Secretory Production Of Recombinant Proteins Research Articles

Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution.

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.

Secretion of a low and high molecular weight \u03b2-glycosidase by Yarrowia lipolytica

BackgroundThe secretory production of recombinant proteins in yeast simplifies isolation and purification but also faces possible complications due to the complexity of the secretory pathway. Therefore, correct folding, maturation and intracellular transport of the recombinant proteins are important processing steps with a higher effort needed for complex and large proteins. The aim of this study was to elucidate the secretion potential of Yarrowia lipolytica for low and high molecular weight β-glycosidases in a comparative cultivation approach.ResultsA low sized β-glucosidase from Pyrococcus furiosus (CelB; 55 kDa) and a large sized β-galactosidase isolated from the metagenome (M1; 120 kDa) were integrated into the acid extracellular protease locus using the CRISPR–Cas9 system to investigate the size dependent secretion of heterologous proteins in Y. lipolytica PO1f. The recombinant strains were cultivated in the bioreactor for 78 h and the extra- and intracellular enzyme activities were determined. The secretion of CelB resulted in an extracellular volumetric activity of 187.5 µkatoNPGal/Lmedium, while a volumetric activity of 2.98 µkatoNPGal/Lmedium was measured during the M1 production. However, when the amount of functional intra- and extracellular enzyme was investigated, the high molecular weight M1 (85%) was secreted more efficiently than CelB (27%). Real-time PCR experiments showed a linear correlation between the transcript level and extracellular activity for CelB, while a disproportional high mRNA level was observed regarding M1. Interestingly, mass spectrometry data revealed the unexpected secretion of two endogenous intracellular glycolytic enzymes, which is reported for the first time for Y. lipolytica.ConclusionThe results of this study provide deeper insights into the secretion potential of Y. lipolytica. A secretion limitation for the low-size CelB was observed, while the large size M1 enzyme was produced in lower amounts but was secreted efficiently. It was shown for the first time that Y. lipolytica is a promising host for the secretion of heterologous high molecular weight proteins (> 100 kDa), although the total secreted amount has to be increased further.

In silico Analysis of Different Signal Peptides for Secretory Production of Arginine Deiminase in Escherichia coli.

Secretory production of recombinant protein in bacterial hosts fulfills several advantages. Selecting an appropriate secretory signal peptide is a critical step in secretory production of different protein. Several patents report the usage of signal peptides for secretory production of recombinant proteins in E. coli. In silico identification of suitable signal peptides is a reliable and cost-effective alternative to experimental approaches. This study was aimed to predict best signal peptides for the secretory production of recombinant arginine deiminase in E. coli. In this study, 30 different signal peptide sequences were retrieved from database. The signal peptide probability, location of cleavage sites, and n, h and c regions were predicted by SignalP 4.1 and Phobius servers. After purging the 30 predicted secretory signal peptides, TorT, bla, NrfA, TolB, PapC, PldA, Lpp were removed. Several physicochemical properties of the remaining potential SPs were determined by ProtParam, PROSO II, and SOLpro servers for theoretically selecting the best candidates. Based on physicochemical properties, the signal peptides of OmpC, OmpF, and DsbA were identified respectively as the promising candidates for efficient secretory production of arginine deiminase in E. coli. Although the computational approach has established itself as a basis of modern biotechnology, the experimental study is necessary to validate its results. The criteria used in this study could be applied to other targets for recombination processes.

In silico Analysis of Different Signal Peptides for the Excretory Production of Recombinant NS3-GP96 Fusion Protein in Escherichia coli

