Production,purification and characterization of non-myristylated human T-cell protein tyrosine kinase in a baculovirus expression system

A non-myristylated form (LCK^M) of the human T-lymphocyte-specific protein tyrosine kinase (LCK) was produced at high levels in a baculovirus expression system (BVES) using two strategies. First, LCK^M was produced by direct expression of a Gly²→ Ala mutant of LCK. Second, LCK was produced as a glutathione S-transferase (GST) fusion, and LCK^M was derived from the fusion protein by cleavage with thrombin. Both recombinant proteins (re-proteins) were produced at 5% of the total protein of infected Spodoptera frugiperda (Sf9) cells and were purified to >95% homogeneity. The enzymatic properties of the re-proteins and their inhibition by protein kinase inhibitors were comparable to the native enzyme (LCK^N) derived from Jurkat cells and wild-type LCK derived from the BVES. The high production levels will facilitate the recovery of large quantities of re-protein for use in biochemical and biophysical studies     

Cleavage and purification of intein fusion proteins using the Streptococcus gordonii spex system

A gram-positive bacterial expression vector using Streptococcus gordonii has been developed for expression and secretion, or surface anchoring of heterologous proteins. This system, termed Surface Protein Expression system or SPEX, has been used to express a variety of surface anchored and secreted proteins. In this study, the Mycobacterium xenopi (Mxe) GyrA intein and chitin binding domain from Bacillus circulans chitinase Al were used in conjunction with SPEX to express a fusion protein to facilitate secretion, cleavage, and purification. Streptococcus gordonii was transformed to express a secreted fusion protein consisting of a target protein with a C-terminal intein and chitin-binding domain. Two target proteins, the C-repeat region of the Streptococcus pyogenes M6 protein (M6) and the nuclease A (NucA) enzyme of Staphylococcus aureus, were expressed and tested for intein cleavage. The secreted fusion proteins were purified from culture medium by binding to chitin beads and subjected to reaction conditions to induce intein self-cleavage to release the target protein. The M6 and NucA fusion proteins were shown to bind chitin beads and elute under cleavage reaction conditions. In addition, NucA demonstrated enzyme activity both before and after intein cleavage.     

Protein expression strategies for identification of novel target proteins

Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cDNAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca2+ ions, the FLAG tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The alpha-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of isolated proteins was in the range of 90%. In the case of unknown cDNAs, the expression product with the highest molecular mass was assumed to represent the correct reading frame. In summary, we consider the YEpFLAG-1 system to be a very efficient tool to overexpress and isolate recombinant proteins in yeast. The expression system enables high throughput production and purification of proteins under physiological conditions, and allows miniaturization into microtiter formats.     

A novel fusion partner for enhanced secretion of recombinant proteins in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.     

Expression of catalytic subunit of bovine enterokinase in the filamentous fungus Aspergillus niger

The cDNA encoding for catalytic subunit of bovine enterokinase (EK(L)), to which the sequence for Kex2 protease cleavage site was inserted, was expressed in the protease deficient filamentous fungus Aspergillus niger AB1.13. Fungal transformants were obtained in which expression of the glucoamylase fusion gene resulted in secretion of the protein into growth medium. Fusion polypeptide was processed to mature EK(L) by endogenous Kex-2 like protease cleavage during secretory pathway. The highest quantity of EK(L), up to 5 mg l(-1), was obtained in soya milk medium. The secreted EK(L) was easily purified from other proteins found in A. niger culture supernatant, using ion exchange and affinity chromatography. The yield of the purified and highly active EK(L) was 1.9 mg l(-1) of culture.     

A novel strategy to over-express and purify homologous proteins from Streptococcus pneumoniae

Functional studies of Streptococcus pneumoniae virulence factors are facilitated by the development of complementation/mutagenesis systems. These methods usually result in poor expression yields; therefore, biochemical and structural/functional characterizations are mostly performed with proteins expressed and purified from heterologous systems (e.g. Escherichia coli). However, heterologous expression does not guarantee correct protein structure and function. In this work, we developed a method to over-express and purify homologous proteins from S. pneumoniae. The system relies on the combined use of the shuttle plasmid pMU1328 and a natural constitutive pneumococcal promoter, P₉₆. Efficient over-expression of secreted, membrane or surface anchored proteins, either wild type or mutant, was achieved. As proof of principle the S. pneumoniae pilus-1 backbone RrgB was successfully purified as a His-tag secreted protein (RrgB-His_SP) from pneumococcal culture supernatants. N-terminal sequencing and mass spectrometry analysis of RrgB-His_SP allowed the determination of the leader sequence cleavage site in pneumococcus, while proteolysis studies confirmed the stability of RrgB-His_SP to trypsin digestion. The data presented here support the use of this novel homologous expression method for all S. pneumoniae proteins for which extensive characterization studies are planned. Moreover, given the promiscuity of the pMU1328 replicon, this system could be used in diverse bacterial species.     

