Screening for soluble expression constructs using cell-free protein synthesis

The SH2 domain of STAT6 was chosen to test the in vitro protein synthesis as a screening tool. Goal of the screening was to obtain constructs which produce soluble protein in E. coli. The expression of 70 different constructs using an E. coli based cell-free system revealed two constructs, which give partly soluble protein. The introduction of two mutations, which had been suggested by a structural based alignment of 20 different SH2 domains lead to increased solubility. The expression of both constructs in E. coli followed by an affinity and size exclusion chromatography resulted in milligram quantities of highly purified protein.     

Optimization of the bioprocessing conditions for scale‐up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression system

Use of transient expression for the rapid, large‐scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale‐up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha‐1‐antitrypsin (rAAT) by co‐infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 ± 8.7)% and (290 ± 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD₆₀₀ of 0.5 and a 0.25‐min vacuum infiltration, and multiple 1‐min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 ± 0.09)% and (4.1 ± 0.29)% of the total soluble protein, respectively, or (90 ± 1.7) and (140 ± 10) mg per kg fresh weight leaf tissue at 6 days post‐induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Optimization of fermentation conditions for the production of human soluble catechol-O-methyltransferase by Escherichia coli using artificial neural network

The aim of this work was to optimize the temperature, pH and stirring rate of the production of human soluble catechol-O-methyltransferase (hSCOMT) in a batch Escherichia coli culture process. A central composite design (CCD) was firstly employed to design the experimental assays used in the evaluation of these operational parameters on the hSCOMT activity for a semi-defined and complex medium. Predictive artificial neural network (ANN) models of the hSCOMT activity as function of the combined effects of these variables was proposed based on this exploratory experiments performed for the two culture media. The regression coefficients (R(2)) for the final models were 0.980 and 0.983 for the semi-defined and complex medium, respectively. The ANN models predicted a maximum hSCOMT activity of 183.73 nmol/h, at 40 °C, pH 6.5 and stirring rate of 351 rpm, and 132.90 nmol/h, at 35 °C, pH 6.2 and stirring rate of 351 rpm, for semi-defined and complex medium, respectively. These results represent a 4-fold increase in total hSCOMT activity by comparison to the standard operational conditions used for this bioprocess at slight scale.     

Enhanced soluble protein expression using two new fusion tags

Production of soluble recombinant proteins is vital for structure-function analysis and therapeutic applications. Unfortunately, when expressed in a heterologous host, such as Escherichia coli, most proteins are expressed as insoluble aggregates. Two new fusion partners have been identified to address these solubility problems. One of the tags was derived from a bacteriophage T7 protein kinase and the other one from a small E. coli chaperone, Skp. We have expressed a panel of insoluble human proteins including Hif1alpha, IL13, and folliculin as fusion proteins using these tags. Most of these fusion proteins were able to be expressed in a soluble form and could be purified by virtue of a Strep-tag II installed at the amino-terminal end of the fusion partners. In addition, we show that some of these proteins remained soluble after removal of the fusion tags by a site-specific protease. The results with these tags compare favorably to results with the most commonly used solubility tags described in the literature. Therefore, these two new fusion tags have the potential to express soluble proteins when fused with many recalcitrant proteins.     

Incorporation of glutamic acid into protein by a soluble system

A heat-labile, non-dialyzable factor(s) in soluble fractions from Escherichia coli strains and Bacillus subtilis was found to incorporate the radioactivity of [14C]glutamic acid into 95 degrees C CCl3COOH-insoluble fraction. Incorporation catalyzed by a partially purified factor from E. coli B required ATP, Mg2+, tRNA, casein, carbonate, and 2-mercaptoethanol. A mixture of nineteen amino acids other than glutamic acid had no effect on the incorporation. Heparin, spermine and monovalent cations were inhibitory. Incorporation proceeded via glutamyl-tRNA. The incorporation from [14C]glutamyl-tRNA required Mg2+, casein, carbonate, and 2-mercaptoethanol, and there was no incorporation from [14C]aspartyl-tRNA. The reaction product was identified as protein. The incorporated moiety was the glutamyl moiety of glutamic acid and it retained a free alpha-amino group in the product protein. The incorporating factor of E. coli B was demonstrated to be glutamyl-tRNA synthetase.     

Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.     

Gene expression response to misfolded protein as a screen for soluble recombinant protein

Proper protein folding is key to producing recombinant proteins for structure determination. We have examined the effect of misfolded recombinant protein on gene expression in Escherichia coli. Comparison of expression patterns indicates a unique set of genes responding to translational misfolding. The response is in part analogous to heat shock and suggests a translational component to the regulation. We have further utilized the expression information to generate reporters responsive to protein misfolding. These reporters were used to identify properly folded recombinant proteins and to create soluble domains of insoluble proteins for structural studies.     

Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus expression system

A method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni-NTA agarose affinity chromatography. In mammalian sources, PrP(C) is attached to the cell membrane by a GPI anchor. However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance. Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media. Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion. Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form. Although affinity-purified PrP from mouse brain or recombinant prion protein (PrP) produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity. These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP. Furthermore, this simple expression system may provide an adequate source for structural, functional, and biochemical analyses of PrP.     

Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis

Aims: To improve the production of sweet‐tasting protein brazzein in Lactococcus lactis using controlled fermentation conditions. Methods and Results: The nisin‐controlled expression system was used for brazzein expression. The concentration of nisin for induction and the optical density (OD) at induction were therefore optimized, together with growth conditions (medium composition, pH, aerobic growth in the presence of hemin). Brazzein was assayed with ELISA on Ni‐NTA plates and Western blot. Use of the M‐17 medium, containing 2·5% glucose, anaerobic growth at pH 5·9 and induction with 40 ng ml^?1 nisin at OD 3·0 led to an approx. 17‐fold increase in brazzein per cell production compared to non‐optimized starting conditions. Aerobic growth in the presence of hemin did not increase the production. Conclusions: Considerable increase in brazzein per cell production was obtained at optimized fermentation conditions. Significance and Impact of the Study: Optimized growth conditions could be used in application of brazzein expression in L. lactis. The importance of pH and OD at induction contributes to the body of knowledge of optimal recombinant protein expression in L. lactis. The new assay for brazzein quantification was introduced.     

A new Trichoplusia ni cell line for membrane protein expression using a baculovirus expression vector system

A new cell line, MSU-TnT4 (TnT4), was established from Trichoplusia ni embryos for use with baculovirus expression vectors and evaluated for its potential for membrane protein production. To evaluate membrane protein synthesis, recombinant baculoviruses were constructed to express the human neurotensin receptor 1 as an enhanced green fluorescent protein (GFP) fusion. TnT4 cells had a doubling time of 21 h and expressed the membrane-GFP fusion protein at approximately twice the level as Sf21 cells from the p10 promoter, as evaluated by GFP intensity. Expression of secreted alkaline phosphatase (SEAP) was similar to that of Sf21 cells. Expression of membrane-GFP fusion proteins in recombinant baculoviruses provides a rapid method for evaluating the potential of new cell lines for the production of membrane proteins using a baculovirus expression vector system (BEVS).     

重组大肠杆菌右旋糖酐蔗糖酶的表达条件优化

通过设计正交实验,考察了培养基中各组分及其浓度对右旋糖酐蔗糖酶工程菌Escherichia coli BL21(DE3)/pET28-dexYG诱导产酶结果的影响。在获得最佳培养条件的基础上,考察温度、蔗糖浓度和pH值对右旋糖酐产量的影响。结果表明:菌浓OD600达到2.0时,加入异丙基硫代-β-D-呋喃半乳糖苷(IPTG)至0.25mmol/L,25°C诱导培养4h,产酶活力最高,达到110.16U/mL,蔗糖浓度对产量的影响比较显著。研究结果得到高效表达的培养条件,为实现该酶的工业化应用打下了基础。     

Construction of non-invasively constitutive expression vectors using a metagenome-derived promoter for soluble expression of proteins