Escherichia coli is one of the simplest hosts which is widely being used to express heterologous proteins. However, without appropriate signal peptide, this host cannot be applied for secretory proteins. Secretory production of recombinant proteins in E. coli has been an issue of interest because of its diverse advantages including cost and time savings, as well as reduction of endotoxin. NS3 from hepatitis C virus (HCV) was chosen as an antigen for vaccine development against HCV virus infections and it connected to gp96 as an adjuvant for stimulating Toll-like receptors (TLRs) to stimulate cytokines secretion by T cells. It was successfully produced in E. coli without using signal peptide previously. In this study, in order to increase the expression level of recombinant NS3-gp96 fusion protein (rNS3-gp96) in periplasmic space, we selected a series of signal peptides. Therefore, to foretell the best signal peptides for expression of NS3-gp96 recombinant protein in E. coli, 52 signal peptides from gram-negative bacteria were chosen and the most important physicochemical features of them were investigated. Therefore, n, h and c regions and signal peptide probability of them were evaluated by signalP software “version 4.1”, and physicochemical features were assessed by ProtParam and PROSO II tools. Eventually, prsK protein, outer membrane pore protein E (phoE), and fimbrial adapter papK protein were determined as the best candidates for the secretory production of rNS3-gp96 in E. coli in our study (with D score 0.899, 0.806, 0.797, respectively). Although, in the experimental investigation, should be considered other influencing parameters.

A Stable, Autonomously Replicating Plasmid Vector Containing Pichia pastoris Centromeric DNA.

The methylotrophic yeast Pichia pastoris is widely used to produce recombinant proteins, taking advantage of this species' high-density cell growth and strong ability to secrete proteins. Circular plasmids containing the P. pastoris-specific autonomously replicating sequence (PARS1) permit transformation of P. pastoris with higher efficiency than obtained following chromosomal integration by linearized DNA. Unfortunately, however, existing autonomously replicating plasmids are known to be inherently unstable. In this study, we used transcriptome sequencing (RNA-seq) data and genome sequence information to independently identify, on each of the four chromosomes, centromeric DNA sequences consisting of long inverted repeat sequences. By examining the chromosome 2 centromeric DNA sequence (Cen2) in detail, we demonstrate that an ∼111-bp region located at one end of the putative centromeric sequence had autonomous replication activity. In addition, the full-length Cen2 sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids in P. pastoris Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entire Cen2 sequence; this episome facilitates genetic manipulation in P. pastoris, providing high transformation efficiency and plasmid stability.IMPORTANCE Secretory production of recombinant proteins is the most important application of the methylotrophic yeast Pichia pastoris, a species that permits mass production of heterologous proteins. To date, the genetic engineering of P. pastoris has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicating Pichia plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as the P. pastoris-specific ARS (PARS1), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences of P. pastoris were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation of P. pastoris is ready to be developed.

Stepwise modifications of genetic parts reinforce the secretory production of nattokinase in Bacillus subtilis.

SummaryNattokinase (NK) is an important serine‐protease with direct fibrinolytic activity involving the prevention of cardiovascular disease as an antithrombotic agent. Dozens of studies have focused on the characterization of intrinsic novel promoters and signal peptides to the secretory production of recombinant proteins in Bacillus subtilis. However, intrinsic genetic elements have several drawbacks, which cannot mediate the production of NK to the desired level. In this study, the genetic elements, which were used to overproduce the recombinant secretory NK, were rationally modified in B. subtilis in a stepwise manner. The first step was to select a suitable signal peptide for the highly efficient secretion of NK. By comparison of the secretory levels mediated by two different signal peptides, which were encoded by the genes of a minor extracellular protease epr (SP epr) and cell‐wall associated protease wapA (SP wapA), respectively, SP wapA was verified as the superior secretory element. Second, P04, which was a synthetic promoter screened from an array of mutants based on the promoter cloned from the operon of a quorum‐sensing associated gene srfA (PsrfA), was paired to SP wapA. The secretory level of NK was obviously augmented by the combination of these two genetic elements. Third, the cis‐acting element CodY‐binding sequence positioned at the 5′UTR was deleted (yielding P08), and thus the secretory level was significantly elevated. The activity of NK, which was defined as fibrinolytic units (FU), reached to a level of 270 FU ml−1. Finally, the superior genetic element composed of P08 and SP wapA was utilized to overproduce NK in the host B. subtilis WB800, which was able to produce the secretory NK at 292 FU ml−1. The strategy established in this study can not only be used to overproduce NK in B. subtilis but also might be a promising pipeline to modify the genetic element for the synthetic secretory system.

