Introduction

Streptomyces are aerobic, filamentous Gram-positive soil bacteria, which produce more than half of the known antibiotics (Hopwood 1999). In addition, living in the soil, Streptomyces secrete many extracellular enzymes to decompose multiple substrates for facilitating their survival (Paradkar et al. 2003). The efficient protein secretion mechanism in Streptomyces has made them very attractive hosts for heterologous protein production (Vrancken and Anne 2009). During the last 20 years, there are many prokaryotic and eukaryotic proteins successfully expressed in Streptomyces, such as Mycobacterium tuberculosis alanine- and proline-rich antigenic protein (APA) (Vallin et al. 2006) and mouse TNF-α (Lammertyn et al. 1997). However, there are still a number of heterologous proteins especially eukaryotic proteins which are produced in Streptomyces only at very low levels, or not at all. For this reason, there is a need for further optimization of this expression system, such as new expression/secretion vectors and adaptation of codon usage of foreign genes.

The interest in the application of Streptomyces as host for the production of heterologous proteins has stimulated the development of an array of expression vectors, which mainly are based on plasmids with a high copy number. Many Streptomyces vectors including pIJ702 are derived from pIJ101, a high copy number plasmid present in S. lividans ISP5434 (Kieser et al. 1982). Herai et al. (2004) reported a new pIJ101-derived P nitA -NitR expression vector, in which expression of the gene of interest can be regulated by an inducer ε-caprolactam. In addition, Hatanaka et al. (2008) reported a pIJ101-derived E. coli-Streptomyces shuttle vector pTONA5, a hyperexpression vector in which a metalloendopeptidase (SSMP) promoter isolated from S. cinnamoneus TH-2 was used. Although some plasmids from other sources such as pFD666 have also been used for heterologous expression (Fink et al. 1991), new expression vectors with integrated features for secretory production of foreign proteins are still being pursued.

pSGL1 is a 7.4 kb plasmid isolated from S. globisporus C-1027 (Li and Li 1992), and its minimal replicon was identified to be within a 2 kb fragment (Hong and Li 1998). pSGL1 and its derived plasmids are compatible with pIJ101-derived plasmids and have a high copy number in Streptomyces, reaching 70–250 copies per genome (Hong and Li 1998). This vector was already successfully used for the efficient expression and secretion of soluble human interleukin-4 receptor in S. lividans TK24 (Zhang et al. 2004). This work suggested that pSGL1 can be a promising skeleton for the construction of new Streptomyces expression vectors.

In this report, we describe the construction of novel E. coli–Streptomyces shuttle vectors pIMB2–4, which can be conveniently used for secretory production of heterologous protein. These vectors employed the replicon of pSGL1, together with a strong promoter, different signal peptides and multiple cloning sites (MCS). These vectors were tested for the production of human interleukin-6 (IL-6), a pleiotropic cytokine. Post-translational modifications such as glycosylation have been shown to have little effect on the bioactivity of IL-6 (Tonouchi et al. 1988), and as a consequence a protein suitable to be expressed in a prokaryotic system. However, when expressed in E. coli IL-6 was produced as inclusion bodies (Tonouchi et al. 1988 and our unpublished data). Therefore, we attempted to express human IL-6 in S. lividans TK24 using novel vectors constructed in this work. Results obtained suggested that pIMB2–4 might be useful new vectors for the secretory production of heterologous proteins in Streptomyces.

Materials and methods

Bacterial strains and growth conditions

E. coli DH5α was used as host for cloning purposes. E. coli ET12567/pUZ8002 (Kieser et al. 2000) was used to transfer DNA into S. lividans by conjugation. Strains were grown either on solid or in liquid Luria–Bertani (LB) medium at 37°C. When applicable, antibiotics were used to select recombinant E. coli strains: 100 μg ampicillin (Ap) ml−1, 50 μg kanamycin (Km) ml−1, 25 μg chloramphenicol (Cm) ml−1 or 50 μg apramycin (Am) ml−1. S. lividans TK24 was used as host strain for heterologous protein production. S. lividans TK24 and its derivatives were grown at 28°C on R2 agar (Kieser et al. 2000) for sporulation, mannitol soya flour (MS) agar (Kieser et al. 2000) for conjugation, in trypticase soy broth (TSB, BD) for isolation of genomic or plasmid DNA. When appropriate, apramycin was added at final concentrations of 50 μg ml−1 to solid medium, and at 10 μg ml−1 to liquid medium. For the production of heterologous protein, a preculture of S. lividans harboring expression plasmid was grown in 50 ml CM medium (Zhang et al. 2004; Qi et al. 2008) at 28°C, 220 rpm, for 48 h. About 30 glass beads (D = 3 mm) were added in the shake-flask to improve the condition of cell growth. 5 ml of this pre-culture was then inoculated into 50 ml of fresh CM medium, and the supernatant was harvest at the indicated time by centrifugation at 3,000 rpm for 20 min.

