Surface display of proteins by Gram-negative bacterial autotransporters

Expressing proteins of interest as fusions to proteins of the bacterial envelope is a powerful technique with many biotechnological and medical applications. Autotransporters have recently emerged as a good tool for bacterial surface display. These proteins are composed of an N-terminal signal peptide, followed by a passenger domain and a translocator domain that mediates the outer membrane translocation of the passenger. The natural passenger domain of autotransporters can be replaced by heterologous proteins that become displayed at the bacterial surface by the translocator domain. The simplicity and versatility of this system has made it very attractive and it has been used to display functional enzymes, vaccine antigens as well as polypeptides libraries. The recent advances in the study of the translocation mechanism of autotransporters have raised several controversial issues with implications for their use as display systems. These issues include the requirement for the displayed polypeptides to remain in a translocation-competent state in the periplasm, the requirement for specific signal sequences and "autochaperone" domains, and the influence of the genetic background of the expression host strain. It is therefore important to better understand the mechanism of translocation of autotransporters in order to employ them to their full potential. This review will focus on the recent advances in the study of the translocation mechanism of autotransporters and describe practical considerations regarding their use for bacterial surface display.     

Trimeric autotransporters: a distinct subfamily of autotransporter proteins

Autotransporter proteins are a large family of gram-negative bacterial extracellular proteins. These proteins have a characteristic arrangement of functional domains, including an N-terminal signal peptide, an internal passenger domain, and a C-terminal translocator domain. Recent studies have identified a novel subfamily of autotransporters, defined by a short trimeric C-terminal translocator domain and known as trimeric autotransporters. In this article, we review our current knowledge of the structural and functional characteristics of trimeric autotransporters, highlighting the distinctions between this subfamily and conventional autotransporters. We speculate that trimeric autotransporters evolved to enable high-affinity multivalent adhesive interactions with host surfaces and circulating host molecules to take place.     

A bacterial expression system revisited for the recombinant production of cystine-rich plant lipid transfer proteins

Non-specific lipid transfer proteins (nsLTPs) are abundant and ubiquitous cystine-rich proteins that are capable, in vitro, of binding lipids and hydrophobic molecules. In view to probe the lipid binding properties of the wheat nsLTP1, mutant variants may represent a powerful tool. To this end, a synthetic gene, encoding a mature wheat nsLTP1 polypeptide, was designed to ensure high level expression in Escherichia coli. The bacterial expression host strain, a translational fusion strategy, and convenient cleavage and purification procedures were optimized to produce in standard fermentation conditions, a significant amount (15 mg/L final yield), of a soluble and correctly folded recombinant nsLTP1. This highly amenable expression system is helpful in order to investigate structure-activity relationships of plant nsLTP.     

Extracellular acidic polysaccharide production by a two-membered bacterial coculture

A two-membered coculture of strains KYM-7 and KYM-8, identified as Cellulomonas cellulans and Agrobacterium tumefaciens, respectively, produced a large amount of an extracellular polysaccharide, designated APK-78, from starch. Each strain in pure culture produced only very little amount of polysaccharide from starch; the coexistence of the two strains from the early stage of cultivation was indispensable for a large amount of polysaccharide to be produced. The polysaccharide APK-78 was acidic and composed of glucose, galactose, succinic acid, and pyruvic acid with a molar ratio of 8.1:1.0:1.7:1.0, indicating that it is a succinoglycan type of polysaccharide.     

Real-time monitoring of recombinant bacterial proteins by mass spectrometry

Genetically altered bacteria manipulated to express green fluorescent protein (GFP) were used in an investigation of real-time monitoring for recombinant protein expression in cell by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). A significant advantage to whole cell MALDI MS is its ability to analyze bacterial cultures without pretreatment other than concentration. This paper describes the simultaneous analysis of overexpressed GFP recombinant Escherichia coli JM101 by MALDI-TOF MS and standard fluorescence measurements. Cells were harvested from liquid culture media during a 12 h GFP induced expression cycle to demonstrate the feasibility of near real-time monitoring of induced protein expression. The results show that although MALDI MS is not as sensitive as fluorescence measurements, expression levels of the targeted protein can easily be determined. Data available only through MALDI MS measurements reveal the presence of both native GFP and GFP-(histidine)(6) proteins. Additionally, biochemical processes not yet fully understood are observed in the presence and absence of ribosomal protein constituents. Thus, the work presented here demonstrates the ability of MALDI MS to monitor and characterize in real time the expression of targeted and unexpected genetically recombinant proteins in active cell cultures.     

Autodisplay: efficient bacterial surface display of recombinant proteins

To display a protein or peptide with a distinct function at the surface of a living bacterial cell is a challenging exercise with constantly increasing impact in many areas of biochemistry and biotechnology. Among other systems in Gram-negative bacteria, the Autodisplay system provides striking advantages when used to express a recombinant protein at the surface of Escherichia coli or related bacteria. The Autodisplay system has been developed on the basis of and by exploiting the natural secretion mechanism of the AIDA-I autotransporter protein. It offers the expression of more than 10⁵ recombinant molecules per single cell, permits the multimerization of subunits expressed from monomeric genes at the cell surface, and allows, after transport of an apoprotein to the cell surface, the incorporation of an inorganic prosthetic group without disturbing cell integrity or cell viability. Moreover, whole cells displaying recombinant proteins by Autodisplay can be subjected to high-throughput screening (HTS) methods such as ELISA or FACS, thus enabling the screening of surface display libraries and providing access to directed evolution of the recombinant protein displayed at the cell surface. In this review, the application of the Autodisplay system for the surface display of enzymes, enzyme inhibitors, epitopes, antigens, protein and peptide libraries is summarised and the perspectives of the system are discussed.     

