Construction of an expression system for aqualysin I in Escherichia coli that gives a markedly improved yield of the enzyme protein

An expression system for aqualysin I from Thermus aquaticus YT-1, a thermophilic serine protease belonging to the proteinase K family, in Escherichia coli is available, but the efficiency of production has been rather low for detailed analysis of the product. We developed a maltose biding protein (MBP)-fused proaqualysin I expression plasmid (pMAQ-c2Delta) in which MBP is attached to the N-terminus of proaqualysin I. MBP appeared effectively to suppress the folding-promoting activity of the N-terminal propeptide when the bacteria were grown at 30 degrees C, leading to a massive accumulation of fusion aqualysin I precursor. The precursor was converted efficiently to mature aqualysin I by heat treatment at 70 degrees C, enabling us to obtain 40 times more aqualysin I than is available using expression systems such as pAQNDeltaC105. By analyzing the product of the pMAQ-c2Delta-derived inactive mutant expression vector, pMAQ-S222A, it was confirmed that aqualysin I was initially expressed as a whole fusion protein and then processed autocatalytically.     

A 38 kDa precursor protein of aqualysin I (a thermophilic subtilisin-type protease) with a C-terminal extended sequence: its purification and in vitro processing

The precursor of aqualysin I, an extracellular subtilisin-type protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, a protease domain, and a C-terminal extended sequence. In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C-terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65°C. The 38 kDa precursor protein is separated as a soluble form from denatured E. coli proteins after heat treatment. Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography. The purified precursor protein gave a single band on SDS-polyacrylamide gels. The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The purified precursor protein was processed to the mature enzyme upon heat treatment. The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C-terminal extended sequence occurs through an intermolecular self-processing mechanism.     

Role of the C-terminal pro-domain of aqualysin I in the maturation and translocation of the precursor across the cytoplasmic membrane in Escherichia coli

Aqualysin I, which is a subtilisin-type, extracellular protease secreted by Thermus aquaticus YT-1, is synthesized as a unique precursor bearing pro-domains at both N- and C-terminus of the mature protease domain as well as an N-terminal signal peptide. To investigate the function of the C-terminal pro-domain in maturation and export pathway of the precursor in E. coli cells, aqualysin I variants were constructed in which deletion mutants of the C-terminal pro-domain lacking its own signal peptide were inserted into pIN-III-ompA3. When E. coli harboring wild type and mutant plasmids were induced by 0.2 mM IPTG, active aqualysin I was produced by heat treatment at 65 °C. Aqualysin I precursors with deletions of more than 5 amino acid residues at the C-terminal end of pro-domain were much more rapidly processed than that of wild type, indicating that the C-terminal pro-domain functions as a inhibitor for processing of aqualysin I precursor. With the wild type, most of aqualysin I was present in membrane fraction (probably the outer membrane), whereas for the truncated mutants, it remained in the cytoplasm, indicating that for deletion mutants, their precursors expressed in cells were not translocated across the cytoplasmic membrane, despite the existence of an N-terminal signal peptide.     

Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.     

Fusion with maltose-binding protein (MBP) affects neither RNA N-glycosidase activity nor immunogenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica

A genomic clone encoding mature karasurin-A (KRNA), a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica, was efficiently expressed in E. coli using an expression cassette vector pMAL-c2. The resultant recombinant KRNA fused with maltose-binding protein (MBP) was recovered from the soluble fraction of the bacterial cells and purified to near homogeneity after one round of the affinity chromatography. Neither the karasurin precursor retaining both N- and C-terminal peptides, nor the protein with the N-terminal peptide was successufully produced even as a MBP-fusion. The protein with its C-terminal peptide was over-produced but was recovered in an insoluble fraction. Both the recombinant MBP-KRNA fusion protein and recombinant KRNA with MBP removed were as active as the native KRNA from root tubers. The immunogenicity of the recombinant KRNA was also unaffected by fusion with MBP.     

Production and Extracellular Secretion of Aqualysin I (a Thermophilic Subtilisin-Type Protease) in a Host-Vector System for Thermus thermophilus

Aqualysin I is synthesized as a large precursor, processed, and secreted into the culture medium by Thermus aquaticus YT-1. An expression plasmid for the aqualysin I gene in T. thermophilus HB27 was constructed. T. thermophilus cells harboring the recombinant plasmid produced correctly processed aqualysin I, and the mature enzyme was secreted into the culture medium.     

