High-throughput system for determining dissolution kinetics of inclusion bodies

Efficient solubilization is a crucial step during inclusion body processing and dissolving conditions were usually empirically established. Here we describe a new methodology for rapid screening of solubilization conditions and evaluation of dissolution kinetics in microtiter plates. Increase of protein in solution over time was directly related to decrease of turbidity measured by absorbance at 600 nm. Dissolution kinetics of inclusion bodies were described by a first-order reaction kinetics, which was used for drug dissolution modeling. Reaction constants were in the range of 0.01–0.03 s^–1 for buffer conditions providing sufficient solubilization power. This method is not limited to the screening of optimal buffer conditions for solubilization and can be applied for studying other parameters involved in the solubility of IBs, such as pI of the protein, influence of fermentation conditions, influence of initial protein concentration, and more.     

Technical refolding of proteins: Do we have freedom to operate?

Expression as inclusion bodies in Escherichia coli is a widely used method for the large-scale production of therapeutic proteins that do not require post-translational modifications. High expression yields and simple recovery steps of inclusion bodies from the host cells are attractive features industrially. However, the value of an inclusion body-based process is dominated by the solubilization and refolding technologies. Scale-invariant technologies that are economical and applicable for a wide range of proteins are requested by industry. The main challenge is to convert the denatured protein into its native conformation at high yields. Refolding competes with misfolding and aggregation. Thus, the yield of native monomer depends strongly on the initial protein concentrations in the refolding solution. Reasonable yields are attained at low concentrations (≤0.1 mg/mL). However, large buffer tanks and time-consuming concentration steps are required. We attempt to answer the question of the extent to which refolding of proteins is protected by patents. Low-molecular mass additives have been developed to improve refolding yields through the stabilization of the protein in solution and shielding hydrophobic patches. Progress has been made in the field of high-pressure renaturation and on-column refolding. Mixing times of the denatured protein in the refolding buffer have been reduced using newly developed devices and the introduction of specific mixers. Concepts of continuous refolding have been introduced to reduce tank sizes and increase yields. Some of the patents covering refolding of proteins will soon expire or have already expired. This gives more freedom to operate.     

Fed-batch production of (S)-flurbiprofen in lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin and its extractive purification

Optically active (S)-flurbiprofen was produced fed-batch-wisely in a lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin (suβ-CD). A highly concentrated 480 mM (S)-flurbiprofen, corresponding to 117.0 g/l, with an enantiomeric excess of 0.98 and conversion yield of 0.48 was obtained. (S)-Flurbiprofen produced in an inclusion complex form with suβ-CD was extractively purified using three-step procedures: decomplexation of (S)-flurbiprofen and residual (R)-flurbiprofen ethyl ester ((R)-FEE) using the ethyl acetate, dissolution of (S)-flurbiprofen from (R)-FEE using a sodium bicarbonate solution, and selective precipitation of (S)-flurbiprofen using 2-propanol. Consequently, an extremely high concentration of 420 mM (S)-flurbiprofen with an optical purity higher than 98% was recovered after purification.     

gAd重组质粒的构建及其在大肠杆菌中的表达

为了建立人脂联素球状结构域(gAd)原核高效表达体系,分离纯化gAd并检测其生物活性,从淋巴细胞中提取人基因组DNA,PCR扩增出含有脂联素编码序列的片段,通过T-A克隆的方法克隆入pMD18-T载体中,然后设计适当的引物引入起始密码、终止密码以及相应的酶切位点,把脂联素球状结构域(gAd)基因亚克隆到表达载体pBV220,将序列鉴定正确的重组质粒转化入大肠杆菌DH5α中,用42℃温控诱导目的蛋白的表达;超声破菌,分离纯化包涵体,8mol/L尿素溶解,采用梯度稀释法复性重组蛋白,动物实验测定其活性。结果在大肠杆菌中成功实现了gAd稳定的以包涵体形式的高效表达,表达量约占全菌蛋白的20%,经聚丙烯酰胺凝胶电泳鉴定分离得到的包涵体具有较高纯度,复性后具有降低家兔血糖和游离脂肪酸的作用。为进一步研究gAd的生物学活性、作用机制奠定了基础。     

