Expression,purification, and characterization of recombinant human H-chain ferritin

Based on their nanocage architectures, ferritins show their potential applications in medical imaging and therapeutic delivery systems. However, the recombinant human H-chain ferritin (rHF) is prone to form inclusion bodies in Escherichia coli. In our study, the cDNA of rHF was cloned into plasmid pET28a under the control of a T7 promoter. Molecular chaperones, including GroES, GroEL, and trigger factor, were coexpressed with rHF to facilitate its correct folding. The results showed that the solubility of rHF was increased more than threefold with the help of molecular chaperones. Taking advantages of its N-terminal His-tag, rHF was then purified with Ni-affinity chromatography. With a yield of 15?mg/L from bacterial culture, the purified rHF was analyzed by circular dichroism spectrometry for its secondary structure. Moreover, the rHF nanocages were characterized by transmission electron microscopy and dynamic light scattering. Our results indicate that rHF is able to self-assemble into nanocages with a narrow size distribution.     

Unique iron binding and oxidation properties of human mitochondrial ferritin: a comparative analysis with Human H-chain ferritin

Ferritins are ubiquitous iron mineralizing and storage proteins that play an important role in iron homeostasis. Although excess iron is stored in the cytoplasm, most of the metabolically active iron is processed in the mitochondria of the cell. Little is known about how these organelles regulate iron homeostasis and toxicity. The recently discovered human mitochondrial ferritin (MtF), unlike other mammalian ferritins, is a homopolymer of 24 subunits that has a high degree of sequence homology with human H-chain ferritin (HuHF). Parallel experiments with MtF and HuHF reported here reveal striking differences in their iron oxidation and hydrolysis chemistry despite their similar diFe ferroxidase centers. In contrast to HuHF, MtF does not regenerate its ferroxidase activity after oxidation of its initial complement of Fe(II) and generally has considerably slower ferroxidation and mineralization activities as well. MtF exhibits sigmoidal kinetics of mineralization more characteristic of an L-chain than an H-chain ferritin. Site-directed mutagenesis reveals that serine 144, a residue situated near the ferroxidase center in MtF but absent from HuHF, is one player in this impairment of activity. Additionally only one-half of the 24 ferroxidase centers of MtF are functional, further contributing to its lower activity. Stopped-flow absorption spectrometry of Fe(II) oxidation by O(2) in MtF shows the formation of a transient diiron(III) mu-peroxo species (lambda(max) = 650 nm) as observed in HuHF. Also, as for HuHF, minimal hydroxyl radical is produced during the oxidative deposition of iron in MtF using O(2) as the oxidant. However, the 2Fe(II) + H(2)O(2) detoxification reaction found in HuHF does not occur in MtF. The structural differences and the physiological implications of the unique iron oxidation properties of MtF are discussed in light of these results.     

用DREAM技术进行全长质粒快速定点突变

利用“设计限制酶辅助突变”(Designed Restriction Enzyme Assisted Mutagenesis, DREAM)进行全长质粒快速定点突变。根据突变位点附近氨基酸靶序列, 以简并密码子进行逆向推导, 这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体(Silent mutants), 这些突变体中包含大量的限制性酶切位点, 选择合适的酶切位点设计引物, 用Phusion超保真DNA聚合酶扩增全长质粒的DNA序列, 得到的PCR产物用T4多聚核苷酸激酶添加5￠磷酸基团后进行平末端连接, 转化大肠杆菌受体菌后用设计的酶切位点进行快速筛选。本研究用该方法成功地纠正了长约8 kb的质粒pcDNA3.1-pIgR中的突变碱基, 从而获得了多聚免疫球蛋白受体(pIgR)的野生型氨基酸序列。以上结果表明: 利用DREAM技术将限制性酶切位点引入目的基因而不改变目的蛋白质的氨基酸序列, 使突变体的筛选简单化; 配合使用高保真和高效率的Phusion DNA聚合酶可以进行长达8 kb的全长质粒的快速突变; 该方法无需使用定点突变试剂盒和特殊的受体菌, 同时避免了核酸杂交以及同位素的使用。     

A novel simple and rapid PCR-based site-directed mutagenesis method

Site-directed mutagenesis (SDM) is a powerful tool for exploring protein structure and function, and several procedures adjusted to specific purposes are still being developed. Herein we describe a straightforward and efficient method with versatile applications for introducing site-specific alterations in any deoxyribonucleic acid (DNA) sequence cloned in a plasmidic expression vector. In this polymerase chain reaction (PCR)-based SDM method, forward and reverse primers are used to amplify the plasmid containing the sequence of interest. The primers are designed so that the desired modifications are introduced at the 5′ end of one of the primers, whereas the other primer starts with the nucleotide at position (−1) of the one to be modified. The PCR is carried out using Pfu DNA polymerase. The blunt-ended PCR-generated DNA fragment is self-ligated and used to transform Escherichia coli. Mutant clones are screened by colony hybridization using the mutagenic primer as probe and the presence of the mutation is confirmed by direct DNA sequencing. This procedure was used efficiently to introduce substitutions, deletions, and insertions in the DNA sequences coding for a recombinant form (scFv) of antibody 107 specific of the human CR3 molecule, the rat α integrin CD11b A-domain and the human CD8β cloned in pPICZαB, pGEX-2T, and CDM8 expression vectors, respectively.     

