Increasing PCR fragment stability and protein yields in a cell-free system with genetically modified Escherichia coli extracts

Escherichia coli cell-free protein synthesis is a highly productive system that can be applied to high throughput expression from polymerase chain reaction (PCR) products in 96-well plates for proteomic studies as well as protein evolution. However, linear DNA instability appears to be a major limitation of the system. We modified the genome of the E. coli strain A19 by removing the endA gene encoding the endonuclease I and replacing the recCBD operon (in which recD encodes the exonuclease V) by the lambda phage recombination system. Using the cell extract from this new strain increased the stability of PCR products amplified from a plasmid containing the cat gene. This resulted in CAT (chloramphenicol acetyltransferase) production from PCR products comparable to that from plasmids (500-600 microg/ml) in a batch reaction. We show that cell-free protein synthesis reactions using PCR products amplified from genomic DNA and extended with the T7 promoter and the T7 terminator give the same high yields of proteins (550 microg/ml) in 96-well plates. With this system, it was possible to rapidly express a range of cytoplasmic and periplasmic proteins.     

Site-directed mutagenesis by combination of homologous recombination and DpnI digestion of the plasmid template in Escherichia coli

A rapid site-directed mutagenesis strategy using homologous recombination and DpnI digestion of the template in Escherichia coli is described. Briefly, inverse polymerase chain reaction amplification of the entire circular plasmid was performed by mutagenic primers with overlapping sequences ( approximately 15 bp) for generating PCR products with approximately 15 bp of homology on the terminal ends. On direct transformation of the amplified PCR products into restriction endonuclease DpnI-expressing E. coli BUNDpnI, homologous recombination occurs in E. coli while the original templates are removed via DpnI digestion in vivo, thus yielding clones harboring mutated circular plasmids. Nearly 100% efficiency was attained when this strategy was used to modify DNA sequences.     

Rapid evolution of beta-glucuronidase specificity by saturation mutagenesis of an active site loop

Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition. We previously used DNA shuffling to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with increased beta-galactosidase activity. Epistatic (synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305, 331-339). Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E. coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-beta-d-xylopyranoside (pNP-xyl). The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS. The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening. One of the evolved proteins exhibited a approximately 200-fold improvement over the wild type in reactivity (k(cat)/K(m)) with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR. Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement. These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique.     

Rapid method for altering bacterial ribosome-binding sequences for overexpression of proteins in Escherichia coli.

In an Escherichia coli expression system, two genes, one from an anaerobic intestinal bacterium and one from E. coli, were overexpressed following the alteration of ribosome-binding (Shine-Dalgarno) sequences. For both genes, the polymerase chain reaction (PCR) was used to modify the ribosome-binding sequence and, at the same time, provide restriction endonuclease sequences at each end of the gene. These restriction endonuclease sequences were used for inserting the DNA into the E. coli plasmid vector pGEM2, which has the T7 promoter upstream from its multiple cloning sites. Each chimeric plasmid, made by ligating the PCR product into pGEM2, was transformed into E. coli strain HMS174(DE3) which, when induced, produces T7 RNA polymerase for regulated overexpression. The gene isolated from the anaerobic intestinal bacterium, a 27-kDa polypeptide gene from Eubacterium sp. strain 12708, when expressed using this system, produced about one-third of the total cell protein as measured in Coomassie-stained protein gels and confirmed by Western blots with rabbit antibody. The E. coli enzyme, a 28.4-kDa tRNA methylation enzyme, was increased fivefold in activity of cell extracts over that of the best previous strain.     

A marker-coupled method for site-directed mutagenesis

A marker-coupled method for site-directed mutagenesis (SDM) has been developed. In this method, target DNA is first cloned into a plasmid vector which carries an inactivated tetracycline-resistance (TcR)-encoding tet gene. Using this cloned plasmid as template, polymerase chain reaction (PCR) is performed with a mutagenic primer and a marker primer. The mutagenic primer contains the desired mutations to be introduced into the target DNA, and the marker primer contains a mutation for restoring the activity of the inactivated tet gene. The PCR product is annealed with a gapped duplex plasmid template, extended and ligated in vitro. The resulting uni-strand-mutated plasmid is converted into the gapped duplex form, transformed into Escherichia coli JM109 and spread on yeast extract/tryptone culture medium + Tc plates. The TcR colonies grown on these plates all carry active tet genes. Due to the 'tight coupling' between the marker primer and the mutagenic primer formed in the PCR product, these TcR colonies should also carry the mutagenic primer, e.g., the desired mutations in the target DNA. In fact, practically all of the TcR colonies have been found to be the desired mutants in the present experiments. Therefore, this method provides a very efficient approach for SDM.     

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method. Joaquin Sanchis, Layla Fernández, and J. Daniel Carballeira contributed equally.     