生物信息学预测与实验验证相结合策略筛选鉴定新的人分泌蛋白基因

使用生物信息学预测结合实验验证的策略筛选鉴定人新的分泌蛋白基因。用SignalP、SOSUI、PSORT和BLAST等程序对UniProt蛋白数据库进行生物信息学分析 ,筛选出用于实验验证的 1 4个功能未知基因。采用RT PCR方法 ,克隆得到 1 4个基因的全长编码序列 ,并构建到真核表达载体pcDNA3.1 ( - ) Myc His质粒。采用蛋白质印迹与免疫荧光分析 ,检测到其中 7个基因的表达。除其中一个在细胞核表达外 ,其余 6个只在细胞质中表达 ;其中的 4个基因的表达产物在细胞培养液中可被检测到 ,鉴定为 4个新的分泌蛋白基因。     

Functional analysis of the Streptomyces lividans type I signal peptidases

Type I signal peptidases are responsible for the proteolytic cleavage of the signal peptide of secreted proteins. In the gram-positive bacterium Streptomyces lividans, four adjacent genes (sipW, sipX, sipY and sipZ) were isolated encoding putative type I signal peptidases. In this work, the different sip genes were cloned and expressed. Subsequently, the Sip proteins were purified to raise antibodies. Although the four Sip proteins share a low degree of sequence similarity and differ significantly in size and pI, anti-Sip antibodies cross-reacted intensively. Functional signal peptidase processing activity for each of these Sip proteins was shown both in vitro and in vivo. The different Sip proteins did not exhibit the same cleavage efficiency on the Bacillus subtilis pre-chitosanase.     

Systematic high-yield production of human secreted proteins in Escherichia coli

Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies.     

The disulphide mapping,folding and characterisation of recombinant Ber e 1, an allergenic protein,and SFA8, two sulphur-rich 2S plant albumins

We have cloned and expressed genes encoding the allergenic brazil nut 2S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were secreted at high levels and that the purified proteins were properly folded. We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity. The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2S albumin family. A model three-dimensional structure of the allergen was generated. During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation. The secreted production of these properly folded sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy.     

Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells

Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence (λ_max = 480 nm). This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, Escherichia coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2–3 mg/L. This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence, indicates that luciferase does not undergo post-translational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis.     

A mammalian expression vector for expression and purification of secreted proteins for structural studies

A mammalian expression vector with features optimized for simple expression and purification of secreted proteins has been developed. This vector was constructed to facilitate X-ray crystallographic studies of cysteine-rich glycoproteins that are difficult to express by other means. Proteins expressed with this vector possess an N-terminal human growth hormone domain and an octahistidine tag separated from the desired polypeptide sequences by a tobacco etch virus protease recognition site. Advantages of this vector are high levels of expression, simple detection and purification of expressed proteins, and reliable cleavage of the fusion protein. Cotransfection of this vector with a dihydrofolate reductase gene allows amplification of expression levels with methotrexate. Over one dozen cysteine-rich secreted proteins have been expressed in sufficient quantity for structural studies using this vector; the structure of at least one of these proteins has been determined.     

A method for the evaluation of the efficiency of signal sequences for secretion and correct N-terminal processing of human parathyroid hormone produced in Escherichia coli.

Expression plasmids have been constructed for evaluation of different signal sequences for secretion and correct amino terminal processing of foreign proteins expressed in Escherichia coli. cDNA representing the N-terminal region (1-37) of human parathyroid hormone was inserted between DNA coding for two different forms of the signal sequence and two IgG binding domains (ZZ) derived from Staphylococcal protein A. The expression products were secreted to the periplasm and even to the growth medium and were easily purified by affinity chromatography using the ZZ part as a specific handle. Further analyses showed that the expression products were correctly processed to the mature protein hPTH(1-37)ZZ in a construct where the wild type signal sequence of Staphylococcus protein A was used. When a mutated signal sequence which lacks the normal cleavage site was employed, the fusion protein was not cleaved. Since signal sequences seem to be processed in the correct way in this system, we conclude that the general design of this type of expression vector is well suited for studying the N-terminal processing and secretion of heterologous proteins in E. coli.     

Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics

Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14(+) blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that approximately 40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca(2+) binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes.     

