Host For Recombinant Protein Production Research Articles

Comparing the Soluble Form of Recombinant Human Insulin-like Growth Factor-1 (rhIGF-1) in Escherichia coli Using Thioredoxin as Fused and Co-expressed Protein.

Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. Escherichia coli is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1. In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli. The rhIGF-1 was expressed in the E. coli Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose-derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process. Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity. It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in E. coli cells.

Synergic kinetic and physiological control to improve the efficiency of Komagataella phaffii recombinant protein production bioprocesses.

The yeast Komagataella phaffii (Pichia pastoris) is currently considered a versatile and highly efficient host for recombinant protein production (RPP). Interestingly, the regulated application of specific stress factors as part of bioprocess engineering strategies has proven potential for increasing the production of recombinant products. This study aims to evaluate the impact of controlled oxygen-limiting conditions on the performance of K. phaffii bioprocesses for RPP in combination with the specific growth rate (μ) in fed-batch cultivations. In this work, Candida rugosa lipase 1 (Crl1) production, regulated by the constitutive GAP promoter, growing at different nominal μ (0.030, 0.065, 0.100 and 0.120 h-1 ) under both normoxic and hypoxic conditions in carbon-limiting fed-batch cultures is analysed. Hypoxic fermentations were controlled at a target respiratory quotient (RQ) of 1.4, with excellent performance, using an innovative automated control based on the stirring rate as the manipulated variable developed during this study. The results conclude that oxygen limitation positively affects bioprocess efficiency under all growing conditions compared. The shift from respiratory to respiro-fermentative metabolism increases bioprocess productivity by up to twofold for the specific growth rates evaluated. Moreover, the specific product generation rate (qp ) increases linearly with μ, regardless of oxygen availability. Furthermore, this hypoxic boosting effect was also observed in the production of Candida antarctica lipase B (CalB) and pro-Rhizopus oryzae lipase (proRol), thus proving the synergic effect of kinetic and physiological stress control. Finally, the Crl1 production scale-up was conducted successfully, confirming the strategy's scalability and the robustness of the results obtained at the bench-scale level.

Antigen surface display in two novel whole genome sequenced food grade strains, Lactiplantibacillus pentosus KW1 and KW2

BackgroundUtilization of commensal bacteria for delivery of medicinal proteins, such as vaccine antigens, is an emerging strategy. Here, we describe two novel food-grade strains of lactic acid bacteria, Lactiplantibacillus pentosus KW1 and KW2, as well as newly developed tools for using this relatively unexplored but promising bacterial species for production and surface-display of heterologous proteins.ResultsWhole genome sequencing was performed to investigate genomic features of both strains and to identify native proteins enabling surface display of heterologous proteins. Basic characterization of the strains revealed the optimum growth temperatures for both strains to be 35–37 °C, with peak heterologous protein production at 33 °C (KW1) and 37 °C (KW2). Negative staining revealed that only KW1 produces closely bound exopolysaccharides. Production of heterologous proteins with the inducible pSIP-expression system enabled high expression in both strains. Exposure to KW1 and KW2 skewed macrophages toward the antigen presenting state, indicating potential adjuvant properties. To develop these strains as delivery vehicles, expression of the mycobacterial H56 antigen was fused to four different strain-specific surface-anchoring sequences.ConclusionAll experiments that enabled comparison of heterologous protein production revealed KW1 to be the better recombinant protein production host. Use of the pSIP expression system enabled successful construction of L. pentosus strains for production and surface display of an antigen, underpinning the potential of these strains as novel delivery vehicles.

Effect of biotin starvation on gene expression in <i>Komagataella phaffii</i> cells

Methylotrophic yeast Komagataella phaffii are widely used in biotechnology as a host for recombinant protein production. Due to the practical significance of this yeast, it is extremely important to properly select the cultivation conditions and optimize the media composition. In this study the effect of biotin starvation on K. phaffii gene expression was investigated at transcriptomic level. It was demonstrated, that K. phaffii cell response to biotin deficiency strongly depends on the carbon source in the medium. In media containing glycerol, biotin deficiency led to activation of genes involved in biotin metabolism, glyoxylate cycle and synthesis of acetyl-CoA in cytoplasm, as well as repression of genes, involved in lipo- and gluconeogenesis. In methanol containing media, biotin deficiency primarily led to repression of genes, involved in protein synthesis, and activation of cell response to oxidative stress.