小麦黄花叶病毒RNA228kDa蛋白基因的表达及表达产物抗血清的制备

根据小麦黄花叶病毒（ Ｗ Ｙ Ｍ Ｖ） 核苷酸序列测定结果,将 Ｗ Ｙ Ｍ Ｖ Ｒ Ｎ Ａ２ 上的２８ ｋ Ｄａ 蛋白基因克隆到ｐ Ｅ Ｔ１１ａ 上,构建了原核表达载体ｐ Ｅ２８３９ 。 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 分析表明,经 Ｉ Ｐ Ｔ Ｇ 诱导,２８ ｋ Ｄａ蛋白基因在大肠杆菌 Ｂ Ｌ２１（ Ｄ Ｅ３）ｐ Ｌｙｓ Ｓ 中得到高效表达。以含表达产物的凝胶为抗原,免疫家兔,首次制备了小麦黄花叶病毒 Ｒ Ｎ Ａ２ 蛋白特异性抗血清。     

A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction

A specific mutagenic change in the cDNA of human protein S was introduced by a modification of the polymerase chain reaction that permits the introduction of a mutation at any position in a double-stranded DNA molecule. The method employed four synthetic oligonucleotide primers. One oligonucleotide contained a single-base mismatch to direct the mutagenesis; the other three oligonucleotides were designed to allow selective amplification of the mutated sequence with Thermus aquaticus polymerase. The mutagenized cDNA was cloned into a plasmid vector and transformed into Escherichia coli RR1 cells for characterization. The desired cytosine to guanine change in the target cDNA was confirmed by the predicted appearance of an AluI restriction site and by dideoxynucleotide sequencing. No other sequence changes were detected within the amplified region. This method of site-specific mutagenesis can be applied to any linear double-stranded DNA large enough for primer annealing and obviates specialized cloning vectors, DNA constructs, and selection techniques. It has the advantage over a recently published PCR technique (R. Higuchi, B. Krummel, and R. Saki (1988) Nucleic Acids Res. 16, 7351-7367) in requiring no diafiltration to remove primers between steps and in requiring only a single mutagenic oligonucleotide to be synthesized for each mutant construct made after the initial one.     

肺炎链球菌去信号肽脂蛋白PsaA锌离子结合特性的初步表征

肺炎链球菌是细菌性肺炎的主要病原体。PsaA是各种肺炎链球菌共有的遗传保守的特异性表面金属结合脂蛋白。通过PCR扩增肺炎链球菌D39不含信号肽的PsaA基因片段,将其通过T4连接酶连接至含6His标签的表达载体PBAD/HisA中,转化表达宿主大肠杆菌Top-10后用L(+)-阿拉伯糖诱导重组蛋白的表达。重组蛋白经亲和镍柱纯化以后,用外切酶-重组肠激酶(REK)去除6His标签。感应偶合电浆质谱(ICP-MS)测得纯化的PsaA蛋白以1:1比例结合金属锌离子。进而,通过圆二色谱法分析金属离子的结合对蛋白二级结构中α-螺旋和β-片层含量的影响,荧光光谱研究蛋白结合锌离子的解离常数及结合当量,为进一步研究该蛋白在体外的金属结合特性及细菌的金属运输及毒力机制提供理论基础。     

人白细胞介素—18基因的克隆及其在大肠杆菌中的表达

利用反转录ＰＣＲ（ＲＴＰＣＲ）法从人肝组织中分离人白介素１８（ｈＩＬ１８）基因。克隆到Ｔｖｅｃｔｏｒｅａｓｙ载体中,经酶切初步鉴定后进行ＤＮＡ序列分析,结果表明与文献报道完全一致。以ｐＢＶ２２０为表达载体,在大肠杆菌中高效表达了ｈＩＬ１８基因,重组蛋白占菌体总蛋白的２７．２％。为进一步研究其生物活性奠定了重要基础。     

汉坦病毒陈株S基因编码区的克隆,序列分析及表达

从汉坦病毒陈株感染的ＶｅｒｏＥ６细胞裂解液中提取病毒ＲＮＡ,经逆转录ＰＣＲ获得病毒Ｓ基因编码区约１．３ｋｂｃＤＮＡ片段,克隆该片段后进行核苷酸序列测定,并与汉坦病毒７６１１８株进行同源性比较,结果二者核苷酸序列同源性为８６％,推导的氨基酸序列同源性为９７％。将该基因片段插入原核表达载体ｐＧＥＸ４Ｔ１,在大肠杆菌中获得高效表达。表达产物为ＧＳＴＮＰ融合蛋白。ＳＤＳＰＡＧＥ检测表达蛋白分子约７２ｋＤ左右。Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ和ＥＬＩＳＡ试验结果表明,表达产物可与多株抗汉坦病毒核蛋白的ＭｃＡｂ发生反应,其抗原表位及ＭｃＡｂ反应谱与７６１１８株相比存在某些差异。     

Constructing DNA by polymerase recombination.