多片段扩增结合Gibson组装法克隆基因定点突变北大核心CSCD

为寻找一种简洁高效的基因定点突变方案,利用Gibson组装技术对重叠延伸PCR法进行简化,并以克隆细胞周期蛋白依赖性激酶4基因单位点与双位点突变为例进行验证。采用与重叠延伸PCR相似的策略扩增含点突变的基因片段,同时采用双酶切制备线性质粒载体,保证质粒载体与基因片段含有一小段重叠序列作为接头。直接将基因片段与线性载体通过Gibson组装法拼接成完整质粒。经转化、筛选与检测,成功得到数个单位点与双位点目标突变体克隆,且阳性率均为100%。由于没有繁琐的多轮PCR扩增和频繁的DNA回收操作,也无需消化原始质粒,该方案避免了很多干扰定点突变的因素,能简便、高效地克隆基因单位点与多位点突变。对比而言,该方案规避了重叠延伸PCR与滚环扩增法的主要缺陷,是一种基因定点突变的良好解决方案。     

利用定点突变法研究精氨酸脱亚胺酶活性的影响机制

精氨酸脱亚胺酶(ADI)是一种针对精氨酸缺陷型癌症(如:肝癌、黑素瘤)的新药,目前处于临床三期试验。文中通过定点突变技术分析了精氨酸脱亚胺酶的特定氨基酸位点对酶活力的影响机制。针对已报道的关键氨基酸残基A128、H404、I410,采用QuikChange法进行定点突变,获得ADI突变株M1(A128T)、M2(H404R)、M3(I410L)和M4(A128T/H404R)。将突变株在大肠杆菌BL21(DE3)中进行重组表达,并对纯化获得的突变蛋白进行酶学性质研究。结果表明,突变位点A128T和H404R对ADI最适pH的提高,生理中性(pH 7.4)条件下的酶活力和稳定性的提高,以及Km值的降低均具有显著的作用。研究结果为阐明ADI的酶活力影响机制和蛋白质的理性改造提供了一定的依据。     

定点突变改造提高纺锤形赖氨酸芽孢杆菌氨基甲酸乙酯水解酶稳定性

氨基甲酸乙酯(Ethyl carbamate,EC)是一种存在于发酵食品和酿造酒精饮料中的潜在致癌物质。利用生物酶法去除食品饮料中的EC是一种较为安全有效的方法。本研究以来源于赖氨酸芽孢杆菌Lysinibacillus fusiformis SC02的氨基甲酸乙酯水解酶为研究对象,采用计算机辅助设计突变位点,构建了其不稳定区域Q328位点的饱和突变体。通过酶学性质分析发现,突变体Q328C和Q328V在40℃下的半衰期分别提高了7.46和1.99倍,Q328R在高温下也有比原酶更好的耐受性。此外,突变体Q328C对乙醇的耐受性和酸耐受性也有所提高。对氨基甲酸乙酯水解酶分子改造的结果表明,通过改造其不稳定区域Q328位点,可以提高酶的热稳定性及对酸和乙醇的耐受性。     

Crystal structure and site-directed mutagenesis of a nitroalkane oxidase from Streptomyces ansochromogenes

Nitroalkane oxidase (NAO) catalyzes neutral nitroalkanes to their corresponding aldehydes or ketones, hydrogen peroxide and nitrite. The crystal structure of NAO from Streptomyces ansochromogenes was determined; it consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern. Site-directed mutagenesis of His¹⁷⁹, which is spatially adjacent to FMN, resulted in the loss of enzyme activity, demonstrating that this amino acid residue is important for catalysis. The crystal structure of mutant H179D-nitroethane was also analyzed. Interestingly, Sa-NAO shows the typical function as nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase. Overall, these results suggest that Sa-NAO is a novel nitroalkane oxidase with TIM barrel structure.     