Efficient production of Thermus protease aqualysin I in Escherichia coli: effects of cloned gene structure and two-stage culture

 The DNA sequence encoding Thermus protease aqualysin I was inserted downstream from a bacteriophage T7 promoter in an expression vector. In the T7 expression system, using a strain lacking an F′ episome, aqualysin I was produced in soluble form without chemical induction. The deletions of part (30 amino acid residues) or all (105 residues) of the C-terminal pro-sequence from the C terminus significantly affected both cellular growth and the production of the enzyme. Complete deletion adversely affected both. In contrast, the 30-residue deletion markedly improved productivity by approximately four times compared to non-deletion, and shortened the time needed for the activation of a precursor to active enzyme. The concentration of inducer isopropyl β-D-thiogalactopyrano-side (IPTG) was varied to examine its effects, and it was found that a low concentration of IPTG improved aqualysin I production. To avoid the inhibitory effects of acetic acid accumulation in the culture medium, the use of other carbon sources besides glucose was examined. When cells were cultivated with glycerol, the acetic acid level remained relatively low, and both good cellular growth and a high level of production were attained. The aqualysin I productivity for a fed-batch culture using two carbon sources, glucose and glycerol, reached more than 150 kU/ml enzymatically active aqualysin I. Received: 19 May 1995/Received revision: 28 July 1995/Accepted: 22 August 1995     

Role of the COOH-terminal pro-sequence of aqualysin I (a heat-stable serine protease) in its extracellular secretion by Thermus thermophilus

Aqualysin I is a subtilisin-type serine protease secreted into the medium by Thermus aquaticus YT-1. Thermus thermophilus cells harboring a plasmid for the aqualysin I precursor secreted pro-aqualysin I with the C-terminal pro-sequence into the culture medium, and the precursor was then processed to the mature enzyme during the cultivation. However, the extracellular levels of aqualysin I in T. thermophilus cells harboring plasmids for deletion mutants as to the C-terminal pro-sequence were about 10–20% in comparison with the level of wild-type. Only the mature enzyme could be detected in the medium, while pro-aqualysin I with the C-terminal pro-sequence could not. These results suggest that the C-terminal pro-sequence of aqualysin I plays an important role in the extracellular secretion of aqualysin I.     

Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase

By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60°C in either single or fusion form can be completely recovered by treatment at 0°C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (R _gfp/A _xyl) at 15°C and 37°C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the R _gfp/A _xyl ratio at 15°C is ten times of that at 37°C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.     

Overexpression of Rhodobacter sphaeroides PufX-bearing maltose-binding protein and its effect on the stability of reconstituted light-harvesting core antenna complex

The PufX protein, encoded by the pufX gene of Rhodobacter sphaeroides, plays a key role in the organization and function of the core antenna (LH1)-reaction centre (RC) complex, which collects photons and triggers primary photochemical reactions. We synthesized a PufX/maltose-binding protein (MBP) fusion protein to study the effect of the PufX protein on the reconstitution of B820 subunit-type and LH1-type complexes. The fusion protein was synthesized using an Escherichia coli expression system and purified by affinity chromatography. Reconstitution experiments demonstrated that the MBP-PufX protein destabilizes the subunit-type complex (20°C), consistent with previous reports. Interestingly, however, the preformed LH1-type complex was stable in the presence of MBP-PufX. The MBP-PufX protein did not influence the preformed LH1-type complexes (4°C). The LH1-type complex containing MBP-PufX showed a unique temperature-dependent structural transformation that was irreversible. The predominant form of the complex at 4°C was the LH1-type. When shifted to 20°C, subunit-type complexes became predominant. Upon subsequent cooling back to 4°C, instead of re-forming the LH1-type complexes, the predominant form remained the subunit-type complexes. In contrast, reversible transformation of LH1 (4°C) and subunit-type complexes (20°C) occurs in the absence of PufX. These results are consistent with the suggestion that MBP-PufX interacts with the LH1α- polypeptide in the subunit (α/β)-type complex (at 20°C), preventing oligomerization of the subunit to form LH1-type complexes.     

A non-covalent NH2-terminal pro-region aids the production of active aqualysin I (a thermophilic protease) without the COOH-terminal pro-sequence in Escherichia coli

The precursor of aqualysin I, an extracellular protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, the protease domain and a C-terminal pro-sequence. In an Escherichia coli expression system, mature and active aqualysin I is formed by treatment at 65 degrees C and the N-pro-sequence is required for its production. Complete deletion of the C-pro-sequence did not affect the production of active aqualysin I, indicating that the C-pro-sequence is not essential. A non-covalent N-pro-region was separately synthesized from the protease domain with or without the C-pro-sequence. In this system, mature and active aqualysin I was detected only when the C-pro-sequence was deleted.     

Improving aquaporin Z expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> by fusion partners and subsequent condition optimization

Aquaporin Z (AqpZ), a typical orthodox aquaporin with six transmembrane domains, was expressed as a fusion protein with TrxA in E. coli in our previous work. In the present study, three fusion partners (DsbA, GST and MBP) were employed to improve the expression level of this channel protein in E. coli. The result showed that, compared with the expression level of TrxA-AqpZ, five- to 40-fold increase in the productivity of AqpZ with fusion proteins was achieved by employing these different fusion partners, and MBP was the most efficient fusion partner to increase the expression level. By using E. coli C43 (DE3)/pMAL-AqpZ, the effects of different expression conditions were investigated systematically to improve the expression level of MBP-AqpZ in E. coli. The high productivity of MBP-AqpZ (200 mg/l) was achieved under optimized conditions. The present work provides a novel approach to improve the expression level of membrane proteins in E. coli.     

