Cloning, expression, purification and characterization of a single chain variable fragment specific to tumor necrosis factor alpha in Escherichia coli

Anti TNF-α molecules have been used as therapeutic agents in a variety of human diseases such as Rheumatoid arthritis, Ankylosing spondylitis, Chron's diseases, Psoriasis, etc., where high levels of TNF-α plays a destructive role. The limitations of the present TNF-α inhibitors in terms of size, tissue penetration and immunogenicity, etc., provoked the search for small anti TNF-α molecules. In the present study, a single chain variable fragment (ScFv) construct was made from a monoclonal antibody of the class IgG raised against TNF-α was used. The anti TNF-α ScFv was well expressed as soluble form in Escherichia coli BL21 (DE3), which was purified to homogeneity by commercial methacrylate monolith-convective interaction media (CIM) supports using two different chemistries, immobilized metal affinity chromatography (IMAC) with copper ions followed by anion exchange chromatography. The anti TNF-α ScFv found to be inhibiting the TNF-α mediated cytotoxicity in MCF-7 cells with an IC₅₀ of 8 μg. Data presented here are promising and encouraging to further optimize anti TNF-α ScFv production in larger scale with higher recovery at a cheaper price for therapeutic purposes.     

抗人VEGF受体Ⅱ基因Ⅲ区单链抗体基因的构建和表达

采用RT-PCR技术从分泌抗人血管内皮生长因子受体Ⅱ（kinase insert domaincontaining receptor,KDR）基因Ⅲ区单克隆抗体Ycom1D3的杂交瘤细胞中克隆出VH和VL可变区基因,通过重叠延伸拼接（spliceoverlap extension）PCR方法在V_H和V_L基因之间引入柔性短肽（Gly₄Ser）₃,体外构建Ycom1D3单链抗体基因Ycom1D3-ScFv）,将其克隆至pAYZ表达载体,在大肠杆菌中表达。SDS-PAGE和Westernblot分析结果表明,Ycom1D3-ScFv在E.coli 16C9中获得表达,重组蛋白的相对分子量为30kD,与预期结果一致。表达产物主要以不溶性包涵体形式存在,经过溶解包涵体,TALON 金属亲合层析基质（TALON metal affinity resin）纯化和体外复性过程,获得了高纯度的单链抗体片段。流式细胞分析结果证实该单链抗体可与人脐静脉内皮细胞结合,保留了鼠源单抗与KDR抗原的特异性结合活性。抗KDRⅢ单链抗体基因Ycom1D3-ScFv的成功构建和功能性表达为靶向诊断治疗及进一步基因工程改造奠定了基础。     

Efficient preparation and site‐directed immobilization of VHH antibodies by genetic fusion of poly(methylmethacrylate)‐binding peptide (PMMA‐Tag)

A PMMA‐binding peptide (PMMA‐tag) was genetically fused with the C‐terminal region of an anti‐human chorionic gonadotropin (hCG) single‐domain antibody (VHH). It was over‐expressed in an insoluble fraction of E. coli cells, and recovered in the presence of 8 M urea via one‐step IMAC purification. Monomeric and denatured PMMA‐tag‐fused VHH (VHH‐PM) was successfully prepared via the reduction and oxidation of VHH‐PM at a concentration less than 1 mg/mL in the presence of 8 M of urea. Furthermore, the VHH‐PM was refolded with a recovery of more than 95% by dialysis against 50 mM TAPS at pH 8.5, because the genetic fusion of PMMA‐tag resulted in a decrease in the apparent isoelectric point (pI) of the fusion protein, and its solubility at weak alkaline pH was considerably increased. The antigen‐binding activities of VHH‐PM in the adsorptive state were 10‐fold higher than that of VHH without a PMMA‐tag. The density of VHH‐PM on a PMMA plate was twice that of VHH, indicating that the site‐directed attachment of a PMMA‐tag resulted in positive effects to the adsorption amount as well as to the orientation of VHH‐PM in its adsorptive state. The preparation and immobilization methods for VHH‐PM against hCG developed in the present study were further applied to VHH‐PMs against four different antigens, and consequently, those antigens with the concentrations lower than 1 ng/mL were detected by the sandwich ELISA. Thus, the VHH‐PMs developed in the present study are useful for preparation of high‐performance and economical immunosorbent for detection of biomarkers. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1563–1570, 2015     