Intact protein analysis for site-directed mutagenesis overexpression products: plasmid-encoded R67 dihydrofolate reductase

Mass spectrometry is currently the method of choice for the analysis of recombinant protein expression products. By combining proteolytic digestion with peptide mapping and tandem mass spectrometry techniques, verification of site-directed mutagenesis products can be obtained. The proteolytic digestion step converts a purified recombinant protein into a mixture that must be reseparated, thus greatly increasing the analysis time associated with the confirmation of site-directed mutagenesis products. Ion/ion reaction chemistry combined with quadrupole ion trap mass spectrometry provides a fast and efficient way to analyze intact proteins for the correct site-directed mutagenesis products, without heavy reliance on the proteolytic digestion step. Analysis of a series of protein variants (I68M, I68Q, Y69F, and Q67Y) from plasmid-encoded R67 dihydrofolate reductase using ion/ion reaction chemistry confirmed the presence of the correct site-directed mutagenesis products. For the I68M mutant, ion/ion separations detected the presence of extensive degradation from the N-terminal end of the protein. In the case of the Q67Y mutant, a mixture of Q67Y and Q67C species was detected by employing tandem mass spectrometry combined with ion/ion reactions. The ion/ion reaction technique was also performed on a partially purified lysate of the Q67Y/C mixture and successfully screened for the presence of both components in a complex mixture. The ion/ion reaction approach achieved the same results as the proteolytic-digestion-based methodology in a much shorter analysis time.     

Heterologous expression and site-directed mutagenesis of endoglucanase CelA from Clostridium thermocellum

The endoglucanase CelA from Clostridium thermocellum was strongly expressed in Bacillus subtilis. The enzyme was purified by Ni²⁺-affinity chromatography. Site-directed substitution of D278 with an asparagine or an alanine residue surprisingly did not decrease the apparent k _cat value. Further substitutions of two other potentially critical residues, Y215 and D152, resulted in a 2-fold decrease in apparent k _cat value for Y215P and complete loss of activity for D152N.     

普鲁兰酶基因在毕赤酵母中组成型表达及定点突变研究

合成Bacillus acidopullulyticus的全长普鲁兰酶基因并在毕赤酵母X-33中进行组成型外分泌表达,重组酶的最适作用温度为60℃,最适作用pH值为4.5~5.0,酶比活力为2.0 U/mg.采用重叠延伸PCR方法对普鲁兰酶基因进行定点突变,实验结果表明,625、626位点Ala、Leu氨基酸突变为Leu、Tyr氨基酸后,该酶的催化效率有所降低,而Gln487Ala的突变对催化效率没有较大的影响.该研究结果为探究关键氨基酸区域对催化效率的影响提供了一定的理论和实验基础.     

Improvement in thermostability and psychrophilicity of psychrophilic alanine racemase by site-directed mutagenesis

A psychrophilic alanine racemase from Bacillus psychrosaccharolyticus has a higher catalytic activity than a thermophilic alanine racemase from Bacillus stearothermophilus even at 60 °C in the presence of pyridoxal 5′-phosphate (PLP), although the thermostability of the former enzyme is lower than that of the latter one [FEMS Microbial. Lett. 192 (2000) 169]. In order to improve the thermostability of the psychrophilic enzyme, two hydrophilic amino acid residues (Glu150 and Arg151) at a surface loop surrounding the active site of the enzyme were substituted with the corresponding residues (Val and Ala) in the B. stearothermophilus alanine racemase. The mutant enzyme (ER150,151VA) showed a higher thermostability, and a markedly lower K_m value for PLP, than the wild type one. In addition, the catalytic activities at low temperatures and kinetic parameters of the two enzymes indicated that the mutant enzyme was more psychrophilic than the wild type one. Thus, the psychrophilic alanine racemase was improved in both psychrophilicity and thermostability by the site-directed mutagenesis. The mutant enzyme may be useful for the production of stereospecifically deuterated NADH and various -amino acids.     

Site-directed mutagenesis of the CP 47 protein of photosystem II: 167W in the lumenally exposed loop C is required for photosystem II assembly and stability

The intrinsic chlorophyll-protein CP 47 is a component of photosystem II which functions in both light-harvesting and oxygen evolution. Using site-directed mutagenesis we have produced the mutant W167S which lies in loop C of CP 47. This strain exhibited a 75% loss in oxygen evolution activity and grew extremely slowly in the absence of glucose. Examination of normalized oxygen evolution traces indicated that the mutant was susceptible to photoinactivation. Analysis of the variable fluorescence yield indicated that the mutant accumulated very few functional PS II reaction centers. This was confirmed by immunoblotting experiments. Interestingly, when W167S was grown in the presence of 20 M DCMU, the mutant continued to exhibit these defects. These results indicate that tryptophan 167 in loop C of CP 47 is important for the assembly and stability of the PS II reaction center.     