人Hepcidin融合表达载体的构建及在大肠杆菌中的表达

为了在大肠杆菌中表达生产hepcidin,根据大肠杆菌密码子偏好性,化学合成了人hepcidin的基因序列,并构建了hepcidin的融合表达载体pET -hpc。pET- hpc在大肠杆菌BL2 1 (DE3)中表达的hepcidin融合蛋白以包涵体形式存在,其N端带有 6个组氨酸。通过优化诱导表达条件,该融合蛋白表达水平显著提高,占总蛋白的 2 5 . 2 %。表达的包涵体经 1 %TritonX 1 0 0洗涤后溶于8mol L尿素,在变性条件下采用金属螯合层析进行纯化,所得融合蛋白纯度大于 95 %。     

Expression of the pyranose 2-oxidase from Trametes pubescens in Escherichia coli and characterization of the recombinant enzyme

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 °C in amount corresponding to 8% of the cell soluble protein (0.6 U mg⁻¹). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 °C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.     

Physicochemical and molecular modeling studies of cefixime–l-arginine–cyclodextrin ternary inclusion compounds

In an attempt to improve the physicochemical properties of cefixime (CEF), its supramolecular inclusion compounds were prepared with β-cyclodextrin (βCD) and hydroxypropyl-β-cyclodextrin (HPβCD) in presence and/or absence of ternary component l-arginine (ARG) using spray drying technique. Initially, the phase solubility studies revealed a stoichiometry of 1:1 molar ratio with an A_L-type of phase solubility curve. The stability constants of binary systems were remarkably improved in presence of ARG, indicating positive effect of its addition. The inclusion complexes were characterized by FTIR, XRPD, DSC, SEM, particle size analysis, and dissolution studies. Further, molecular mechanic (MM) calculations were performed to investigate the possible orientations of CEF inside βCD cavity in presence and/or absence of ternary component. In case of physicochemical studies, the ternary systems performed well as a result of comprehensive effect of ternary complexation and particle size reduction achieved by a spray drying technology.     

Quantum mechanical study of the inclusion process of adamantanol isomers by l-tryptophan-modified-β-cyclodextrin

The complexation processes of 1 and 2-adamantanol with l-tryptophan-β-cyclodextrin have been studied using ab initio Hartree–Fock and density functional theory levels. For the host: guest inclusion processes, the up mode with the OH group of the alcohol oriented towards the secondary rim, is found to be in qualitative agreement with the experimental finding. A molecular recognition mechanism is proposed based on the host: guest relative dipole orientation. For the complex with the 2-Ada isomer the host and guest the dipoles are parallels favoring the interaction energy and. This mechanism can explain the small energy difference for the processes involving the adamantanol isomers and modified cyclodextrins.     

可溶性HLA-A*0203-BSP原核表达载体的构建及其在大肠杆菌中的高效表达

目的：构建HLA-A*0203重链胞外域羧基端融合生物素化酶BirA底物肽(BSP)的融合蛋白（HLA-A*0203-BSP）的原核表达载体并在大肠杆菌中进行表达。方法：以RT-PCR方法从HLA-A2+ 供者外周血单个核细胞(PBMC)中克隆HLA-A*0203重链基因的cDNA并测序鉴定,然后以PCR方法构建HLA-A*0203-BSP的原核表达载体,在大肠杆菌BL21(DE3)菌株中诱导表达并以免疫印迹鉴定。结果：DNA测序显示,从3名HLA-A2+ 供者PBMC中克隆的cDNA中,只有从供者2获得编码HLA-A*0203重链基因的cDNA。将编码重链胞外域1-276的序列和编码BSP的序列融合,构建HLA-A*0203-BSP融合蛋白的原核表达载体并经测序验证。该融合蛋白在BL21(ED3)中获得高效表达,约占菌体总蛋白的30％;产物相对分子质量约为34 kD,与理论大小一致。Western印迹分析显示融合蛋白完全存在于包涵体中。结论：成功克隆HLA-A*0203重链基因的cDNA,构建HLA-A*0203-BSP融合蛋白的原核表达载体,并在大肠杆菌中获得高效表达,为制备HLA-A*0203四聚体打下基础。