The production of recombinant pharmaceutical proteins in plants

Imagine a world in which any protein, either naturally occurring or designed by man, could be produced safely, inexpensively and in almost unlimited quantities using only simple nutrients, water and sunlight. This could one day become reality as we learn to harness the power of plants for the production of recombinant proteins on an agricultural scale. Molecular farming in plants has already proven to be a successful way of producing a range of technical proteins. The first plant-derived recombinant pharmaceutical proteins are now approaching commercial approval, and many more are expected to follow.     

Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using Escherichia coli

Applied Microbiology and Biotechnology - As a common expression host, Escherichia coli has received more and more attention due to the recently developed secretory expression system, which offers...     

重组肠道细菌作为产乙醇生物催化剂的研究进展

以木质纤维素水解液发酵生产的燃料酒精作为一种清洁的可再生能源引起人们极大的关注。本文介绍了燃料酒精工业发展的最新动态,并对多种产乙醇重组肠道细菌的研究进展作了综述。     

Recent advances in production of recombinant spider silk proteins

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Highlights? Spider silk a biopolymer of great strength, toughness, and elasticity as well as biodegradability and biocompatibility. ? Various host systems ranging from bacteria to animal systems have been employed for the production of recombinant spider silk proteins. ? Ultra-high molecular weight spider silk protein showing Kevlar strength could be produced in E. coli by systems metabolic engineering. ? Transgenic silkworms producing recombinant or chimera spider silk have great potential for actual production in large scale.     

Insect cell culture for industrial production of recombinant proteins

Insect cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production. Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. Here we review some of the recent developments in protein expression by insect cells and their potential application in large-scale culture. Our current knowledge of insect cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media. The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing. Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells. Methods for proteolysis control, the optimal inhibitors and culture and storage conditions which affect proteolysis are discussed. Finally, engineering issues related to high-density culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale culture.     

Plant cell cultures for the production of recombinant proteins

The use of whole plants for the synthesis of recombinant proteins has received a great deal of attention recently because of advantages in economy, scalability and safety compared with traditional microbial and mammalian production systems. However, production systems that use whole plants lack several of the intrinsic benefits of cultured cells, including the precise control over growth conditions, batch-to-batch product consistency, a high level of containment and the ability to produce recombinant proteins in compliance with good manufacturing practice. Plant cell cultures combine the merits of whole-plant systems with those of microbial and animal cell cultures, and already have an established track record for the production of valuable therapeutic secondary metabolites. Although no recombinant proteins have yet been produced commercially using plant cell cultures, there have been many proof-of-principle studies and several companies are investigating the commercial feasibility of such production systems.     

Actinomycetes as host cells for production of recombinant proteins

Actinomycetes (Actinobacteria) are highly attractive as cell factories or bioreactors for applications in industrial, agricultural, environmental, and pharmaceutical fields. Genome sequencing of several species of actinomycetes has paved the way for biochemical and structural analysis of important proteins and the production of such proteins as recombinants on a commercial scale. In this regard, there is a need for improved expression vectors that will be applicable to actinomycetes. Recent advancements in gene expression systems, knowledge regarding the intracellular environment, and identification and characterization of plasmids has made it possible to develop practicable recombinant expression systems in actinomycetes as described in this review.     

Extracellular production of phospholipase A2 from Streptomyces violaceoruber by recombinant Escherichia coli

Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber was successfully produced extracellularly in an active form by using a recombinant strain of Escherichia coli. The PLA(2) gene, which was artificially synthesized with optimized codons for E. coli and fused with pelB signal sequence, was expressed in E. coli using pET system. Most of the enzyme activity was detected in the culture supernatant with negligible activity in the cells. The recombinant enzyme was purified to homogeneity from the culture supernatant simply by ammonium sulfate precipitation and an anion exchange chromatography. The purified enzyme showed a specific activity comparable to that of the authentic enzyme. The recombinant enzyme had the same N-terminal amino acid sequence to that of the mature protein, indicating the correct removal of the signal peptide. An inactive PLA(2) with a mutation at the catalytic center was also secreted to the culture medium, suggesting that the observed secretion was not dependent on enzymatic activity. A simple screening method for the PLA(2)-producing colonies was established by detecting clear zone formation around the colonies on agar media containing lecithin. This is the first example of direct extracellular production of active PLA(2) by recombinant E. coli.     

Automatic laboratory-scale fed-batch procedure for production of recombinant proteins using inducible expression systems of Escherichia coli

Summary An automatic fed-batch procedure for the production of recombinant proteins in Escherichia coli was developed. Using glycerol as carbon source and by controlling the growth rate by using feed-forward algorithm, enabled high specific expression level (10–20 % of total cell protein) at high cell densities (20 g dry wt/l) to be achieved: rat and human soluble catechol-O-methyltransferase, calf prochymosin, and human troponin C were expressed with nearly 50-fold higher volumetric yield compared to the conventional (batch) procedures.