Construction of secretory expression vectors

The minimal replicon of pSGL1 (Li and Li 1992) was digested with SalI and SacI and subcloned into the SalI–SacI digested pUC19E (Supplementary Table 1). The resulted plasmid pUC19E-SG was digested with SacI and PstI to obtain the fragment containing the minimal replicon of pSGL1. pOJ446 (Kieser et al. 2000) was digested with EcoRI and then partially digested with PstI to obtain the fragment containing oriT of RK2 for conjugation and aac(3)IV gene for apramycin resistance. pBluescript II KS(+) (Stratagene) was digested with SacI and EcoRI to obtain the fragment carrying ori (pUC) and ampicillin resistance gene bla. These three fragments were ligated together to generate pIMB1.

The ermE* promoter (Bibb et al. 1994) fragment was amplified using primers 1 and 2 (Supplementary Table 2) from pL646 plasmid (Hong et al. 2007), and then ligated into the pGEM-T (Promega) resulting in pGEM-ermE*p. The fragment containing the melC1 (the first ORF in the melanin operon of S. antibioticus) gene (Bernan et al. 1985) including the promoter melC1p and signal sequence SP MelC1 was amplified with primers 3 and 4 (Supplementary Table 2) from pUC19-melC1 (Hong et al. 2003), and then ligated into pGEM-T resulting in pGEM-melC1p-SP MelC1. ermE*p fragment from pGEM-ermE*p and melC1p-SP MelC1 fragment from pGEM-melC1p-SP MelC1 were ligated together into the HindIII–EcoRI gap of pIMB1 resulting in plasmid pIMB2. The multiple cloning sites (MCS) of pIMB2, including NdeI, SacII, BglII, ClaI, BclI, EcoRV and EcoRI, was acquired through incorporation of primer 4 (Supplementary Table 2). The fragment containing cagA [an apoprotein gene in S. globisporus C-1027 (Sakata et al. 1992)] promoter cagAp and its wild type signal sequence SP CagA(TTA) was amplified from S. globisporus C-1027 total DNA with primer 5 and 6 (Supplementary Table 2), and ligated into pGEM-T resulting in pGEM-cagAp-SP CagA(TTA). The cagAp-SP CagA(TTA) fragment from pGEM-cagAp-SP CagA(TTA) and ermE*p fragment were ligated together into the HindIII-EcoRI gap of pIMB1 as such obtaining plasmid pIMB3. The rare leucine codon TTA in SP CagA(TTA) was replaced by the preferred codon CTG through overlapping PCR using primer 7 and 8 (Supplementary Table 2). The fragment including cagAp and mutated signal sequence SP CagA(CTG) were ligated together into the HindIII-EcoRI gap of pIMB1, resulting in plasmid pIMB4 similarly as pIMB3. Through incorporation of primer 6 pIMB3 and pIMB4 also obtained a MCS consisting of NdeI, SacII, EcoRV, BglII and EcoRI (Supplementary Table 2).

For cloning human IL-6 cDNA, total RNA of human peripheral blood mononuclear cells was extracted, and then was reversely transcribed into cDNA. Sequence for mature IL-6 coding region was amplified by PCR using primer 9 (Supplementary Table 2) containing the NdeI site and primer 10 (Supplementary Table 2) containing the BamHI site downstream of the stop codon of IL-6 coding region. The resultant fragments were cloned into pGEM-T and confirmed by sequencing. The IL-6 cDNA fragments were then digested with NdeI and BamHI, and then ligated into the NdeI-BglII (isocaudamer of BamHI) gaps of pIMB2, pIMB3 and pIMB4 respectively, resulting in IL-6 expression plasmids pIMB2-IL6, pIMB3-IL6 and pIMB4-IL6.