Genome-wide identification and in-silico analysis of papain-family cysteine protease encoding genes in Tetrahymena thermophila

Tetrahymena thermophila is a promising host for recombinant protein production, but its utilization in biotechnology is mostly limited due to the presence of intracellular and extracellular papain-family cysteine proteases (PFCPs). In this study, we employed bioinformatics approaches to investigate the T. thermophila PFCP genes and their encoded proteases (TtPFCPs), the most prominent protease family in the genome. Results from the multiple sequence alignment, protein modeling, and conserved motif analyses revealed that all TtPFCPs showed considerably high homology with mammalian cysteine cathepsins and contained conserved amino acid motifs. The total of 121 TtPFCP-encoding genes, 14 of which were classified as non-peptidase homologs, were found. Remaining 107 true TtPFCPs were divided into four distinct subgroups depending on their homology with mammalian lysosomal cathepsins: cathepsin L-like (TtCATLs), cathepsin B-like (TtCATBs), cathepsin C-like (TtCATCs), and cathepsin X-like (TtCATXs) PFCPs. The majority of true TtPFCPs (96 out of the total) were in TtCATL-like peptidase subgroup. Both phylogenetic and chromosomal localization analyses of TtPFCPs supported the hypothesis that TtPFCPs likely evolved through tandem gene duplication events and predominantly accumulated on micronuclear chromosome 5. Additionally, more than half of the identified TtPFCP genes are expressed in considerably low quantities compared to the rest of the TtPFCP genes, which are expressed at a higher level. However, their expression patterns fluctuate based on the stage of the life cycle. In conclusion, this study provides the first comprehensive in-silico analysis of TtPFCP genes and encoded proteases. The results would help designing an effective strategy for protease knockout mutant cell lines to discover biological function and to improve the recombinant protein production in T. thermophila.

Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains.

Komagataella phaffii (Pichia pastoris) is a widely known microbial host for recombinant protein production and an emerging model organism in fundamental research. The development of new materials and techniques on this yeast improves heterologous protein synthesis. One of the most prominent ways to enhance protein production efficiency is to select K. phaffii strains with multiple expression cassettes and generate MutS strains using various vectors. In this study, we demonstrate approaches to expand the applications of pPICZ series vectors. Procedures based on PCR amplification and in vivo cloning allow rapid exchange of selectable markers. The combination of PCR amplification with split-marker-mediated transformation allows the development of K. phaffii MutS strains with two expression cassettes using pPICZ vectors. Both PCR-based approaches were applied to efficiently produce interleukin-2 mimetic Neo-2/15 in K. phaffii. The described techniques provide alternative ways to generate and improve K. phaffii strains without the need for obtaining new specific vectors or additional cloning of expression cassettes.

Heterologous Expression of Plantaricin 423 and Mundticin ST4SA in Saccharomyces cerevisiae

Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX). The native and codon-optimised variants of the plaA and munST4SA genes were cloned into episomal expression vectors containing either the S. cerevisiae alpha mating factor (MFα1) or the Trichoderma reesei xylanase 2 (XYNSEC) secretion signal sequences. The recombinant peptides retained their activity and stability, with the MFα1 secretion signal superior to the XYNSEC secretion signal for both bacteriocins. An eight-fold increase in activity against Listeria monocytogenes was observed for MunX after codon optimisation, but not for PlaX-producing strains. After HPLC-purification, the codon-optimised genes yielded 20.9 mg/L of MunX and 18.4 mg/L of PlaX, which displayed minimum inhibitory concentrations (MICs) of 108.52 nM and 1.18 µM, respectively, against L. monocytogenes. The yields represent a marked improvement relative to an Escherichia coli expression system previously reported for PlaX and MunX. The results demonstrated that S. cerevisiae is a promising host for recombinant bacteriocin production that requires a simple purification process, but the efficacy is sensitive to codon usage and secretion signals.

