Green fluorescent protein and factorial approach: an effective partnership for screening the soluble expression of recombinant proteins in Escherichia coli

We report how the combined use of protein expression reporter green fluorescent protein (GFP), and of an incomplete factorial approach (“InFFact”) made of 12 combinations of different states of three expression variables (bacterial strains, culture media and expression temperatures) created a convenient tool for screening the soluble expression of recombinant proteins in Escherichia coli (E. coli).In the first part of this work, we used two recombinant proteins that could be easily detected by Western blotting in the soluble fraction of E. coli lysate in most of the 12 InFFact combinations. When these proteins were fused to GFP and used in the same experiment (“InFFact-GFP”), fluorescence signals proved as sensitive and reliable as those provided by Western blotting. A trend analysis based on Western blot signals or on fluorescence allowed finding expression conditions for successfully scaling up the production of both proteins. Thus, GFP allowed InFFact trend analysis to be performed without gel electrophoresis or Western blotting.In the second part, we compared the results obtained by InFFact and InFFact-GFP when two other recombinant proteins were used which, in contrast with the proteins used in the first part, were barely detectable by Western blotting. Surprisingly, InFFact-GFP but not InFFact was able to find expression conditions for successfully scaling up the production of both proteins, suggesting that GFP could increase the solubility of the fusion partner.In conclusion, GFP allowed InFFact to be performed without gel electrophoresis and with at least the same sensitivity and specificity as that of Western blotting.     

An automated high-throughput screening method for the identification of high-yield, soluble protein variants using cell-free expression and systematic truncation

A highly automated method for rapidly identifying soluble protein variants with good expression yields has been developed. This method is based on a commercially available in vitro protein expression system. It consists of two polymerase chain reactions (PCR) followed by in vitro protein expression and protein quantification by dot blot. The PCR protocols have been improved and optimized to allow automation using commercial fluid handling devices. A PCR primer design program has also been implemented to streamline protein variant design. This automated protocol is highly reliable and has tremendously improved the throughput of expression screening as compared to conventional cell-based methods and manual in vitro methods. We have applied this method to 32 problematic targets from the TB Structural Genomics Consortium. Experimental results of these studies are reported.     

Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening

Proteins are the main reagents for structural, biomedical, and biotechnological studies; however, some important challenges remain concerning protein solubility and stability. Numerous strategies have been developed, with some success, to mitigate these challenges, but a universal strategy is still elusive. Currently, researchers face a plethora of alternatives for the expression of the target protein, which generates a great diversity of conditions to be evaluated. Among these, different promoter strength, diverse expression host and constructs, or special culture conditions have an important role in protein solubility. With the arrival of automated high-throughput screening (HTS) systems, the evaluation of hundreds of different conditions within reasonable cost and time limits is possible. This technology increases the chances to obtain the target protein in a pure, soluble, and stable state. This review focuses on some of the most commonly used strategies for the expression of recombinant proteins in the enterobacterium Escherichia coli, including the use of HTS for the production of soluble proteins.     

Expression and purification of the recombinant His-tagged GST-CD38 fusion protein using the baculovirus/insect cell expression system

CD38 is a type II transmembrane glycoprotein found in myriad mammalian tissues and cell types. It is known for its involvement in the metabolism of cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate, two nucleotides with calcium mobilizing activity independent of inositol trisphosphate. CD38 itself has been shown to have clinical significance in certain diseases with possible utilization in diagnostic and prognostic applications. Previous studies on several autoimmune diseases have shown the usefulness of recombinant CD38 protein expressed from Escherichia coli and Pichia pastoris in the detection of autoantibodies to CD38 via Western blot and ELISA. In this study, we produced a 6 x His-tagged GST-CD38 fusion protein using a recombinant baculovirus/insect cell expression technique that was purified as a soluble protein. The fusion protein was purified to homogeneity by affinity and gel filtration chromatography steps. It has an apparent molecular mass of 56 kDa on SDS-PAGE gel stained with Coomassie blue and was recognized on Western blots by antibodies against human CD38 as well as the polyhistidine tag. Peptide mass fingerprinting analysis confirmed the identity of human CD38 in the fusion protein.     

重组毕赤酵母甲醇利用表型与基因拷贝数对外源基因表达的影响

毕赤酵母是目前最优秀的外源蛋白表达系统之一。本文着重对重组毕赤酵母甲醇利用表型（Mut+型、MutS型和Mut-型）、基因剂量对外源蛋白高效表达的影响机理进行综述。MutS型的比生长速率和蛋白产率比Mut+型低、发酵周期长、副产物（如乙醇、乙酸等）形成速率不同。外源基因拷贝数对外源蛋白的影响主要有三种情况：（1）高基因拷贝数对外源蛋白表达水平有明显的正效应作用;（2）基因拷贝数增加反而降低了表达水平,即负效应作用;（3）重组蛋白表达与基因剂正相关,之后则表现负相关关系,这可能与外源蛋白翻译后加工有关（如二硫键形成、折叠等）,而与分子伴侣共表达可促进外源蛋白的高表达。     

Design of improved membrane protein production experiments: quantitation of the host response

Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial-and-error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high-performance bioreactors under tightly-defined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly-controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology.     