Expression of soluble and functional proteins has been one of the critical challenges to many aspects of synthetic biology, metabolic and protein engineering. Among the current methods for expression of target proteins, constitutive expression systems offer several advantages over inducible systems, which require a chemical or physical inducer. In a previous study, a G196 DNA fragment containing constitutive promoters was mined from the soil metagenome and evaluated for the expression of target proteins in the functional and soluble state. In this study, we further improved this system by constructing a series of constitutive expression vectors, pCEM (using the CEM promoter trimmed from G196), pCEMT (incorporating rrnB T1 and T2 terminator into the downstream region of MCS in pCEM) and pRCEMT (grafting the cis-acting region of pCEMT into a low-copy-number plasmid). Subsequently, genes encoding GFPuv, esterase 1767 and β-glucosidase were subcloned into the resulting vectors, and their expression level and solubility were compared with those of IPTG-inducible vector systems pQE30 and pTrc99A. The extent of homogeneity and the ratio of the soluble fraction in the pRCEMT vector were relatively higher, without any delay of growth rate, than that of the pQE30 or pTrc99A. These results indicate that new expression vectors with moderate constitutive function could more easily lead to a homogenous population of cells expressing target proteins than those with conventionally inducible promoters.     

Screening factors effecting a response in soluble protein expression: formalized approach using design of experiments

We have integrated high-throughput expression and purification with quantitative protein analysis to identify factors influencing protein production. Application of high-throughput expression and purification, combined with automated gel capillary electrophoresis, allowed the quantitative analysis of multiple expression variables in a single experiment. An experimental design utilizing multiple factorial screens was employed to identify single factors and interactions having a significant impact on expression. As a test case, expression of the nonstructural protein NS3 from different hepatitis C virus genotypes (1b, 2a, and 3a) was examined in Escherichia coli. The 1b genotype of NS3 produced the highest level of expression, which was then further optimized using response surface modeling to give a four-fold increase in soluble protein levels. The quantitative and statistical approach presented has the capability of rapidly identifying interactions among experimental variables, leading to more reliable prediction of protein expression. We propose that this technique has universal application in the production of recombinant proteins, providing a powerful tool for the optimization of protein expression.     

拟态弧菌重组蛋白OmpUa的表达条件优化及其抗原性分析

目的分析重组蛋白OmpUa的表达形式和抗原性,优化拟态弧菌OmpUa基因工程重组菌（pMALo4X-OmpUa／TB1）的表达条件。方法诱导基因重组工程菌表达,分别采用SDS-PAGE和Westernblot分析重组蛋白OmpUa的表达形式及其抗原性。采用正交试验设计L9（34）方案,通过SDS-PAGE和电泳图谱光密度扫描分析培养基种类、诱导温度、诱导时间和诱导剂浓度等因素对重组蛋白表达的影响。结果重组蛋白OmpUa主要以可溶性形式表达,具有良好的抗原性。基因工程重组菌接种LB培养基,37℃振荡培养3h时加入0。5mmoL／L IPTG诱导4h,即可获得高表达量的重组OmpUa蛋白。结论摸索出拟态弧菌OmpUa基因工程重组菌的最佳表达条件,为下一步大量制备OmpUa诊断性抗原奠定了基础。     

Expression of a versatile DC-targeting fusion protein using an Adenovirus expression system

The importance of viral and tumour vaccines in eliciting elicit strong CD8+ T-cell responses has been widely acknowledged. Strategies exploring ways to enhance CD8+ T-cell responses have been developed, including targeting of vaccine antigens to dendritic cell (DC) receptors to access to the cross presentation pathway. Many DC endocytic receptors could potentially lead to augmented CD8+ T-cell responses if antigens were targeted directly to them, however only a few receptors have been explored because current targeting reagents are limited in the number of receptors that they are able to target. Consequently, this study describes the production and purification of a streptavidin-fusion protein that provides a versatile and efficient means to target antigen to more than one DC receptor. A model antigen gene, CMV pp65, and a streptavidin core gene, were spliced together using an overlap-extension PCR technique. The resulting fusion gene was cloned into a vector allowing expression in an Adenovirus-based expression system. Expression was verified and optimised before Ni-NTA affinity chromatography purification. Evaluation of pp65-streptavidin immunogenicity revealed that it elicits similar levels of CD8+ T-cell proliferative responses as pp65 and is able to effectively target specific DC receptors when used in addition to biotinylated receptor-specific antibodies. Additionally, enhancement of CD8+ T-cell responses was shown after directing pp65-strep to selected DC receptors in preliminary in vitro experiments. Collectively, this highlights the ease of production of a streptavidin-fusion protein, and demonstrates its use as a promising strategy to evaluate numerous DC receptors as potential targets in vaccine strategies.