Developing a metabolic model‐based fed‐batch feeding strategy for Pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway

Pichia pastoris is a commonly used microbial host for recombinant protein production. It is mostly cultivated in fed-batch mode, in which the environment of the cell is continuously changing. Hence, it is vital to understand the influence of feeding strategy parameters on the intracellular reaction network to fine-tune bioreactor performance. This study used dynamic flux balance analysis (DFBA) integrated with transcriptomics data to simulate the recombinant P. pastoris (Muts ) growth during the induction phase for three fed-batch strategies, conducted at constant specific growth rates (μ-stat). The induction phase was split into equal time intervals, and the correlated reactions with protein yield were identified in the three fed-batch strategies using the Pearson correlation coefficient. Subsequently, principal component analysis (PCA) was applied to cluster induction phase time intervals and identify the role of correlated reactions on metabolic differentiation of time intervals. It was found that increasing fluxes through the methanol dissimilation pathway increased protein yield. By adding a methanol assimilation pathway inhibitor (HgCl2 ) to the shake flask medium growing on glycerol: methanol mixture (10%: 90%, v/v), the protein titre increased by 60%. As per DFBA, the higher the methanol to biomass flux ratio (Rmeoh/Δx ), the higher the protein yield. Finally, a novel feeding strategy was developed to increase the amount of Rmeoh/Δx compared to the three feeding strategies. The concentration of recombinant human growth hormone (rhGH), used as the model protein, increased by 16% compared to the optimal culture result obtained previously (800 mg L-1 to 928 mg L-1 ), while production yield improved by 85% (24.8 mg gDCW -1 to 46 mg gDCW -1 ).

Central carbon metabolite profiling reveals vector-associated differences in the recombinant protein production host Escherichia coli BL21

The Gram-negative bacterium Escherichia coli is the most widely used host for recombinant protein production, both as an industrial expression platform and as a model system at laboratory scale. The recombinant protein production industry generates proteins with direct applications as biopharmaceuticals and in technological processes central to a plethora of fields. Despite the increasing economic significance of recombinant protein production, and the importance of E. coli as an expression platform and model organism, only few studies have focused on the central carbon metabolic landscape of E. coli during high-level recombinant protein production. In the present work, we applied four targeted CapIC- and LC-MS/MS methods, covering over 60 metabolites, to perform an in-depth metabolite profiling of the effects of high-level recombinant protein production in strains derived from E. coli BL21, carrying XylS/Pm vectors with different characteristics. The mass-spectrometric central carbon metabolite profiling was complemented with the study of growth kinetics and protein production in batch bioreactors. Our work shows the robustness in E. coli central carbon metabolism when introducing increased plasmid copy number, as well as the greater importance of induction of recombinant protein production as a metabolic challenge, especially when strong promoters are used.

Promoters and introns as key drivers for enhanced gene expression in Saccharomyces cerevisiae.

The transcription of genes in the yeast Saccharomyces cerevisiae is governed by multiple layers of regulatory elements and proteins, cooperating to ensure optimum expression of the final protein product based on the cellular requirements. Promoters have always been regarded as the most important determinant of gene transcription, but introns also play a key role in the expression of intron-encoding genes. Some introns can enhance transcription when introduced either promoter-proximal or embedded in the open reading frame of genes. However, the outcome is seldom predictable, with some introns increasing or decreasing transcription depending on the promoter and reporter gene employed. This chapter provides an overview of the general structure and function of promoters and introns and how they may cooperate during transcription to allow intron-mediated enhancement of gene expression. Since S. cerevisiae is a suitable host for recombinant protein production on a commercial level, stronger and more controllable promoters are in high demand. Enhanced gene expression can be achieved via promoter engineering, which may include introns that increase the efficacy of recombinant expression cassettes. Different models for the role of introns in transcription are briefly discussed to show how these intervening sequences can actively interact with the transcription machinery. Furthermore, recent examples of improved protein production via the introduction of promoter-proximal introns are highlighted to showcase the potential value of intron-mediated enhancement of gene expression.