SDS-PAGE and Western blot analysis for recombinant IL-6

Crude fermentation sample and purified recombinant protein were detected by SDS-PAGE and Western blot. The samples were loaded onto 12% SDS-PAGE gels and protein bands were visualized with Coomassie brilliant blue R-250 after electrophoresis. For Western blotting, proteins were separated by 12% SDS-PAGE and then blotted onto PVDF membrane (Millipore) using a semi-dry electroblotter (Bio-Rad). IL-6 was detected using mouse monoclonal anti-human IL-6 antibody (R&D Systems). Alkaline phosphate (AP) conjugate horse anti-mouse IgG (H + L) (Zhongshan Jinqiao) was used as secondary antibody. Immunoreactive bands were visualized using NBT/BCIP (Promega) substrate for AP.

ELISA of recombinant IL-6

The human IL-6 Quantikine immunoassay kit (R&D Systems), which employed the quantitative sandwich enzyme immunoassay technique, was used to detect IL-6 in the culture medium. A monoclonal antibody specific for IL-6 has been pre-coated onto a 96-well microplate. To each well, 100 μl of assay diluent which contains buffered protein, and 100 μl of IL-6 standard or sample were added. After incubation for 2 h at room temperature, the microplate was washed four times with 400 μl wash buffer. After adding 200 μl of IL-6 conjugate, which contained poly-antibody against IL-6 conjugated with horseradish peroxidase to each well, the microplate was incubated for 2 h at room temperature. The microplate was washed four times with 400 μl wash buffer, and then 200 μl of substrate solution was added to each well and kept at room temperature for 20 min. 50 μl of stop solution was added to each well, and the absorbance at 450 nm was recorded as measurement of the reaction.

Purification of the recombinant IL-6 protein

The ÄKTA explorer system (GE Healthcare) was used for the purification of the recombinant IL-6. The lyophilized protein sample obtained through ammonium sulfate precipitation (90% saturation) was dissolved in buffer A (50 mM Tris–HCl, pH 8.5). After filtration with 0.4 μm membrane, the sample was loaded on a 1 ml cation exchange resin Q sepharose fast flow (GE Healthcare) column which was pre-equilibrated with at least 5 column volumes of buffer A. The column was washed with 5 column volumes buffer A and then eluted with about 100 column volumes buffer B (50 mM Tris–HCl, 1 M NaCl, pH 8.5) using a stepwise gradient (0–100%, the concentration of buffer B was increased by 2% when the absorbance at 280 nm returned to the baseline level) with a flow rate at 0.5 ml/min. The recombinant IL-6 was eluted from the cation exchange column at 8% buffer B fractions monitored at 280 nm. The purity of the recombinant protein was evaluated by 12% SDS-PAGE followed by Coomassie brilliant blue staining. The recombinant protein was dissolved in buffer C (50 mM Tris–HCl, 150 mM NaCl, pH 8.5) and then loaded on a superdex™ 75 10/300 (GE Healthcare) gel filtration column which was pre-equilibrated with buffer C. The flow rate was maintained at 0.3 ml/min and the eluant was monitored at 280 nm. The purified product was lyophilized and kept at −20°C.

HPLC analysis of purified recombinant IL-6

Reversed-phase HPLC of recombinant IL-6 was performed with C-8 column (ZORBAX 300SB-C8, 4.6 × 50 mm, Agilent) using prominence LC-20A HPLC system (Shimadzu). A linear gradient of 20–80% (vol/vol) acetonitrile in water containing 0.1% (vol/vol) trifluoroacetic acid (TFA) for 30 min served as mobile phase at a flow rate of 1.0 ml/min. Chromatograms were recorded by UV detection at 280 nm.

N-terminal amino acid sequence analysis

The N-terminal amino acid residues of the purified recombinant IL-6 were identified by Edman degradation sequencing using Applied Biosystems Procise 491 (Applied Biosystems).

Biological activity assay

The purified recombinant IL-6 activity was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) proliferation assay using IL-6-dependent 7TD1 cells, which belong to murine B-cell hybridoma. 7TD1 cells (4 × 104 cells ml−1) were seeded into a 96-well plate and cultured in the presence of four-fold serial dilutions of the IL-6 samples at 37°C, and recombinant human IL-6 (R&D product) was applied as standard control. After culturing for 72 h, 20 μl MTT was added to each well, and crystals of formazan were dissolved with 100 μl 10% SDS-10 mM HCl 4 h later. Cell growth was assessed by the absorbance at 570 nm. The results were processed with four parameters regression method, and the recombinant IL-6 bioactivity was calculated by comparing with the standard curve.