Combinatory use of cell-free protein expression,limited proteolysis and mass spectrometry for the high-throughput protein domain identification

The structural domains of proteins have often been identified through the use of limited proteolysis. In structural genomics studies, it is necessary to carry this out in a high-throughput manner. Here, we constructed a novel high-throughput system, which consists of cell-free protein expression and one-step affinity purification, followed by limited proteolysis using a unique new method, referred to “on beads method”. All these steps were carried out on 96-well plate formats and completed in two days, even by manual handling. The merits of the new method versus the conventional one are as follows: (1) experimental times are reduced, (2) the sample preparation for limited proteolysis experiments is simplified, and (3) both protein purification and limited digestion can be performed “in situ” on the same sample plate. This preparation method is therefore suitable for highly automated, proteolytic analyses coupled to mass spectrometry techniques at a micro-scale protein expression level. The resulting protease-resistant fragments were analyzed by MALDI-TOF-MS and protein domains of 34 mouse cDNA products were identified with this system.     

Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale

AMP-activated protein kinase (AMPK) is responsible for sensing of the cell’s energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the γ2 regulatory subunit have been shown to cause hypertrophic cardiomyopathy (HCM) with conduction abnormalities. As part of a study to examine the role of AMPK in the heart, we tested whether specific domains of the thick filament component cardiac myosin binding protein-C (cMyBP-C) were good in vitro AMPK substrates. The commercially available pET28a expression vector was used to generate a recombinant form of the cMyBP-C C8 domain as a fusion protein with a hexahistidine tag. In vitro phosphorylation with activated kinase showed that the purified fusion protein was a good AMPK substrate, phosphorylated at a similar rate to the control SAMS peptide and with phosphate incorporation specifically in serine residues. However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector. As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.     

Automated, high-throughput platform for protein solubility screening using a split-GFP system

Overproduction of soluble and stable proteins for functional and structural studies is a major bottleneck for structural genomics programs and traditional biochemistry laboratories. Many high-payoff proteins that are important in various biological processes are “difficult to handle” as protein reagents in their native form. We have recently made several advances in enabling biochemical technologies for improving protein stability (), allowing stratagems for efficient protein domain trapping, solubility-improving mutations, and finding protein folding partners. In particular split-GFP protein tags are a very powerful tool for detection of stable protein domains. Soluble, stable proteins tagged with the 15 amino acid GFP fragment (amino acids 216–228) can be detected in vivo and in vitro using the engineered GFP 1–10 “detector” fragment (amino acids 1–215). If the small tag is accessible, the detector fragment spontaneously binds resulting in fluorescence. Here, we describe our current and on-going efforts to move this process from the bench (manual sample manipulation) to an automated, high-throughput, liquid-handling platform. We discuss optimization and validation of bacterial culture growth, lysis protocols, protein extraction, and assays of soluble and insoluble protein in multiple 96 well plate format. The optimized liquid-handling protocol can be used for rapid determination of the optimal, compact domains from single ORFS, collections of ORFS, or cDNA libraries.     

Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems

Sixty-three proteins of Pseudomonas aeruginosa in the size range of 18-159 kDa were tested for expression in a bacterial cell-free system. Fifty-one of the 63 proteins could be expressed and partially purified under denaturing conditions. Most of the expressed proteins showed yields greater than 500 ng after a single affinity purification step from 50 microl in vitro protein synthesis reactions. The in vitro protein expression plus purification in a 96-well format and analysis of the proteins by SDS-PAGE were performed by one person in 4 h. A comparison of in vitro and in vivo expression suggests that despite lower yields and less pure protein preparations, bacterial in vitro protein expression coupled with single-step affinity purification offers a rapid, efficient alternative for the high-throughput screening of clones for protein expression and solubility.     