Advances in Synthetic Biology Tools and Engineering of Corynebacterium glutamicum as a Platform Host for Recombinant Protein Production

Advances in Synthetic Biology Tools and Engineering of Corynebacterium glutamicum as a Platform Host for Recombinant Protein Production

Insights into the quality of recombinant proteins produced by two different Bombyx mori expression systems

The silkworm, Bombyx mori, is an attractive host for recombinant protein production due to its high expression efficiency, quality, and quantity. Two expression systems have been widely used for recombinant protein production in B. mori: baculovirus/silkworm expression system and transgenic silkworm expression system. Both expression systems enable high protein production, but the qualities of the resulting recombinant proteins have not been well evaluated. In this study, we expressed bovine interferon γ (IFN-γ) using the two systems and examined the quality of the resulting proteins in terms of N-glycosylation and protein cleavage. Both expression systems successfully produced IFN-γ as an N-glycoprotein. Although the production in the baculovirus/silkworm expression system was much more efficient than that in the transgenic silkworm expression system, unexpected variants of IFN-γ were also produced in the former system due to the different N-glycosylation and C-terminal truncations. These results indicate that while high protein production could be achieved in the baculovirus/silkworm expression system, unintentional protein modification might occur, and therefore protein expression in the transgenic silkworm expression system is preferable from the point-of-view of N-glycosylation of the recombinant protein and evasion of unexpected attack by a protease in B. mori.

Second-Generation Escherichia coli SuptoxR Strains for High-Level Recombinant Membrane Protein Production.

Escherichia coli is one of the most widely utilized hosts for recombinant protein production, including that of membrane proteins (MPs). We have recently engineered a specialized E. coli strain for enhanced recombinant MP production, termed SuptoxR. By appropriately co-expressing the effector gene rraA, SuptoxR can suppress the high toxicity, which is frequently observed during the MP-overexpression process, and, at the same time, enhance significantly the cellular accumulation of membrane-incorporated and properly folded recombinant MP. The combination of these two beneficial effects results in dramatically enhanced volumetric yields for various prokaryotic and eukaryotic MPs. Here, we engineered second-generation SuptoxR strains with further improved properties, so that they can achieve even higher levels of recombinant MP production. We searched for naturally occurring RraA variants with similar or improved MP toxicity-suppressing and production-promoting effects to that of the native E. coli RraA of the original SuptoxR strain. We found that the RraA proteins from Proteus mirabilis and Providencia stuartii can be even more potent enhancers of MP productivity than the E. coli RraA. By exploiting these two newly identified RraAs, we constructed two second-generation SuptoxR strains, termed SuptoxR2.1 and SuptoxR2.2, whose MP-production capabilities often surpass those of the original SuptoxR significantly. SuptoxR2.1 and SuptoxR2.2 are expected to become widely useful expression hosts for recombinant MP production in bacteria.

Predicting high recombinant protein producer strains of Pichia pastoris MutS using the oxygen transfer rate as an indicator of metabolic burden

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a widely used host for recombinant protein production. In this study, a clonal library of P. pastoris MutS strains (S indicates slow methanol utilization) was screened for high green fluorescent protein (GFP) production. The expression cassette was under the control of the methanol inducible AOX promoter. The growth behavior was online-monitored in 48-well and 96-well microtiter plates by measuring the oxygen transfer rate (OTR). By comparing the different GFP producing strains, a correlation was established between the slope of the cumulative oxygen transfer during the methanol metabolization phase and the strain’s production performance. The correlation corresponds to metabolic burden during methanol induction. The findings were validated using a pre-selected strain library (7 strains) of high, medium, and low GFP producers. For those strains, the gene copy number was determined via Whole Genome Sequencing. The results were consistent with the described OTR correlation. Additionally, a larger clone library (45 strains) was tested to validate the applicability of the proposed method. The results from this study suggest that the cumulative oxygen transfer can be used as a screening criterion for protein production performance that allows for a simple primary screening process, facilitating the pre-selection of high producing strains.