Determining subcellular localization of novel drug targets by transient transfection in COS cells

Genomics-based approaches are increasingly being used to identify disease-associated genes that represent potential new drug targets. As a first step in the validation of genes of unknown function, we describe a method for rapidly determining the subcellular localization of the gene product. If an immunotherapeutic approach is being considered, it is of particular interest to identify targets that are either on the cell-surface or secreted. Transient expression in COS cells combined with immunofluorescent staining provides a semi-high throughput method for determining the subcellular localization of multiple targets in parallel. COS cells are ideal for this purpose since: (i) they transfect easily; (ii) the high levels of expression that can be achieved transiently allow detection after 24 h; and (iii) the relatively large size and spread morphology of these cells allows the subcellular organelles to be easily visualized. To evaluate the system, we show prototype staining patterns for known cytoplasmic,secreted, Golgi-associated, endoplasmic reticulum-associated, and plasma membrane proteins, as well as data for novel targets. The localization of novel secretory and cell-surface proteins as determined by immunofluorescent staining, was confirmed by independent methods. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dihydrofolate reductase as a new "affinity handle".

Dihydrofolate reductase (DHFR) has been demonstrated to be a versatile "affinity handle" for expression of recombinant proteins. The DHFR "handle" has advantages not only in terms of efficiency of expressing the fusion protein as a soluble form but also in stabilizing unstable polypeptides and facilitating purification of the expressed protein by means of methotrexate-bound affinity chromatography and by making use of the enzyme activity. Fifteen genes encoding different lengths of polypeptides of 5 to 44 amino acids were chemically synthesized and introduced into expression vectors, pTP70-1 or its derivatives. All the polypeptide genes were efficiently expressed in Escherichia coli cells as fusion proteins which show DHFR activity. The respective fusion proteins were highly purified from cell-free extracts by monitoring the DHFR activity at each purification step. The use of methotrexate-bound affinity chromatography was very effective. In order to cut out the polypeptides, the purified fusion proteins were treated with either BrCN or site-specific protease according to the spacer sequence. The objective polypeptide was purified by means of a reversed-phase high-pressure liquid chromatography (HPLC) system. Specific cleavage of the purified fusion protein actually yielded very few peptide fragments, so the assignment and isolation of the objective polypeptide were carried out without difficulty.     

Highly efficient protein expression and purification using bacterial hemoglobin fusion vector

Recently developed bacterial hemoglobin (VHb) fusion expression vector has been widely used for the production of many target proteins due to its distinctive properties of expressing fusion protein with red color which facilitates visualization of the steps in purification, and increasing solubility of the target proteins. However, after intensive use of the vector, several defects have been found. In this report, we present a modified VHb fusion vector (pPosKJ) with higher efficiency, in which most of the defects were eliminated. First, it was found that thrombin protease often digests target protein as well as inserted thrombin cleavage site, so it was replaced by a TEV cleavage site for more specific cleavage of VHb from target protein. Second, a glycine-rich linker sequence was inserted between 6x his-tag and VHb to improve the affinity of 6x his-tag to Ni-NTA resin, resulting in higher purity of eluted fusion protein. Third, EcoRI and XhoI restriction sites located elsewhere in the vector were removed to make these restriction sites available for the cloning of target protein coding genes. A pPosKJ vector was fully examined with an anti-apoptotic BCL-2 family member of Caenorhabditis elegans, CED-9. A C-terminal VHb fusion expression vector (pPosKJC) was also constructed for stable expression of target proteins that may be difficult to express with an N-terminal fusion. Vaccinia-related kinase 1 (VRK1) was also successfully expressed and purified using the vector with high yield. Taken together, we suggest that the VHb fusion vector may be well suited for high-throughput protein expression and purification.     

The parasitome of the phytonematode Heterodera glycines

Parasitism genes expressed in the esophageal gland cells of phytonematodes encode secretions that control the complex process of plant parasitism. In the soybean cyst nematode, Heterodera glycines, the parasitome, i.e., the secreted products of parasitism genes, facilitate nematode migration in soybean roots and mediate the modification of root cells into elaborate feeding cells required to support the growth and development of the nematode. With very few exceptions, the identities of these secretions are unknown, and the mechanisms of cyst nematode parasitism, therefore, remain obscure. The most direct and efficient approach for cloning parasitism genes and rapidly advancing our understanding of the molecular interactions during nematode parasitism of plants is to create gland cell-specific cDNA libraries using cytoplasm microaspirated from the esophageal gland cells of various parasitic stages. By combining expressed sequence tag analysis of a gland cell cDNA library with high throughput in situ expression localization of clones encoding secretory proteins, we obtained the first comprehensive parasitome profile for a parasitic nematode. We identified 51 new H. glycines gland-expressed candidate parasitism genes, of which 38 genes constitute completely novel sequences. Individual parasitome members showed distinct gland cell expression patterns throughout the parasitic cycle. The parasitome complexity discovered paints a more elaborate picture of host cellular events under specific control by the nematode parasite than previously hypothesized.