Results

Construction of novel secretory expression vectors pIMB2–4

Fragments containing the replicons for plasmid maintenance in Streptomyces and E. coli, promoter, signal peptides and selection markers were incorporated to construct a novel series of E. coli–Streptomyces shuttle vectors pIMB2–4 (Fig. 1). The minimum replicon of pSGL1 was employed as Streptomyces replication origin, and the E. coli replication origin was obtained from plasmid pBluescript II KS(+) (Stratagene) for convenient gene cloning in E. coli. pIMB2–4 also contain the RK2 oriT obtained from pOJ446 (Kieser et al. 2000) which allows conjugation between E. coli and Streptomyces. In these vectors, ermE*p (Bibb et al. 1994) is used as a strong constitutive promoter for the expression of the target protein. The melC1 promoter-signal sequence (Bernan et al. 1985) was located downstream of ermE*p for protein secretion in pIMB2. As for pIMB3 and pIMB4, cagA promoter-signal sequence (Sakata et al. 1992) was employed in the same way as for pIMB2. The only difference between pIMB3 and 4 is that a rare codon TTA for leucine in the CagA signal peptide (SPCagA) coding sequence was changed to CTG by site-directed mutagenesis. Gene cloning may be facilitated with the introduced multiple cloning sites (MCS), which is located just downstream the signal peptide cleavage site. The vectors pIMB2–4 contain the aac(3)IV gene originating from pOJ446, giving apramycin resistance, and bla corresponding to ampicillin resistance from pBluescript II KS(+) for selection in Streptomyces and E. coli, respectively. The complete nucleotide sequences of pIMB2–4 were confirmed by sequencing and that of pIMB4 has been submitted to the GenBank (accession number HM756283).

Fig. 1
figure 1

The map of expression vectors pIMB2–4. ori (pSGL1), origin for Streptomyces plasmid pSGL1 replication; rep (pSGL1), replicase gene of pSGL1; bla ampicillin-resistance gene; aac(3)IV apramycin-resistance gene; ori (pUC), pUC origin of replication in E. coli; oriT, origin of DNA plasmid transfer during conjugation; ermE*p ermE gene mutated promoter; SP MelC1 DNA sequence encoding the signal peptide of MelC1; SP CagA(TTA) wild type DNA sequence encoding the signal peptide of CagA, includes a rare leucine codon TTA; SP CagA(CTG) DNA sequence encoding the signal peptide of CagA, of which TTA was changed to CTG; MCS multiple cloning sites

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Expression of human interleukin-6 (IL-6) in S. lividans TK24

Mature human IL-6 coding sequence was amplified from cDNA of peripheral blood mononuclear cells, and then inserted into NdeI–BglII digested pIMB2–4 to generate pIMB2-IL6, pIMB3-IL6 and pIMB4-IL6 respectively. These recombinant IL-6 expression plasmids were conjugated from E. coli into S. lividans TK24, resulting in recombinant expression strains S. lividans [pIMB2-IL6], S. lividans [pIMB3-IL6] and S. lividans [pIMB4-IL6]. S. lividans [pIMB2] harboring the vector pIMB2 was used as a control. These S. lividans recombinant strains were cultured in CM medium at 28°C for 72 h. No apparent difference in the growth characteristics, such as dry weight of mycelia and pH of culture medium, was observed among these strains (data not shown). The secreted proteins in the culture medium after ammonium sulfate precipitation as well as the proteins in the cell lysates were determined by SDS-PAGE and Western blot analysis. A clear IL-6 specific band of about 20 kDa was detected in both S. lividans [pIMB3-IL6] and [pIMB4-IL6] culture medium samples (Fig. 2a, b lanes 3 and 4) but undetectable in their cell lysates (Fig. 2c, lanes 3 and 4), suggesting that the recombinant human IL-6 is expressed and secreted efficiently in both recombinant S. lividans strains. No IL-6 specific band was observed in S. lividans [pIMB2-IL6] culture medium (Fig. 2a, b lane 2), and Western blot detected an approximately 23 kDa IL-6 specific band intracellular (Fig. 2c, lane 2), which was consistent with the molecular weight of the IL-6 precursor. These results suggested that the MelC1 signal peptide (SPMelC1) was inefficient for IL-6 secretion in S. lividans TK24.

