The pURI family of expression vectors: a versatile set of ligation independent cloning plasmids for producing recombinant His-fusion proteins

A family of restriction enzyme- and ligation-independent cloning vectors has been developed for producing recombinant His-tagged fusion proteins in Escherichia coli. These are based on pURI2 and pURI3 expression vectors which have been previously used for the successful production of recombinant proteins at the milligram scale. The newly designed vectors combines two different promoters (lpp(p)-5 and T7 RNA polymerase ?10), two different endoprotease recognition sites for the His?-tag removal (enterokinase and tobacco etch virus), different antibiotic selectable markers (ampicillin and erythromycin resistance), and different placements of the His?-tag (N- and C-terminus). A single gene can be cloned and further expressed in the eight pURI vectors by using six nucleotide primers, avoiding the restriction enzyme and ligation steps. A unique NotI site was introduced to facilitate the selection of the recombinant plasmid. As a case study, the new vectors have been used to clone the gene coding for the phenolic acid decarboxylase from Lactobacillus plantarum. Interestingly, the obtained results revealed markedly different production levels of the target protein, emphasizing the relevance of the cloning strategy on soluble protein production yield. Efficient purification and tag removal steps showed that the affinity tag and the protease cleavage sites functioned properly. The novel family of pURI vectors designed for parallel cloning is a useful and versatile tool for the production and purification of a protein of interest.     

The use of continuous cultures for selection and isolation of microorganisms producing extracellular enzymes adapted to extreme environments

Microorganisms producing extracellular enzymes with special properties can be selected and isolated by growing them in continous culture and using as the growth-limiting substrate a substance which must be broken down by the enzyme of interest. A diffusion layer around the microbial cell will cause gradients in the concentrations of enzymes and the hydrolyzed substrate. This will cause differences in growth rates between the parent cells and those mutant cells which produce an enzyme better adapted to the selective environment provided.     

Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins

The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example.     

MAAP: a versatile and universal tool for genome analysis

Multiple arbitrary amplicon profiling (MAAP) uses one or more oligonucleotide primers (5 nt) of arbitrary sequence to initiate DNA amplification and generate characteristic fingerprints from anonymous genomes or DNA templates. MAAP markers can be used in general fingerprinting as well as in mapping applications, either directly or as sequence-characterized amplified regions (SCARs). MAAP profiles can be tailored in the number of monomorphic and/or polymorphic products. For example, multiple endonuclease digestion of template DNA or the use of mini-hairpin primers can enhance detection of polymorphic DNA. Comparison of the expected and actual number of amplification products produced with primers differing in length, sequence and GC content from templates of varying complexity reveal severe departures from theoretical formulations with interesting implications in primer-template interaction. Extensive primer-template mismatching can occur when using templates of low complexity or long primers. Primer annealing and extension appears directed by an 8 nt 3-terminal primer domain, requires sites with perfect homology to the first 5–6 nt fom the 3 terminus, and involves direct physical interaction between amplicon annealing sites.     

The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins.

A fusion tag, called FLAG and consisting of eight amino acids (AspTyrLysAspAspAspAspLys) including an enterokinase-cleavage site, was specifically designed for immunoaffinity chromatography. It allows elution under non-denaturing conditions [Bio/Technology, 6 (1988) 1204]. Several antibodies against this peptide have been developed. One antibody, denoted as M1, binds the peptide in the presence of bivalent metal cations, preferably Ca(+). Elution is effected by chelating agents. Another strategy is competitive elution with excess of free FLAG peptide. Antibodies M2 and M5 are applied in this procedure. Examples demonstrating the versatility, practicability and limitations of this technology are given.     