靶向人源BCMA的嵌合抗原受体T细胞构建及体外抗肿瘤活性研究

摘要 目的：通过对CAR结构的ScFv单链可变区进行改造,构建并筛选具有更强杀伤肿瘤细胞功能的新型靶向人源B细胞成熟抗原（BCMA）的嵌合抗原受体 (CAR)-T细胞。方法：构建靶向人源BCMA的CAR分子,用逆转录病毒载体包装成功后转导健康志愿者的T细胞,制备Anti-BCMA-CAR-T细胞。将Anti-BCMA-CAR-T细胞作为观察组,普通T细胞作为对照组,将其与RPMI-8226细胞共培养,采用CFSE染色的T细胞增殖实验观察两组体外增殖能力。采用荧光素酶化学发光实验检测两组细胞在不同效靶比（1:8、1:4、1:2、1:1、2:1、4:1）对RPMI-8226细胞的杀伤效率,采用流式细胞术检测两组细胞在不同效靶比（1:4、1:2、1:1、2:1、4:1）对RPMI-8226细胞的杀伤效率。结果：CFSE检测结果显示,与对照组比较,观察组FITC信号明显左移,表明T细胞增殖能力越强。流式细胞术检测结果显示,相同效靶比时,观察组对RPMI-8226细胞的杀伤效率均高于对照组（P均<0.05）;荧光素酶化学发光实验结果显示,相同效靶比时,观察组对RPMI-8226细胞的杀伤效率均高于对照组（P均<0.05）。在效靶比为4:1时,CAR170-T（未经改造的传统的ScFv）细胞和CAR174-T（经改造的ScFv）细胞的杀伤效率分别达到了88.5±0.3 %和98.5±0.7 %。结论：通过对CAR结构的ScFv单链可变区进行改造后成功构建出的新型靶向BCMA的CAR-T细胞,它能保持较强的增殖活性且具有更强的杀伤肿瘤细胞的能力。     

Mutational analysis of protein solubility enhancement using short peptide tags

Protein aggregation is a common phenomenon. The preparation of highly concentrated protein samples, typically required for biophysical measurements, often involves a time consuming and tedious testing of solvent conditions for improving protein solubility. Here, in a systematic analysis, we have determined the increase in solubility upon the addition of SEP-tags (solubility enhancement peptide tags) containing, one, three, and five lysines or arginines (or six arginines) to either the N or C terminus of our low solubility model protein, bovine pancreatic trypsin inhibitor variant, BPTI-22 (a BPTI variant containing 22 alanines). As anticipated, the BPTI-22 solubility increased in direct relation to the number of charged residues contained in the SEP-tag, and without altering either the activity or the structure of the protein. The largest solubility increases were of 4.2-, 4.8-, and 6.2-folds produced by the addition, at the C terminus, of five lysine (BPTI-22-C5K), five and six arginine residues (BPTI-22-C5R and BPTI-22-C6R), respectively. The increased solubility of the tagged BPTI-22 yielded higher quality NMR spectra (hetero single quantum correlation HSQC spectra; with respect of the signal-to-noise and line shapes) in a much shorter time than for the untagged BPTI-22. Furthermore, tagged samples remained soluble for over ten days, as observed by their HSQC spectra. We believe that lysine- and arginine-based SEP-tags may provide an effective and versatile method for enhancing protein solubility.     

A favorable solubility partner for the recombinant expression of streptavidin

Recombinant streptavidin is extremely difficult to express at high levels in the cytoplasm of Escherichia coli without the formation of inclusion bodies. Fusing a solubility enhancing partner to an aggregation prone protein is a widely used tool to circumvent inclusion body formation. Here, we use streptavidin as a target protein to test the properties of N-terminal fragments of translation initiation factor IF2 from E. coli as a solubility partner. Domain I (residue 1-158) of IF2 is superior to the well-established solubility partners maltose-binding protein (MBP) and NusA for soluble expression of active streptavidin. The number of active streptavidin molecules isolated by chromatography is increased threefold when domain I is used as solubility partner as compared to MBP or NusA. The relatively small size, high expressivity, and extreme solubility make domain I of IF2 an ideal partner for streptavidin and may also prevent other recombinant proteins such as ScFv antibodies from being expressed as insoluble aggregates in the cytoplasm of E. coli.     