One-Step In Vivo Assembly of Multiple DNA Fragments and Genomic Integration in Komagataella phaffii.

The methylotrophic yeast species Komagataella phaffii (synonym: Pichia pastoris) is widely used as a host for recombinant protein production. Although several genetic engineering techniques are being employed on K. phaffii, advanced methods such as in vivo DNA assembly in this yeast species are required for synthetic biology applications. In this study, we established a technique for accomplishing one-step in vivo assembly of multiple DNA fragments and genomic integration in K. phaffii. To concurrently achieve an accurate multiple DNA assembly and a high-efficient integration into the target genomic locus in vivo, a K. phaffii strain, lacking a non-homologous end joining-related protein, DNA ligase IV (Dnl4p), that has been reported to improve gene targeting efficiency by homologous recombination, was used. Using green fluorescent protein along with the lycopene biosynthesis, we showed that our method that included a Dnl4p-defective strain permits direct and easy engineering of K. phaffii strains.

Enhancing Extracellular Pullulanase Production in Bacillus subtilis Through dltB Disruption and Signal Peptide Optimization.

Bacillus subtilis has many attributes that make it a popular host for recombinant protein production. Although its protein production ability has been enhanced through protease gene disruption, residual proteases like quality control HtrA and HtrB can limit protein yield by degrading inadequately folded proteins present during overexpression. In this study, two strategies were employed to increase production of industrial enzyme pullulanase: enhancing extracellular pullulanase folding and optimizing its signal peptide. The hypothesis was that disruption of dltB gene of expression host B. subtilis WS9 would enhance recombinant extracellular folding by increasing cation binding to the cell's outer envelope. Consistent with this hypothesis, disrupting dltB enhanced pullulanase production by 49% in shake-flask cultures. The disruption also enhanced extracellular α-CGTase and β-CGTase production by 25% and 35%, respectively. Then, more effective signal peptide for pullulanase production was identified through high-throughput screening of 173 unique B. subtilis signal peptides. Replacing the native signal peptide of pullulanase with that encoded by ywtF increased extracellular pullulanase activity by an additional 12%. Three-liter fermenter scale-up production yielded the highest extracellular pullulanase activity reported to date: 8037.91 U·mL-1. This study highlights the usefulness of dltB deletion and signal peptide optimization in enhancing extracellular protein production.

Evolving Escherichia coli Host Strains for Efficient Deuterium Labeling of Recombinant Proteins Using Sodium Pyruvate-d3.

Labeling of proteins with deuterium (2H) is often necessary for structural biology techniques, such as neutron crystallography, NMR spectroscopy, and small-angle neutron scattering. Perdeuteration in which all protium (1H) atoms are replaced by deuterium is a costly process. Typically, expression hosts are grown in a defined medium with heavy water as the solvent, which is supplemented with a deuterated carbon source. Escherichia coli, which is the most widely used host for recombinant protein production, can utilize several compounds as a carbon source. Glycerol-d8 is often used as a carbon source for deuterium labelling due to its lower cost compered to glucose-d7. In order to expand available options for recombinant protein deuteration, we investigated the possibility of producing a deuterated carbon source in-house. E. coli can utilize pyruvate as a carbon source and pyruvate-d3 can be made by a relatively simple procedure. To circumvent the very poor growth of E. coli in minimal media with pyruvate as sole carbon source, adaptive laboratory evolution for strain improvement was applied. E. coli strains with enhanced growth in minimal pyruvate medium was subjected to whole genome sequencing and the genetic changes were revealed. One of the evolved strains was adapted for the widely used T7 RNA polymerase overexpression systems. Using the improved strain E. coli DAP1(DE3) and in-house produced deuterated carbon source (pyruvic acid-d4 and sodium pyruvate-d3), we produce deuterated (>90%) triose-phosphate isomerase, at quantities sufficient enough for large volume crystal production and subsequent analysis by neutron crystallography.