Antisense-mediated exon skipping: a versatile tool with therapeutic and research applications

Antisense-mediated modulation of splicing is one of the few fields where antisense oligonucleotides (AONs) have been able to live up to their expectations. In this approach, AONs are implemented to restore cryptic splicing, to change levels of alternatively spliced genes, or, in case of Duchenne muscular dystrophy (DMD), to skip an exon in order to restore a disrupted reading frame. The latter allows the generation of internally deleted, but largely functional, dystrophin proteins and would convert a severe DMD into a milder Becker muscular dystrophy phenotype. In fact, exon skipping is currently one of the most promising therapeutic tools for DMD, and a successful first-in-man trial has recently been completed. In this review the applicability of exon skipping for DMD and other diseases is described. For DMD AONs have been designed for numerous exons, which has given us insight into their mode of action, splicing in general, and splicing of the DMD gene in particular. In addition, retrospective analysis resulted in guidelines for AON design for DMD and most likely other genes as well. This knowledge allows us to optimize therapeutic exon skipping, but also opens up a range of other applications for the exon skipping approach.     

CRISPR: a versatile tool for both forward and reverse genetics research

Human genetics research employs the two opposing approaches of forward and reverse genetics. While forward genetics identifies and links a mutation to an observed disease etiology, reverse genetics induces mutations in model organisms to study their role in disease. In most cases, causality for mutations identified by forward genetics is confirmed by reverse genetics through the development of genetically engineered animal models and an assessment of whether the model can recapitulate the disease. While many technological advances have helped improve these approaches, some gaps still remain. CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated), which has emerged as a revolutionary genetic engineering tool, holds great promise for closing such gaps. By combining the benefits of forward and reverse genetics, it has dramatically expedited human genetics research. We provide a perspective on the power of CRISPR-based forward and reverse genetics tools in human genetics and discuss its applications using some disease examples.     

A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

Background  

Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures.     

Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview

Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.     

PSysCal: a parallel tool for calibration of ecosystem models

The methods used for ecosystem modelling are generally based on differential equations. Nowadays, new computational models based on concurrent processing of multiple agents (multi-agents) or the simulation of biological processes with the Population Dynamic P-System models (PDPs) are gaining importance. These models have significant advantages over traditional models, such as high computational efficiency, modularity and its ability to model the interaction between different biological processes which operate concurrently. By this, they are becoming useful for simulating complex dynamic ecosystems, untreatable with classical techniques. On the other hand, the main counterpart of P-System models is the need for calibration. The model parameters represent the field measurements taken by experts. However, the exact values of some of these parameters are unknown and experts define a numerical interval of possible values. Therefore, it is necessary to perform a calibration process to fit the best value of each interval. When the number of unknown parameters increases, the calibration process becomes computationally complex and storage requirements increase significantly. In this paper, we present a parallel tool (PSysCal) for calibrating next generation PDP models. The results shown that the calibration time is reduced exponentially with the amount of computational resources. However, the complexity of the calibration process and a limitation in the number of available computational resources make the calibration process intractable for large models. To solve this, we propose a heuristic technique (PSysCal+H). The results show that this technique significantly reduces the computational cost, it being practical for solving large model instances even with limited computational resources.     

Modelling and optimization of a recombinant BHK-21 cultivation process using hybrid grey-box systems

In this work a model-based optimization study of fed-batch BHK-21 cultures expressing the human fusion glycoprotein IgG1-IL2 was performed. It was concluded that due to the complexity of the BHK metabolism it is rather difficult to develop a kinetic model with sufficient accuracy for optimization studies. Many kinetic expressions and a large number of parameters are involved resulting in a complex identification problem. For this reason, an alternative more cost-effective methodology based on hybrid grey-box models was adopted. Several model structures combining the a priori reliable first principles knowledge with black-box models were investigated using data from batch and fed-batch experiments. It has been reported in previous studies that the BHK metabolism exhibits modulation particularities when compared to other mammalian cell lines. It was concluded that these mechanisms were effectively captured by the hybrid model, this being of crucial importance for the successful optimization of the process operation. A method was proposed to monitor the risk of hybrid model unreliability and to constraint the optimization results to acceptable risk levels. From the optimization study it was concluded that the process productivity may be considerably increased if the glutamine and glucose concentrations are maintained at low levels during the growth phase and then glutamine feeding is increased.     