恶性转化的成纤维细胞表皮生长因子受体的研究

本文用受体的放射性配基结合分析方法观察了C_3H小鼠胚胎成纤维细胞C_3H_(10)T1/2 CL8(简称NC_3H_(10))和~3H-TdR恶性转化的C_3H_(10)T1/2CL8(简称TC_3H_(10))的表皮生长因子受体(EGFR)。结果表明细胞恶性转化前后的EGFR都存在高亲和力和低亲和力两种结合位点,细胞恶性转化后能结合表皮生长因子的EGFR结合位点减少,Western blotting和受体的亲和交联分析表明EGFR的分子量为170kD,是单链多肽。     

人源天然ScFv噬菌体抗体库的构建及鉴定

噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本,通过提取淋巴细胞、逆转录－PCR（RT PCR）、抗体可变区基因的扩增、重叠PCR获得单链抗体（ScFv）基因,将ScFv克隆入噬粒载体,通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性,用5种不同抗原对其进行筛选,均获得了特异性噬菌体抗体的不同富集,表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。     

Radiosynthesis and evaluation of IGF1R PET ligand [11C]GSK1838705A

Radiosynthesis and evaluation of [¹¹C]GSK1838705A in mice using microPET and determination of specificity in human GBM UG87MR cells are described herein. The radioligand was synthesized by reacting desmethyl-GSK1838705A with [¹¹C]CH₃I using GE FX2MeI module in ～5% yield (EOS), >95% radiochemical purity and a specific activity of 2.5 ± 0.5 Ci/μmol. MicroPET imaging in mice indicated that [¹¹C]GSK1838705A penetrated blood brain barrier (BBB) and showed retention of radiotracer in brain. The radioligand exhibited high uptake in U87MG cells with >70% specific binding to IGF1R. Our experiments suggest that [¹¹C]GSK-1838705A can be a potential PET radiotracer for the in vivo quantification of IGF1R expression in GBM and other brain tumors.     

Co-expression of Dsb proteins enables soluble expression of a single-chain variable fragment (scFv) against human type 1 insulin-like growth factor receptor (IGF-1R) in E. coli

Type 1 insulin-like growth factor receptor (IGF-1R) is a promising therapeutic target for cancer treatment. A single-chain variable fragment (scFv) against human IGF-1R forms inclusion body when expressed in periplasmic space of E. coli routinely. Here, we described that co-expression of appropriate disulfide bonds (Dsb) proteins known to catalyze the formation and isomerization of Dsb can markedly recover the soluble expression of target scFv in E. coli. A 50 % recovery in solubility of the scFv was observed upon co-expression of DsbC alone, and a maximum solubility (80 %) was obtained when DsbA and DsbC were co-expressed in combination. Furthermore, the soluble scFv present full antigen-binding activity with IGF-1R, suggesting its correct folding. This study also suggested that the selection of Dsb proteins should be tested case-by-case if the approach of co-expression of Dsb system is adopted to address the problem of insoluble expression of proteins carrying Dsb.     

Evaluation of EGFR-targeted radioimmuno-gold-nanoparticles as a theranostic agent in a tumor animal model

This study evaluated the tumor targeting and therapeutic efficacy of a novel theranostic agent ¹³¹I-labeled immuno-gold-nanoparticle (¹³¹I-C225-AuNPs-PEG) for high epidermal growth factor receptor (EGFR)-expressed A549 human lung cancer. Confocal microscopy demonstrated the specific uptake of C225-AuNPs-PEG in A549 cells. ¹³¹I-C225-AuNPs-PEG induced a significant reduction in cell viability, which was not observed when incubated with AuNPs-PEG and C225-AuNPs-PEG. MicroSPECT/CT imaging of tumor-bearing mice after intravenous injection of ¹²³I-C225-AuNPs-PEG revealed significant radioactivity retention in tumor suggested that ¹³¹I-labeled C225-conjugated radioimmuno-gold-nanoparticles may provide a new approach of targeted imaging and therapy towards high EGFR-expressed cancers.     