Optimization of Protease Extraction from Horse Mango (Mangifera foetida Lour) Kernels by a Response Surface Methodology

The thermophilic protease aqualysin I (AQI) gene (aqul), derived from Thermus aquaticus YT-I, was inserted under the control of the bacteriophage T7 promoter in an expression plasmid. The plasmid was introduced into two strains of E. coli JMI09 (DE3), one carrying and one lacking an F’ episome, which carries the lacI_q gene. Upon cultivation the strain carrying an F’ episome produced AQI as an insoluble fusion protein (74 kDa) with the T7 gene 10 protein. This insoluble protein could not be processed into mature AQI by heat treatment and thus it had no proteolytic activity. On the other hand, when the strain lacking an F’ episome was used as a host cell for aqul expression, non-induced, or leaky, expression occurred, and AQI was produced in a soluble form. This soluble protein could be processed into active AQI by heat treatment. Moreover, when a low concentration of IPTG (0.0125 mM) was added, the amount of active AQI was 2.7 times greater than that produced in a batch culture without induction.     

Expression and purification of recombinant proteins by fusion to maltose-binding protein

The pMAL vectors provide a method for purifying proteins from cloned genes by fusing them to maltose-binding protein (MBP, product of malE), which binds to amylose. The vectors use the tac promoter and the translation initiation signals of MBP to give high-level expression of the fusion, and an affinity purification for MBP to isolate the fusion protein. The pMAL polylinkers carry restriction sites to insert the gene of interest, and encode a site for a specific protease to separate MBP from the target protein after purification. Vectors with or without the malE signal sequence can be used, to express the protein cytoplasmically for the highest level of production or periplasmically to help in proper folding of disulfide-bonded proteins.     

High-Level Expression of Soluble Protein inEscherichia coliUsing a His6-Tag and Maltose-Binding-Protein Double-Affinity Fusion System

Using the maltose-binding protein (MBP) fusion vector pMAL-c1 from C. V. Mainaet al.(1988,Gene74, 365–373), we have constructed expression vectors which contain a sequence encoding six consecutive histidine residues (His₆-tag) at the 3′ end of the MBP-encoding malE gene which is followed by either a thrombin-binding site (LVPRGS) or a factor Xa-binding site (IEGR). The benefits of this approach include: (a) high expression levels of soluble MBP fusion proteins (exceeding 2% of the total cellular protein), (b) high-quality purification of proteins under various conditions (high salt, low salt, denaturing, nondenaturing, etc.), and (c) two alternative protease cleavage sites to test for the most efficient cleavage of each fusion protein. We also constructed these MBP–His₆-tag expression vectors with alternative selection markers (Amp^r, Kan^r) and alternative promoters (tac, T7). Using these constructs, we expressed and purified several proteins of which we present two, penicillin-binding protein PBP2a and UDP-N-acetylmuramate:l-alanine ligase (MurC), and compare their expression level and purity with other expression systems. We also discuss the use of minimal media with supplements versus rich media and cell growth strategies to optimize the protein yield in general and for isotope labeling.     

Stability of Thermostable Enzyme,Aqualysin I ; a Subtilisin-type Serine Protease from Thermus aquaticus YT-1

We characterized the heat stability and detergent stabilities of aqualysin I, produced by Thermus aquaticus YT-1, and compared them with those of fungal proteinase K and Bacillus subtilisin.

Aqualysin I displayed excellent heat and detergent stabilities. Proteinase K, another Cys-containing enzyme, was less stable than aqualysin I. All these enzymes maintained activities in the presence of urea or Tween-20.     

Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins

It is difficult to imagine any strategy for high-throughput protein expression and purification that does not involve genetically engineered affinity tags. Because of its ability to enhance the solubility and promote the proper folding of its fusion partners, Escherichia coli maltose-binding protein (MBP) is a particularly useful affinity tag. However, not all MBP fusion proteins bind efficiently to amylose resin, and even when they do it is usually not possible to obtain a sample of adequate purity after a single affinity step. To address this problem, we endeavored to incorporate supplemental affinity tags within the framework of an MBP fusion protein. We show that both the nature of the supplemental tags and their location can influence the ability of MBP to promote the solubility of its fusion partners. The most promising configurations for high-throughput protein expression and purification appear to be a fusion protein with a biotin acceptor peptide (BAP) on the N-terminus of MBP and/or a hexahistidine tag (His-tag) on the C-terminus of the passenger protein. Abbreviatoins: BAP, biotin acceptor peptide; EDTA, ethelenediaminetetraacetic acid; IPTG, isopropyl--d-thiogalactopyranoside; MBP, E. coli maltose-binding protein; GFP; green fluorescent protein; Ni-NTA, nickel-nitrilotriacetic acid; ORF, open reading frame; PCR; polymerase chain reaction; R5, polyarginine tag; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TEV, tobacco etch virus; WT, wild-type