Neoglycoenzymes: a versatile tool for lectin detection in solid-phase assays and glycohistochemistry

Carbodiimide-mediated coupling of p-aminophenyl glycosides to a naturally nonglycosylated enzyme yields a neoglycoenzyme. This compound combines inherent enzymatic activity with synthetically conferred ligand properties to lectins. Appropriate choice of the ligand allows custom-made synthesis to reliably detect various types of lectins. To exemplify practical applications of this class of compounds, glycosylated bacterial beta-galactosidase has been employed to quantitate plant lectins, immobilized on plastic surfaces as well as on nitrocellulose. Competitive inhibition by specific sugar ascertained the dependence of binding on protein--carbohydrate interactions. In view of lectins as tools, a sandwich lectin-binding assay for high mannose-type glycoprotein detection has been modified to principally facilitate wide application to other lectin-reactive sugar chains by introducing the neoglycoenzyme. In addition to lectin determination in solid-phase assays, neoglycoenzymes allow one to glycohistochemically localize endogenous lectins in tissue prints and tissue sections with a minimum number of steps. This nonradioactive, rapid, sensitive, and convenient assay concept, based on conjugation of a ligand to an enzyme with maintenance of its receptor-binding activity, may find extended application beyond lectinology in receptor analysis.     

Comparison of cell-based and cell-free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies

We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell-based and cell-free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N-labeled proteins to be analyzed by 1H-15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell-based and two involved wheat germ cell-free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H-15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC+/- (protein partially disordered or not in a single stable conformational state), HSQC- (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X- for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell-free platform offers the advantage of greater genome coverage for NMR-based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X-ray structure determination.     

AMPer: a database and an automated discovery tool for antimicrobial peptides

MOTIVATION: Increasing antibiotics resistance in human pathogens represents a pressing public health issue worldwide for which novel antibiotic therapies based on antimicrobial peptides (AMPs) may offer one possible solution. In the current study, we utilized publicly available data on AMPs to construct hidden Markov models (HMMs) that enable recognition of individual classes of antimicrobials peptides (such as defensins, cathelicidins, cecropins, etc.) with up to 99% accuracy and can be used for discovering novel AMP candidates. RESULTS: HMM models for both mature peptides and propeptides were constructed. A total of 146 models for mature peptides and 40 for propeptides have been developed for individual AMP classes. These were created by clustering and analyzing AMP sequences available in the public sources and by consequent iterative scanning of the Swiss-Prot database for previously unknown gene-coded AMPs. As a result, an additional 229 additional AMPs have been identified from Swiss-Prot, and all but 34 could be associated with known antimicrobial activities according to the literature. The final set of 1045 mature peptides and 253 propeptides have been organized into the open-source AMPer database. AVAILABILITY: The developed HMM-based tools and AMP sequences can be accessed through the AMPer resource at http://www.cnbi2.com/cgi-bin/amp.pl     

Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures

Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. Although expression of GFP in plants has been widely reported, research on the use of GFP in plant cell cultures for bioprocess applications has been limited. In this study, the suitability of GFP as a secretory reporter and a useful tool in plant cell bioprocess optimization was demonstrated. GFP was produced and secreted from suspension cells derived from tobacco that was transformed with a binary vector containing mgfp5-ER cDNA, a modified GFP for efficient sorting to the endoplasmic reticulum, under control of the CaMV 35S promoter. For cell line gfp-13, extracellular and intracellular GFP accumulated to 15.4 and 29.4 mg x 1(-1), respectively. Extracellular GFP accounted for 30.9% of the total extracellular protein. The molecular mass of extracellular GFP was nearly identical to that of a recombinant GFP standard, indicating cleavage of the signal sequence. Neomycin phosphotransferase II, a cytosolic selection marker, was found almost exclusively in cellular extracts with less than 2% in the extracellular medium. These results suggest that extracellular GFP is most likely the result of secretion rather than nonspecific leakage from cells. Furthermore, medium fluorescence intensity correlated nicely with extracellular GFP concentration supporting the use of GFP as a quantitative secretory reporter. During the batch cultivation, culture GFP fluorescence also followed closely with cell growth. A medium feeding strategy was then developed based on culture GFP fluorescence that resulted in improved biomass as well as GFP production in a fed-batch culture.