Improvement of biological and pharmacokinetic features of human interleukin-11 by site-directed mutagenesis

Recombinant human interleukin-11 (rhIL-11) has been shown to increase platelet counts in animals and humans and is the only drug approved for its use in chemotherapy-induced thrombocytopenia (CIT). However, due to its serious side effects, its clinical use has been limited. The current work presents significantly improved efficacy of rhIL-11 via knowledge based re-designing process. The interleukin-11 mutein (mIL-11) was found to endure chemical and proteolytic stresses, while retaining the biological activity of rhIL-11. The improved efficacy of mIL-11 was evident after subcutaneous administration of mIL-11 and rhIL-11 in the rodent and primate models. More than three-fold increase in maximum plasma concentration (C_max) and area-under-the curve (AUC) was observed. Furthermore, three-fold higher increase in the platelet counts was obtained after seven consecutive daily subcutaneous mIL-11 injections than that with rhIL-11. The mIL-11 demonstrated not only improved stability but also enhanced efficacy over the currently used rhIL-11 regimen, thereby suggesting less toxicity.     

Improving the binding affinity of an antibody using molecular modeling and site-directed mutagenesis

Activated Factor X releases F1.2, a 271-amino acid peptide, from the amino terminus of prothrombin during blood coagulation. A nine-amino acid peptide, C9 (DSDRAIEGR), corresponding to the carboxyl terminus of F1.2 was synthesized and used to produce a monoclonal antibody, TA1 (K(D)) 1.22 x 10(-6) M). To model the TA1 antibody, we entered the sequence information of the cloned TA1 Fv into the antibody modeling program, ABM, which combines homology methods, conformational search procedures, and energy screening and has proved to be a reliable and reproducible antibody modeling method. Using a novel protein fusion procedure, we expressed the C9 peptide fused to the carboxyl terminus of the PENI repressor protein from Bacillus licheniformis in Escherichia coli. We constructed fusion proteins containing alanine substitutions for each amino acid in the C9 epitope. Binding studies, using the C9 alanine mutants and TA1, and spatial constraints predicted by the modeled TA1 binding cleft enabled us to establish a plausible conformation for C9 complexed with TA1. Furthermore, based on binding results of conservative amino acid substitutions in C9 and mutations in the antibody, we were able to refine the complex model and identify antibody mutations that would improve binding affinity.     

Mismatch repair during pneumococcal transformation of small deletions produced by site-directed mutagenesis

Summary The genetic behaviour of short non-homologous regions has been studied during transformation of Streptococcus pneumoniae. Amethopterin-resistant mutants belonging to the amiA locus were used for these investigations. Five mutants deleted for 1–5 bp were obtained by oligonucleotide-direcrted mutagenesis. Their efficiency of transformation was measured using recipient strains either able to excise and repair mismatched bases (Hex⁺) or Hex^- derivatives. Deletions or insertions of 1 and 2 bp are fully recognized by the Hex system, and are efficiently repaired whereas 3-bp deletions or insertions are only partially excised and repaired. The efficiency of repair is inversely related to the size of the non-homology. Markers with 5-bp deletions or insertions are poorly repaired and thus transform at very high frequency: similar results are obtained in reciprocal crosses. It is proposed that 1-or 2-bp deletions or insertions are included in the heteroduplex structure as transition mutations. The Hex system would detect only small deviations from the normal DNA structure.     

Sigma-class glutathione transferase from Xenopus laevis: molecular cloning, expression, and site-directed mutagenesis

The structural gene for glutathione transferase (XlGSTS1-1) in the amphibia Xenopus laevis has been cloned from an embryo library and its nucleotide sequence has been determined. Open reading frame analysis indicated that xlgsts1 gene encodes the smallest protein of sigma class GST so far identified as being composed of only 194 amino acid residues. The recombinant XlGSTS1-1 shows a narrow range of substrate specificity as well as a significantly lower 1-chloro-2,4-dinitrobenzene conjugation capacity than that of squid sigma class GST. To compare the structural and functional differences between the squid and amphibian enzymes, several site-specific mutations were introduced in XlGSTS1-1, i.e., Ser100Asn, Phe102Tyr, Trp143Leu, Phe146Leu, and Trp148Cys. The results obtained indicate that Trp143 and Trp148 are more important determinants for the structural stability of XlGSTS1-1 rather than for its substrate specificity.     

Isolation of Cry1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site-directed mutagenesis system

Abstract A site-directed mutagenesis method was designed and used to create Cry1Ab mutant proteins in two of the five highly conserved blocks present in the Cry protein family. Region 1 comprises the central α-helix 5 of domain I and has been implicated in the pore formation activity of the toxin. Substitution of arginine by serine at position 173 (R173S) affects neither structural integrity nor toxicity. Region 2 comprises the major part of the domain I/domain II interface, characterized by the presence of numerous hydrogen bonds and electrostatic interactions. Mutations in the salt bridge formed by aspartic acid 242 and arginine 265 (D242N, D242C, R265C, and D242C/R265C) resulted in structurally unstable mutant proteins as is shown by their increased protease sensitivity and lack of biological activity.     

Improving the solubility of anti‐LINGO‐1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis

Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti‐LINGO‐1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.