Fig. 2
figure 2

Expression analysis of IL-6 in recombinant S. lividans strains. Proteins in the culture medium precipitated by ammonium sulfate, and the proteins in the cell lysates were detected by 12% SDS-PAGE and Western blot. Arrows indicate the band corresponding to the recombinant IL-6 protein. a SDS-PAGE analysis of recombinant proteins in the culture medium. b Western blot analysis of recombinant IL-6 in the culture medium. c Western blot analysis of IL-6 in the cell lysates. Lane M pre-stained protein marker; lane 1 S. lividans [pIMB2]; lane 2 S. lividans [pIMB2-IL6]; lane 3 S. lividans [pIMB3-IL6]; lane 4 S. lividans [pIMB4-IL6]

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To evaluate the secretion efficiency of recombinant IL-6, ELISA was used for quantitative measurement of IL-6 secreted by the S. lividans recombinant strains at different culture time. IL-6 reached a maximum expression level at 72 h (Fig. 3a, b), and by that time up to 0.50 and 0.61 mg/l IL-6 was detected in the culture media of S. lividans [pIMB3-IL6] and [pIMB4-IL6] respectively. However, no secreted IL-6 was detected in the culture media of S. lividans [pIMB2-IL6] (data not shown). These results demonstrated that replacing the rare codon TTA with CTG in SPCagA coding sequence led to a relatively higher IL-6 protein production.

Fig. 3
figure 3

Western blot and ELISA of recombinant IL-6 secreted by S. lividans [pIMB3-IL6] and [pIMB4-IL6] at different culture time. a Western blot analysis of recombinant IL-6 in the culture medium. A representative of three independent experiments was shown here. b ELISA analysis of IL-6. The human IL-6 Quantikine immunoassay kit was used to detect IL-6 in the culture medium. The values represent the means of three independent experiments (Mean ± standard error)

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Purification of recombinant IL-6

The recombinant IL-6 protein expressed and secreted by S. lividans [pIMB4-IL6] was purified through a series of steps including ammonium sulfate precipitation, cation exchange chromatography and gel filtration chromatography. The progress of purification was monitored by densitometric scanning of Coomassie brilliant blue stained SDS-PAGE gels (Fig. 4a) and Western blotting (Fig. 4b). The amount of recombinant IL-6 was about 0.5% of the total protein from ammonium sulfate precipitation fraction. After Q-sepharose cation exchange column, recombinant IL-6 reached about 24% of the total protein content in the pooled fractions. Further purification was carried out by superdex HR 75 column, and HPLC analysis showed that the purity of the recovered recombinant IL-6 was 90% (Fig. 4c). The profile of purification was summarized in Table 1. The biological activity of the purified recombinant IL-6 was determined by measuring the proliferation of IL-6-dependent 7TD1 cells by MTT assay, and its potency reached 7.7 × 106 U/mg.

Fig. 4
figure 4

Purification of recombinant IL-6 protein. a SDS-PAGE of proteins in different purification steps. The recombinant IL-6 protein secreted in S. lividans [pIMB4-IL6] at 72 h was purified through a series of steps. Lane M pre-stained protein marker; lane 1 the crude sample precipitated by ammonium sulfate; lane 2 sample purified after Q Sepharose FF chromatography; lane 3 sample purified after Superdex™ 75 10/300 chromatography; lane 4 0.5 μg IL-6 standard (R&D). b Western blot analysis of purified recombinant IL-6. Lane 1 S. lividans [pIMB2]; lane 2 purified recombinant IL-6. c HPLC analysis of purified recombinant IL-6

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Table 1 Purification of recombinant IL-6 secreted by S. lividans [pIMB4-IL6]
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The N-terminal amino acid residues of the purified recombinant IL-6 were determined as His-Met-Val-Pro-Pro-Gly-Glu-Asp-Ser by the Edman degradation method. Two additional residues (His-Met) at the N-terminal were identified when compared to the mature human IL-6, which was introduced by the Nde I site (CATATG). The result confirmed that no N-terminal degradation occurred in the process of protein expression and purification.

Discussion

Heterologous expression has been widely applied as an effective tool for the production of proteins of biopharmaceutical and therapeutic interest. Streptomyces have high secretion ability, and have been an attractive prokaryotic host for expression of heterologous proteins. Although there are a few commonly used expression plasmids available, sufficient choice of convenient vectors still lacks in Streptomyces expression system. In this work, we constructed a series of novel E. coli-Streptomyces shuttle expression vectors pIMB2–4 derived of pSGL1, which has a high copy number and is compatible with pIJ101 (Hong and Li 1998). These vectors contain a strong constitutive promoter and efficient signal peptide sequence for directing secretory expression of foreign proteins. They also contain the oriT of RK2 for conjugative transferring plasmid from E. coli to Streptomyces, which may save time and effort compared to protoplast transformation.