Development an effective system to expression recombinant protein in E. coli via comparison and optimization of signal peptides: Expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study

BackgroundThermostable lipases from microbial sources have been substantially overexpressed in E. coli, however, these enzymes are often produced with low-level enzymatic activity and mainly in the form of inclusion bodies. Several studies have reported that the secretory production of recombinant proteins fused their N-terminus to a signal peptide has been employed to resolve the problem. In general, the feasibility of this approach largely depends on the secretory pathway of signal peptide and the type of target protein to be secreted. This study was performed to compare and optimize signal peptides for efficient secretion of thermostable lipase lipBJ10 from Pseudomonas fluorescens BJ-10. Meanwhile, a comparative study between this method and cytoplasmic secretion was implemented in secreting soluble and active lipases.ResultsFusion expression using six signal peptides, i.e., PelB and five native E. coli signal peptides, as fusion partners produced more soluble and functional recombinant lipBJ10 than non-fusion expression. Recombinant lipBJ10, fused to these six diverse signal peptides, was secreted into the periplasm in E. coli. The total lipase activity in all cases of fusion expression was higher than those in non-fusion expression. The relative activity peaked when lipBJ10 was fused to DsbA, yielding a value 73.3 times greater than that of the non-fusion protein. When DsbA was used as the fusion partner, the highest activity (265.41 U/ml) was achieved with the least formation of inclusion bodies; the other four E. coli signal peptides, to some extent, led to low activity and insoluble inclusion bodies. Therefore, DsbA is the optimal signal peptide partner to fuse with lipBJ10 to efficiently produce soluble and functional protein.ConclusionWe found that fusing to these signal peptides, especially that of DsbA, can significantly decrease the formation of inclusion bodies and enhance the function and solubility of lipBJ10 compared to non-fusion lipBJ10. Our results reported here can provide a reference for the high-level expression of other lipases with respect to a possible industrial application.

Efficient Extracellular Expression of Phospholipase D in Escherichia Coli with an Optimized Signal Peptide

New secretion vectors containing the synthetic signal sequence (OmpA’) was constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli Phospholipase D structural gene (Accession number:NC_018658) fused to various signal sequence were expressed from the Lac promoter in E. coli Rosetta strains by induction with 0.4mM IPTG at 28°C for 48h. SDS-PaGe analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of Phospholipase D (PLD) not only to the medium but also remained in periplasm of E. coli with OmpA’ signal sequence at the N-terminus of PLD. Thus the study on the effects of various surfactants on PLD extracellular production in Escherichia coli in shake flasks revealed that optimal PLD extracellular production could be achieved by adding 0.4% Triton X-100 into the medium. The maximal extracellular PLD production and extracellular enzyme activity were 0.23mg ml-1 and 16U ml-1, respectively. These results demonstrate the possibility of efficient secretory production of recombinant PLD in E. coli should be a potential industrial applications.

Enhanced secretion of recombinant proteins via signal recognition particle (SRP)-dependent secretion pathway by deletion of rrsE in Escherichia coli.

Although signal recognition particle (SRP)-dependent secretion pathway, which is characterized by co-translational translocation, helps prevent cytoplasmic aggregation of proteins before secretion, its limited capacity for the protein secretion is a major hurdle for utilizing the pathway as an attractive route for secretory production of recombinant proteins. Therefore, we developed an Escherichia coli mutant, whose efficiency of secretion via the SRP pathway was dramatically increased. First, we developed a novel FACS-based screening system by combining a periplasmic display system (PECS) and direct fluorescent labeling with the organoarsenic compound, FlAsH-EDT2 . With this screening system, transposon-insertion library of E. coli was screened, and then we isolated mutants which exhibited higher protein production through the SRP pathway than the parental strain. From the genetic analysis, we found that all isolated mutants had the same mutation-disruption of the 16S rRNA gene (rrsE). The positive effect of rrsE deficiency on protein secretion via the SRP pathway was successfully demonstrated using various model proteins including endogenous SRP-dependent proteins, antibodies, and G protein-coupled receptor. For the large-scale production of IgG and GPCR, we performed fed-batch cultivation with the rrsE-deficient mutant, and very high yields of IgG (0.4 g/L) and GPCR (1.4 g/L) were obtained. Biotechnol. Bioeng. 2016;113: 2453-2461. © 2016 Wiley Periodicals, Inc.