一株针对人EGFR的单链抗体克隆与哺乳细胞表达

目的:制备特异性抗人表皮生长因子受体(EGFR)的单链抗体(sc Fv),鉴定其生物学活性,为进一步研究基于单链抗体的免疫治疗奠定基础。方法:从分泌抗人EGFR单克隆抗体的杂交瘤细胞系提取总RNA,利用5'RACE技术扩增轻链和重链可变区(VL、VH)基因,构建具有VL、VH基因的单链抗体基因,并将构建的单链抗体基因克隆到真核细胞表达载体pc DNA3.1中进行表达和鉴定。ELISA鉴定单链抗体对抗原的特异性;Fortebio检测抗原抗体间的亲和力,流式细胞术检测单链抗体结合肺癌细胞系天然EGFR的功能活性。结果:获得唯一的轻重链可变区序列VL、VH,成功构建EGFR-sc Fv,特异性与天然EGFR蛋白结合,亲和力达3.22×10-9mol/L。结论:成功构建了抗人EGFR单链抗体,为肺癌免疫导向治疗研究奠定了基础。     

抗CD5单链抗体基因克隆和表达

以分泌抗CD5单克隆抗体的杂交瘤细胞poly(A) mRNA为模板,通过RTPCR扩增出抗CD5单克隆抗体的重链可变区(VH)和轻链可变区(VL) cDNA片段组装出抗CD5单链抗体(ScFv) cDNA片段。该ScFv片段被克隆到pCANTAB 5E载体上,以E.coli TG1为宿主,进行噬菌体表面呈现。通过Molt4细胞表面分子CD抗原,对噬菌体表面呈现的ScFv进行免疫亲和富集筛选。经细胞ELISA鉴定,得到4株高亲和力克隆。DNA序列分析得知,单链抗体全长732碱基,其中VH为339碱基,VL为300碱基。抗CD5 ScFv在E.coli HB2151中以可溶形式分泌表达,产物主要分布于周质之中,占周质中总蛋白的20%。     

Growth patterns and age validation from otolith ring deposition in New Zealand longfin eels Anguilla dieffenbachii recaptured after 10 years at large

In 1998, 9500 juvenile New Zealand longfin eels Anguilla dieffenbachii (mean total length, L_T, 42 cm) captured from the lower Clutha River were transferred upstream to Lake Hawea, a high‐country oligotrophic lake in the same catchment where recruitment of juvenile eels has been prevented by hydroelectric dams since 1958. A total of 2010 of the transferred A. dieffenbachii were tagged with coded wire tags. Ten years later in 2008, the A. dieffenbachii population in Lake Hawea was sampled resulting in 399 recaptures (distinguishable by the presence of tags and by L_T from the remnant resident population of large old A. dieffenbachii) of the 1998 transfers; 79 (19·2%) of the recaptured fish had tags compared with 21·3% at release, indicating good tag retention and low mortality due to tagging. All recaptured tagged A. dieffenbachii were female. Mean annual growth over the 10 years since release was 3·80 cm year^?1 for all recaptures and 3·65 cm year^?1 for tag recaptures, and both were significantly greater than the estimate of 2·38 cm year^?1 at release. After release, mean condition (K) increased significantly (P < 0·001) for all recaptures and tag recaptures. Annual length growth increment was linear. Tag recaptures showed significant increases in somatic growth rate post‐transfer, and otoliths from the 2008 recaptured A. dieffenbachii were examined to see whether any similar enhanced growth after transfer was incorporated into the otolith structure that would serve as a date stamp. Measurement of otolith ring radii indicated that an increase in the radius occurred on most otoliths corresponding to the year after transfer. Because there was 9 years of completed growth following the observed growth inflection on the otoliths, this was strong evidence that opaque rings were formed annually.     

Tailoring of recombinant FDH: effect of histidine tag location on solubility and catalytic properties of Chaetomium thermophilum formate dehydrogenase (CtFDH)

Abstract

Several protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD⁺ dependent formate dehydrogenase from Chaetomium thermophilum (CtFDH) can be utilized for interconversion of formate to carbon dioxide coupled with the conversion of NAD⁺ to NADH. In this study, N-terminal His tagged CtFDH (N-CtFDH) and C-terminal His tagged CtFDH (C-CtFDH) was constructed to learn the effect of His tag location on the activity and kinetic parameters of the enzyme. The solubility of proteins was not affected by tag position, however, an interference on the N-terminal region caused a deterioration in specific activity and the kinetic ability of enzyme. The obtained results indicated that the C-terminus of the enzyme is an appropriate region for tag engineering. The C-CtFDH has an approximately three-fold larger specific activity and two-fold higher catalytic efficiency than N-CtFDH. The results suggest that insertion of a His-tag at the N-terminal or C-terminal end of CtFDH has different effects on the protein and the N-terminal fragment of the protein is crucial for the function of CtFDH.     