For improving heterologous protein expression in Streptomyces, one of the effective ways is to increase the expression capacity of the host. Overexpression of some host proteins involved in expression or secretion, may lead to an increased production of foreign proteins. For example, Vrancken et al. (2007) overexpressed phage-shock protein A (PspA) in S. lividans TK24, and ultimately improved the secretory expression of several heterologous proteins in this way. Until now, most commonly used Streptomyces expression plasmids are derived from pIJ101 (Kieser et al. 1982), so it is necessary to develop vectors which can coexist with pIJ101 derived plasmids. Novel vectors pIMB2–4 derived from pSGL1 compatible with pIJ101 enlarge the number of existing vectors as such providing more options for the choice of expression plasmids. These vectors may also be useful in improving the production of the existing Streptomyces expression system which harbors pIJ101 derived plasmids.

In order to obtain high expression of foreign genes, an efficient promoter is needed. A strong constitutive promoter ermE*p, which has been widely used to direct high level expression of foreign genes in S. lividans (Schmitt-John and Engels 1992), was introduced into the vectors pIMB2–4. For protein secretion two different signal peptides were used in pIMB2–4. The SPMelC1 has been used previously for the successful secretion of salmon calcitonin (Hong et al. 2003) and human glucagons (Qi et al. 2008) in Streptomyces. CagA is the apoprotein of antitumor antibiotic C-1027 in S. globisporus C-1027, and its promoter and signal peptide have been used to direct soluble human interleukin-4 receptor secretion in S. lividans TK24 (Zhang et al. 2004). In this work, recombinant human IL-6 was secreted into the culture medium guided by SPCagA, while no recombinant IL-6 accumulated intracellularly, demonstrating that SPCagA was efficient for recombinant IL-6 secretion. However, under the guidance of SPMelC1, the secretion of IL-6 was unsuccessful, and IL-6 was detected as a precursor intracellularly. This result suggested that different signal peptides vary greatly in secretion efficiency for a specific foreign protein, and the fusion of a signal sequence with proved high efficiency to secrete foreign protein will not certainly export another foreign protein effectively. Adopting different signal peptides in each vector respectively, pIMB2–4 may provide different options for heterologous protein secretion in Streptomyces.

In the process of protein export, the signal peptide of protein precursor is cleaved by the signal peptidase (SPase). The numbers and types of the amino acids between the signal peptide and mature foreign protein might have some influence on SPase processing. Lammertyn et al. (1997) kept two amino acids (Glu-Ala) of Vsi mature protein after the signal peptide cleavage site, which enhanced the accurate processing of the recombinant heterologous protein mTNF-α. In this work, for convenient construction of expression plasmids, multiple cloning sites were placed downstream of signal peptide and thus there were additionally two amino acids (His-Met) coded by Nde I between signal peptide and mature IL-6. Amino acid sequence analysis revealed that the SPCagA was correctly recognized and cleaved off at the right site, suggesting that the insertion of His-Met did not affect the SPase cleaving efficiency.

Streptomycetes are characterized by a high GC-content genome (>70%) which results in a highly-biased codon-usage pattern. A rare leucine codon TTA is involved in regulating the expression of secondary metabolism related genes. In addition, the TTA codons within genes are tended to be close to the start of protein coding sequence, which might be more effective in affecting the translation of mRNAs (Leskiw et al. 1991). Ueda et al. (1993) have reported that, when preferred codons CTG and CTC for leucine in Streptomyces were changed to TTA, the expression of ssl gene was decreased. In this work, although the codons of IL-6 were not optimized, when a TTA at the start of SPCagA coding sequence was replaced with CTG through site-directed mutagenesis, the result showed that this mutation led to a relatively higher level of IL-6 expression. These results suggested that codon optimization of recombinant genes could be an efficient approach to improve their expression in Streptomyces, and pIMB4 may therefore provide a better choice for other heterologous protein expression in Streptomyces.

Conclusion

Here we report the construction of a series of novel vectors pIMB2–4, which were shown to be convenient for protein secretory production in Streptomyces. Secretory production of active human IL-6 was obtained in S. lividans TK24 using pIMB3 and pIMB4 as vectors, in which enhanced IL-6 expression was achieved when the rare codon TTA of SPCagA coding sequence was substituted by the preferred codon CTG. Since these vectors were compatible with pIJ101, they can be applied in combination with available pIJ101 derived vectors, which raises the prospect of a wider application.