An overview and future prospects of recombinant protein production in Bacillus subtilis.

Bacillus subtilis is a well-characterized Gram-positive bacterium and a valuable host for recombinant protein production because of its efficient secretion ability, high yield, and non-toxicity. Here, we comprehensively review the recent studies on recombinant protein production in B. subtilis to update and supplement other previous reviews. We have focused on several aspects, including optimization of B. subtilis strains, enhancement and regulation of expression, improvement of secretion level, surface display of proteins, and fermentation optimization. Among them, optimization of B. subtilis strains mainly involves undirected chemical/physical mutagenesis and selection and genetic manipulation; enhancement and regulation of expression comprises autonomous plasmid and integrated expression, promoter regulation and engineering, and fine-tuning gene expression based on proteases and molecular chaperones; improvement of secretion level predominantly involves secretion pathway and signal peptide screening and optimization; surface display of proteins includes surface display of proteins on spores or vegetative cells; and fermentation optimization incorporates medium optimization, process condition optimization, and feeding strategy optimization. Furthermore, we propose some novel methods and future challenges for recombinant protein production in B. subtilis.Key points• A comprehensive review on recombinant protein production in Bacillus subtilis.• Novel techniques facilitate recombinant protein expression and secretion.• Surface display of proteins has significant potential for different applications.

Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens

Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of ‘difficult-to-express’ bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd.’s Clostridium difficile vaccine programme. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. Further, proteolytic cleavage of Ant2-3 was observed. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. However, screening of different buffer/additives showed that the addition of 1–15 mM dithiothreitol alone decreased the formation of insoluble aggregates and improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3, and solubility-based prediction tools were employed to determine the role of hydrophobicity and charge on protein production. The results showed that a large non-polar region (containing hydrophobic amino acids) was detected on the surface of Ant2 structures, whereas positively charged regions (containing lysine and arginine amino acids) were observed for Ant3, both of which were associated with poor protein solubility. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression and stability of single and potentially multi-domain (or fusion) antigens in bacterial expression systems.

Enhancing extracellular production of recombinant proteins in Escherichia coli by co-expressing with a haloarchaeal protein containing a putative LolA-like domain.

Escherichia coli represents one of the most widely used hosts for recombinant protein production, but its limited capacity for producing extracellular proteins is often cited as a drawback. NJ7G_0991 is an extracellular protein of the haloarchaeon Natrinema sp. J7-2 and comprises a signal peptide, a putative LolA-like domain, and a C-terminal domain of unknown function. Here, we found that the full-length (0991) and the C-terminal domain-deletion variant (0991ΔC) of NJ7G_0991, but not its signal peptide-deletion variant (0991ΔS), were efficiently released into the culture supernatant of E. coli without extensive cell lysis as determined by β-galactosidase activity assay. After lysozyme treatment, E. coli cells producing 0991 or 0991ΔC, but not 0991ΔS, were converted from rod-shaped forms to spheres, suggesting that the secretion of 0991 or 0991ΔC into the periplasm leads to an increase of outer membrane permeability of E. coli. A pelB signal peptide was fused to the N-terminus of the LolA-like domain, and the resulting variant PelB-0991ΔC could be released into the culture supernatant of E. coli more efficiently than 0991ΔC. By using PelB-0991ΔC as a co-expression partner, the extracellular production level of a recombinant thermostable subtilase WF146 could be enhanced by up to 14-fold, and the extracellular concentration of an active site variant of WF146 (WF146-SA) reached up to 129 mg/l. To the best of our knowledge, this is the first report on archaeal protein-based co-expression system for extracellular production of recombinant proteins in E. coli. KEY POINTS: • The haloarchaeal protein NJ7G_0991 can be efficiently released into the culture supernatant of E. coli. • The recombinant NJ7G_0991 increases the outer membrane permeability of E. coli. • The LolA-like domain of NJ7G_0991 can be used as a co-expression partner to improve extracellular production of recombinant proteins in E. coli.