

Display of heterologous proteins on the surface of Lactococcus lactis using the H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix

Aims: The aim of this study was to develop a cell‐surface display system for foreign antigens on the surface of a Lactococcus lactis strain using an H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix. Methods and Results: To construct a cell‐surface display pACL1 vector, a derivative of pSECE1 vector, we amplified the H and W domain of the cell‐surface proteinase Prt B from Lact. bulgaricus using specific primers and then cloned it into a site downstream of the secretion signal sequence in the pSECE1 vector. The new system, designed for cell‐surface display of recombinant proteins on L. lactis, was evaluated by the expression and display of the FliC protein of Salmonella enterica serovar Enteritidis as a reporter gene (pALC1:FliC). The expression of the FliC protein by the transformed cells was analysed by Western blot analysis, and the localization of FliC on the cell surface was confirmed by immunofluorescence microscopy and flow cytometry analysis. A specific band corresponding in size (approx. 110 kDa) to FliC plus the anchor residues was detected by anti‐FliC antibody in the cell extract of L. lactis H61 harbouring pALC1:FliC, but not L. lactis H61 harbouring pALC1. In addition, flow cytometry and immunofluorescence microscopy revealed FliC‐specific positive signals and a significant increase of fluorescence, respectively, in cells harbouring pALC1:FliC compared with that in control cells harbouring the parental pALC1 plasmid. These findings demonstrated that FliC was successfully displayed on the cell surface by the anchor domain of PrtB. Conclusions: A pALC1 vector using the H and W domain of PrtB from Lact. bulgaricus as an anchoring matrix can be used to successfully display the FliC protein on the surface of L. lactis. Significance and Impact of the Study: This novel way of displaying heterologous proteins on the cell surface of L. lactis using the PrtB anchor domain should prove useful for the delivery of antigens and other proteins.     

Improving the stability of an antibody variable fragment by a combination of knowledge-based approaches: validation and mechanisms

Numerous approaches have been described to obtain variable fragments of antibodies (Fv or scFv) that are sufficiently stable for their applications. Here, we combined several knowledge-based methods to increase the stability of pre-existing scFvs by design. Firstly, the consensus sequence approach was used in a non-stringent way to predict a large basic set of potentially stabilizing mutations. These mutations were then prioritized by other methods of design, mainly the formation of additional hydrogen bonds, an increase in the hydrophilicity of solvent exposed residues, and previously described mutations in other antibodies. We validated this combined method with antibody mAbD1.3, directed against lysozyme. Fourteen potentially stabilizing mutations were designed and introduced into scFvD1.3 by site-directed mutagenesis, either individually or in combinations. We characterized the effects of the mutations on the thermodynamic stability of scFvD1.3 by experiments of unfolding with urea, monitored by spectrofluorometry, and tested the additivity of their effects by double-mutant cycles. We also quantified the individual contributions of the resistance to denaturation ([urea](1/2)) and cooperativity of unfolding (m) to the variations of stability and the energy of coupling between mutations by a novel approach. Most mutations (75%) were stabilizing and none was destabilizing. The progressive recombination of the mutations into the same molecule of scFvD1.3 showed that their effects were mostly additive or synergistic, provided a large overall increase in protein stability (9.1 kcal/mol), and resulted in a highly stable scFvD1.3 derivative. The mechanisms of the mutations and of their combinations involved variations in the resistance to denaturation, cooperativity of unfolding, and likely residual structures of the denatured state, which was constrained by two disulfide bonds. This combined method should be applicable to any recombinant antibody fragment, through a single step of mutagenesis.