Polymerase-mediated recombination based on DNA polymerase chain reactions (PCRs) has been used to carry out directed joining at a present point of two DNA fragments initially contained in a plasmid and a single-stranded synthetic DNA. The process includes copying of these fragments by PCR with generation of an overlapping homologous region. Such overlap of 12 base pairs in length was found to be sufficient to provide further DNA joining also by use of PCR.     

Ligase-free subcloning: a versatile method to subclone polymerase chain reaction (PCR) products in a single day.

Often, it is convenient to subclone polymerase chain reaction (PCR) products into a plasmid vector for subsequent replication in bacteria, but conventional subcloning methods often fail. We report a rapid and versatile method to subclone PCR products directionally into a specific site of virtually any plasmid vector. The procedure requires only four primers, does not require DNA ligase, and may be accomplished in a single day. Ligase-free subcloning is performed by incorporating into the PCR primers sequences at the 5' ends that result in PCR products whose 3' ends are complementary to the 3' ends of the recipient linearized plasmid. The PCR product and the linearized plasmid are spliced together in a second PCR reaction in which Taq polymerase extends the complementary overlapping 3' ends (ligation by overlap extension). Denaturation followed by heterologous reannealing and cyclization results in a cyclic recombinant plasmid with two nicks that may be used directly to transform competent Escherichia coli. In our hands, ligase-free subcloning is rapid, and offers many advantages over existing strategies.     

猪大肠杆菌耐热肠毒素和热敏肠毒素基因的融合及表达

用聚合酶链反应扩增出猪源大肠杆菌编码ST前体（proST）和LT的B亚单位(LTB)成熟多肽的序列,再通过套式PCR将proST编码序列3′端和LTB编码序列5′端融合,并置于同一阅读框内,得到ST和LTB的融合基因,将此序列克隆到pGEMT质粒中,序列分析后,亚克隆到表达载体pQE30中,在大肠杆菌细胞中得到表达,表达的融合蛋白同时具有ST和LTB的抗原性,且无ST和LT的生物毒性。     

An efficient bipartite PCR technique to introduce specific changes in large plasmids

Amplifying an entire double-stranded plasmid by an inverse polymerase chain reaction (PCR) using a pair of tail-to-tail primers is a particularly efficient approach for introducing changes into DNA sequences. However, the approach generally works best for plasmids less than 5 Kb and it can be difficult to amplify the large multicomponent vectors that are used for protein expression in various eukaryotic cells. We have therefore adopted an alternative approach in which two smaller PCR products are generated and then ligated to produce the complete plasmid. A mutagenic primer is used to introduce the desired change and each reaction includes one of a pair of tail-to-tail primers from within an antibiotic resistance gene contained on the plasmid so that the two PCR products contain complementing parts of the complete gene. Ligating the two products generates various combinations but only the correctly ligated molecules recreate the antibiotic resistance gene and are able to replicate in Escherichia coli. When combined with methods to minimize the carryover of template plasmid, this can be an efficient way of introducing mutations into large plasmids.     

High-level expression of the Streptomyces clavuligerus isopenicillin N synthase gene in Escherichia coli.

The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein.     

High-level expression of the Streptomyces clavuligerus isopenicillin N synthase gene in Escherichia coli.

The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein.     

结核分枝杆菌Rv3369基因的表达和纯化

在大肠杆菌中高效表达结核分枝杆菌Rv3369蛋白,获得纯化的重组蛋白rRv3369。通过聚合酶链反应(Polymerase chain reaction,PCR)扩增Rv3369基因;以质粒pET28a为表达载体,构建重组质粒,转化大肠杆菌BL21(DE3);以异丙基硫代半乳糖苷(IPTG)诱导表达目的蛋白,通过SDS-PAGE鉴定rRv3369在大肠杆菌中的表达,确定rRv3369在大肠杆菌中的表达形式;采用Ni-NTA His.Bind Resin来纯化重组蛋白。重组质粒pET28a-Rv3369中目的基因测序结果与报道序列相同。分子量约19.5kDa,表达量约占菌体总蛋白的18%,纯化后的重组蛋白样品经SDS-PAGE和激光密度扫描分析表明其纯度为90%以上,每100mL培养菌可获得1.56mg左右的重组蛋白。用亲和层析法纯化的重组蛋白纯度较好。     

来源于Bacillus subtilis的中性植酸酶基因的克隆及在大肠杆菌中的表达

通过ＰＣＲ的方法从Bacillus subtilis基因组中克隆了中性植酸酶基因nphy,ＤＮＡ全序列分析表明其结构基因全长１１５２个核苷酸（编码３８３个氨基酸）,５′端有一编码２６个氨基酸的信号肽序列。去除信号肽编码序列的nphy克隆到大肠杆菌ＩＰＴＧ诱导表达载体pTYB40上,在大肠杆菌中得到了高效表达,表达量达到大肠杆菌可溶性蛋白的４０％以上,表达产物具有生物学活性,证实了克隆到的中性植酸酶的基因有正常的生物学功能。