Biotechnology and Bioengineering: Volume 113, Number 1, January 2016

Cover Legend New platform for the secretory production of recombinant proteins in Corynebacterium glutamicum. (Image courtesy of Dr. Sung Sun Yim, Jae Woong Choi, Roo Jin Lee, Yong Jae Lee, and Dr. Ki Jun Jeong.) image

Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum.

Corynebacterium glutamicum, which has been for long an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large-scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α-amylase, and camelid antibody fragment (VHH). For large-scale production, fed-batch cultivations were also conducted and high yields were successfully achieved--as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α-amylase), and 1.57 g/L (VHH)--in the extracellular medium. From the culture media, all model proteins could be simply purified by one-step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum.

Comparison of the large-scale periplasmic proteomes of the Escherichia coli K-12 and B strains

Escherichia coli typically secretes many proteins into the periplasmic space, and the periplasmic proteins have been used for the secretory production of various proteins by the biotechnology industry. However, the identity of all of the E. coli periplasmic proteins remains unknown. Here, high-resolution periplasmic proteome reference maps of the E. coli K-12 and B strains were constructed and compared. Of the 145 proteins identified by tandem mass spectrometry, 61 proteins were conserved in the two strains, whereas 11 and 12 strain-specific proteins were identified for the E. coli K-12 and B strains, respectively. In addition, 27 proteins exhibited differences in intensities greater than 2-fold between the K-12 and B strains. The periplasmic proteins MalE and OppA were the most abundant proteins in the two E. coli strains. Distinctive differences between the two strains included several proteins that were caused by genetic variations, such as CybC, FliC, FliY, KpsD, MglB, ModA, and Ybl119, hydrolytic enzymes, particularly phosphatases, glycosylases, and proteases, and many uncharacterized proteins. Compared to previous studies, the localization of many proteins, including 30 proteins for the K-12 strain and 53 proteins for the B strain, was newly identified as periplasmic. This study identifies the largest number of proteins in the E. coli periplasm as well as the dynamics of these proteins. Additionally, these findings are summarized as reference proteome maps that will be useful for studying protein secretion and may provide new strategies for the enhanced secretory production of recombinant proteins.

Optimisation of signal peptide for recombinant protein secretion in bacterial hosts

Escherichia coli-the powerhouse for recombinant protein production-is rapidly gaining status as a reliable and efficient host for secretory expression. An improved understanding of protein translocation processes and its mechanisms has inspired and accelerated the development of new tools and applications in this field and, in particular, a more efficient secretion signal. Several important characteristics and requirements are summarised for the design of a more efficient signal peptide for the production of recombinant proteins in E. coli. General approaches and strategies to optimise the signal peptide, including the selection and modification of the signal peptide components, are included. Several challenges in the secretory production of recombinant proteins are discussed, and research approaches designed to meet these challenges are proposed.

Secretory Production of Recombinant Proteins in Escherichia coli

Extracellular production of heterologous proteins using the Escherichia coli cell factory offers several advantages over intracellular production and mammalian culture. Properly folded proteins can be rapidly accumulated in the culture media, and downstream processes for isolation and purification can be much simplified. Efforts to enhance the secretory production of target proteins can be largely classified as selection and modification of the signal peptide, co-expression of proteins to assist translocation and folding, improvement of periplasmic release, and protection of target proteins from degradation and contamination. Here, we review recent patents on the secretory production of recombinant proteins in E. coli.

Secretory Production of Recombinant Proteins by Streptomyces

Bacterial systems are widely applied as production platforms for proteins of biopharmaceutical or therapeutic interest and industrial enzymes. Among these prokaryotic systems, streptomycetes are attractive host cells because several strains of these Gram-positive bacteria have a high innate secretion capacity and extensive knowledge on their fermentation is available. A survey of the literature and our own experience suggests that several proteins are secreted to commercially acceptable levels. However, many heterologous proteins, most often of eukaryotic origin, are currently only poorly secreted by this host, indicating the need for further optimization of Streptomyces as a production host. In this review, the considerable efforts and strategies made in recent years aimed at improving streptomycetes as a host for the production of recombinant proteins will be discussed.