基因设计对重组蛋白表达的影响研究进展

利用异源表达系统生产重组蛋白已成为现代基因工程和生物工程研究热点和重点。但是研究者发现并非所有的基因都能在异源宿主中高效表达,除了宿主、分泌途径、启动子等因素外,基因自身的序列也蕴含了多种影响蛋白表达的因素,如密码子偏爱性,密码子对偏爱性,GC含量,mRNA二级结构,mRNA稳定性等。从基因设计的角度对影响蛋白表达的因素和方法进行了综述,尤其是对密码子优化和密码子对优化,详细讨论了与传统基因优化理念截然不同的密码子协调化及密码子对协调化等最新进展。     

The efficiency of pooling mRNA in microarray experiments

In a microarray experiment, messenger RNA samples are oftentimes pooled across subjects out of necessity, or in an effort to reduce the effect of biological variation. A basic problem in such experiments is to estimate the nominal expression levels of a large number of genes. Pooling samples will affect expression estimation, but the exact effects are not yet known as the approach has not been systematically studied in this context. We consider how mRNA pooling affects expression estimates by assessing the finite-sample performance of different estimators for designs with and without pooling. Conditions under which it is advantageous to pool mRNA are defined; and general properties of estimates from both pooled and non-pooled designs are derived under these conditions. A formula is given for the total number of subjects and arrays required in a pooled experiment to obtain gene expression estimates and confidence intervals comparable to those obtained from the no-pooling case. The formula demonstrates that by pooling a perhaps increased number of subjects, one can decrease the number of arrays required in an experiment without a loss of precision. The assumptions that facilitate derivation of this formula are considered using data from a quantitative real-time PCR experiment. The calculations are not specific to one particular method of quantifying gene expression as they assume only that a single, normalized, estimate of expression is obtained for each gene. As such, the results should be generally applicable to a number of technologies provided sufficient pre-processing and normalization methods are available and applied.     

The pST44 polycistronic expression system for producing protein complexes in Escherichia coli

Protein complexes are responsible for key biological processes, but methods to produce recombinant protein complexes for biochemical and biophysical studies are limited. We have developed a second generation Escherichia coli polycistronic expression system which improves on the modularity of our original pST39 polycistronic system. This pST44 expression system simplifies the construction of polycis-tronic plasmids, particularly of variant plasmids expressing deletion or point mutations in any subunit. To facilitate purification of the expressed complex, we have prepared a suite of 72 plasmids which allows individual subunits to be tagged at the N- or C-terminus with six permanent or cleavable peptide affinity tags. We demonstrate these new features in a detailed deletion analysis of a three protein yeast Piccolo NuA4 histone acetyltransferase complex, and in the affinity purification of a human Piccolo NuA4 complex. We also utilize the modular design to show that the order of expression of the three subunits along the polycistronic plasmid does not affect the reconstitution of the yeast Piccolo complex in E. coli.     

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.     

Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.     

新型载体蛋白P64K在大肠杆菌中的可溶性表达与纯化

用常规PCR方法从脑膜炎球菌中克隆出P64K基因,构建重组质粒pET28a-P64K转化BL21(DE3)宿主菌,经眦诱导表达筛选P64K蛋白的高表达菌株。结果证实P64K蛋白为胞内可溶性表达,表达量约占胞内总蛋白的30％。重组P64K蛋白经Butyl疏水柱、G200分子筛柱和Q阴离子柱三步层析后纯度达到95％以上,为今后进一步的功能和应用研究打下了良好的基础。     

Some factors influencing the proportion of periplasmic hepatitisB virus pre-S2 antigen in the recombinant yeastHansenula polymorpha

Summary A central composite design (CCD) was used to evaluate, for the purpose of future process optimization, the influence of pH, yeast extract and ammonium chloride concentrations on the proportion of periplasmic hepatitisB pre-S2 antigen in the recombinant yeastHansenula polymorpha. Each factor was tested at five levels, and a second order polynomial model for the proportion of periplasmic antigen was fitted to the treatment combinations. pH showed the greatest effect: the proportion of periplasmic antigen was greatly increased at the higher pH levels. At the higher pH levels used, the proportion of periplasmic antigen was enhanced by a high concentration of ammonium chloride. Additional experiments have confirmed both the validity of the selected model and the optimal conditions found. A significant correlation was found between the proportion of periplasmic antigen and the total yield of antigen. These results indicated that is should be possible to modulate the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast.     

芹菜韧皮部蛋白基因的分离与表达分析

韧皮部蛋白在维持植物形态,物质转运以及植物伤口保护等方面起着重要作用。本研究以地域来源和性状特性差异均较大的两个芹菜品种‘六合黄心芹’和‘美国西芹’为试验材料,利用RT-PCR技术获得这两种芹菜韧皮部蛋白基因的cDNA序列。结果显示：这两种芹菜来源的韧皮部蛋白基因全长均为546 bp,编码181个氨基酸。两者核苷酸序列有3个位点的不同,分别为：88G/A、399T/C和489T/C;在氨基酸序列上有一个位点的不同,为30T/A。预测其蛋白质分子量为19 kD,pI值为9.18。‘六合黄心芹’和‘美国西芹’的韧皮部蛋白与忽地笑等植物的韧皮部蛋白相似度较高,在保守位置分别具有5个亮氨酸残基和4个色氨酸残基。实时定量PCR表达分析表明,该基因主要在芹菜的茎和根等部位表达,具有明显的组织特异性。