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 10⁴ receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.     

Residue fractionation and decomposition: The significance of the active fraction

This paper describes an incubation experiment with homogeneously ¹⁴C labeled maize-straw and its insoluble fraction. The role of the soluble fraction in the decomposition process was assessed, using three independently measured characteristics: (1) fractionation of the maize-straw, resulting in kinetically different fractions; (2) microbial biomass C and its ¹⁴C activity determined by a fumigation extraction method, and (3) the ¹⁴C activity of the released CO₂-C. The fumigation extraction method was proved to be useful from 9 days after the application of the maize-straw onwards. The fractionation method yielded a soluble (48%), a (hemi) cellulosic (47%), and a lignin fraction (1%). Nine days after addition of either the complete residue or its insoluble fraction, the microbial biomass C increased from 53 to 337 and 217 mg C kg^-1 dry soil, respectively. Similar values were maintained up to day 40. The large increase in microbial activity was accompanied by a N-immobilization of 65 and 29 mg N Kg^-1 dry soil for the maize-straw treatment and its insoluble fraction, respectively, resulting in biomass C/N values of 5.5 and 5.6 A genuine priming effect (10 and 7% of the total CO₂-C production) on the mineralization of native soil organic C was caused by an increase in decomposition of the native C rather than by an increase in turnover of the microbial biomass in the soil amended with maize straw. The soluble fraction caused a priming effect on the decomposition of the less decomposable cell-wall fraction. Calculations by nonlinear regression confirmed this observation.     

鼠源抗HFRSV衣壳蛋白McAbF3株单链抗体基因克隆构建及表达

为了获得原抗ＨＦＲＳＶ衣壳蛋白ＭｃＡｂＦ３＾１株轻链可变区基因,由连接肽体外连接获得单链抗体基因,在大肠杆菌中表达,从鼠源抗ＨＦＲＳＶ衣壳蛋白ＭｃＡｂＦ３株细胞中分离总ＲＮＡ,以ｏｌｉｇｏ（ｄＴ）１８为引物逆转录成ｃＤＮＡ,通过ＰＣＲ扩增出抗体的轻链（ＶＬ）和重链可变区（Ｖ１１）基因,由连接本外连接获得单链抗体（ＳｅＦｖ）基因。将此单链抗体（ＳｅＦｖ）基因插入原核表达载体ＰＥＴ２８ａ,经大肠杆菌（     

A novel recombinant system for functional expression of myonecrotic snake phospholipase A(2) in Escherichia coli using a new fusion affinity tag

A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A₂ ([Lys⁴⁹]PLA₂), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and showed better quantitative yield when folded correctly. The solubility of the recombinant congerin II-tagged BPII increased up to >90% in E. coli strain JM109 when coexpressed with the molecular chaperones GroEL, GroES, and trigger factor (Tf). The tag protein was cleaved by digestion with restriction protease, such as α-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for α-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity.     

5-(4-((4-[18F]fluorobenzyl)oxy)-3-methoxybenzyl)pyrimidine-2,4-diamine: A selective dual inhibitor for potential PET imaging of Trk/CSF-1R

The tropomyosin receptor kinases (TrkA/B/C) and colony-stimulating factor-1 receptor (CSF-1R) represent highly pursued oncological therapeutic targets. The 2,4-diaminopyrimidine inhibitor GW2580 (9) has been previously reported as a highly selective low nanomolar TrkB/TrkC/CSF-1R inhibitor. In this study, fluorinated derivatives of 9 were designed, synthesized and evaluated in enzymatic assays. The highly potent inhibitor 10 was identified, which retained the selectivity profile of the non fluorinated lead compound 9, and the radiosynthesis of [¹⁸F]10 was developed. The results obtained from the biological evaluation of 10 and the radiosynthesis of [¹⁸F]10 support further investigation of this tracer as a potential PET imaging probe for TrkB/TrkC and CSF-1R.