The extracellular proteomes of Escherichia coli B and K-12 strains and its application in the secretory production of recombinant proteins

The extracellular proteomes of Escherichia coli B and K-12 strains and its application in the secretory production of recombinant proteins

Strategy To Approach Stable Production of Recombinant Nattokinase in Bacillus subtilis

Bacillus subtilis (B. subtilis) is widely accepted as an excellent host cell for the secretory production of recombinant proteins. In this study, a shuttle vector was constructed by fusion of Staphylococcus aureus (S. aureus) plasmid pUB110 with Escherichia coli (E. coli) plasmid pUC18 and used for the expression of nattokinase in B. subtilis. The pUB110/pUC-based plasmid was found to exhibit high structural instability with the identification of a DNA deletion between two repeated regions. An initial attempt was made to eliminate the homologous site in the plasmid, whereas the stability of the resulting plasmid was not improved. In an alternative way, the pUC18-derived region in this hybrid vector was replaced by the suicidal R6K plasmid origin of E. coli. As a consequence, the pUB110/R6K-based plasmid displayed full structural stability, leading to a high-level production of recombinant nattokinase in the culture broth. This was mirrored by the detection of a very low level of high molecular weight DNAs generated by the plasmid. Moreover, 2-fold higher nattokinase production was obtained by B. subtilis strain carrying the pUB110/R6K-based plasmid as compared to the cell with the pAMbeta1-derived vector, a plasmid known to have high structural stability. Overall, it indicates the feasibility of the approach by fusing two compatible plasmid origins for stable and efficient production of recombinant nattokinase in B. subtilis.

PspA overexpression in Streptomyces lividans improves both Sec- and Tat-dependent protein secretion

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its innate capacity to secrete proteins at high level in the culture medium. In this report, we evaluated the importance of the phage-shock protein A (PspA) homologue on the protein secretion yield in Streptomyces lividans. The PspA protein is supposed to play a role in the maintenance of the proton motive force (PMF). As the PMF is an energy source for both Sec- and Tat-dependent secretion, we evaluated the influence of the PspA protein on both pathways by modulating the pspA expression. Results indicated that pspA overexpression can improve the Tat-dependent protein secretion as illustrated for the Tat-dependent xylanase C and enhanced green fluorescent protein (EGFP). The effect on Sec-dependent secretion was less pronounced and appeared to be protein dependent as evidenced by the increase in subtilisin inhibitor (Sti-1) secretion but the lack of increase in human tumour necrosis factor (hTNFalpha) secretion in a pspA-overexpressing strain.

Comparison of the Sec and Tat secretion pathways for heterologous protein production by Streptomyces lividans

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its natural ability to secrete high levels of active proteins into the culture broth and the availability of extensive fermentation knowledge. In bacterial expression systems, heterologous protein secretion has, so far, almost exclusively been investigated using signal peptides that direct the secretion to the Sec pathway. In this study, we assessed the possibility of the Streptomyces lividans twin-arginine translocation (Tat) pathway to secrete the human proteins tumor necrosis factor (TNF) α and interleukin (IL) 10 by fusing the coding sequences of mature hTNFα and hIL10 to the twin-arginine signal peptides of S. lividans xylanase C (XlnC) and Streptomyces antibioticus tyrosinase. Both proteins were secreted and this secretion was blocked in the Δ tatB and Δ tatC single mutants, indicating that the transport of hTNFα and hIL10 could be directed through the Tat pathway. Secretion levels of hTNFα and hIL10, however, were lower for Tat-dependent than for Sec-dependent transport using the Sec-dependent signal peptide of the Streptomyces venezuelae subtilisin inhibitor. Surprisingly, Sec-dependent transport was enhanced in the tatB deletion strain. This was especially interesting in the case of hIL10, where Sec-dependent transport of hIL10 was at least 15 times higher in the Δ tatB mutant than in the wild-type strain.

Secretory and extracellular production of recombinant proteins using Escherichia coli.

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, there are often problems in recovering substantial yields of correctly folded proteins. One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. The secretory production of recombinant proteins has several advantages, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding. In addition to the well-established Sec system, the twin-arginine translocation (TAT) system has recently been employed for the efficient secretion of folded proteins. Various strategies for the extracellular production of recombinant proteins have also been developed. For the secretory production of complex proteins, periplasmic chaperones and protease can be manipulated to improve the yields of secreted proteins. This review discusses recent advances in secretory and extracellular production of recombinant proteins using E. coli.