The vector pEJBmD1.3scFv for the production and the export of the lysozyme specific single chain Fv (scFv) antibody fragment was constructed from the B. megaterium expression vector pHIS1525 25. To obtain pEJBmopSplipA codon usage optimized DNA encoding the signal peptide sp_lipA of B. megaterium lipase A was integrated and a residual E. coli tetracycline gene fragment was removed from the vector. The scFv gene fragment encoding the murine anti-hen egg white lysozyme antibody D1.3 3031 was amplified by polymerase chain reaction (PCR) from the vector pHAL1-D1.3scFv, a pHAL1-D1.3 variant 32, and cloned into pEJBmopSplipA, resulting in the vector pEJBMD1.3scFv. The structure of the vector pEJBmD1.3scFv is given in figure 1. Efficient cloning of gene fragments encoding antibody fragments in E. coli DH10B was sustained by the cotransformation of pMMEc4 encoding the xylose repressor gene xylR under control of an arabinose promoter. Continuous xylR expression led to the succesful repression of the otherwise leaky B. megaterium xylA promoter in E. coli. After transformation of B. megaterium with this vector and the induction of gene expression with xylose, antigen binding by culture supernatant was confirmed by lysozyme ELISA (experimental setup as shown in Fig. 2) (data not shown). However, the initial yields were low and required significant optimization.

The impact of the temperature on the xylose induced production of scFv in B. megaterium was evaluated first. Eight different temperatures from 23°C to 45°C were analyzed using LB medium for cultivation and a gene expresion time of 24 h. The yields of functional scFv were estimated by antigen enzyme-linked immunosorbent assay (ELISA) on lysozyme. The best yield of functional D1.3 fragments was obtained at a cultivation temperature of 41°C (Fig. 2). Interestingly, in contrast to E. coli secretion systems, yields did not drop significantly at even higher temperatures than 37°C.

D1.3 scFv production at 41°C for 18 h was induced in minimal media M9 or A5, LB medium and the rich media 2xTY and TB, respectively. The secreted scFv protein was analyzed for antigen binding function by antigen ELISA (Fig. 3A) and for its molecular mass and potential degradation by SDS polyacrylamid gel electrophoresis (SDS-PAGE) and immunobloting (Fig. 3B). The minimal media did not allow production of functional scFv. The use of LB medium resulted in a low production of functional scFvs. Significant production of funtional scFvs was observed using the rich medium 2xTY. The highest production was achieved by using TB medium which is the richest of the used media. Here, the immunoblot indicated some degradation products.

The optimal expression time for scFvs in B. megaterium was determined at 41°C using TB medium for activation. Samples were taken from the supernatant between 0 and 48 h after induction of the recombinant gene expression with xylose and analyzed by antigen ELISA (Fig. 4A) and immunoblot (Fig. 4B). Functional scFv fragments were detectable 6 h after induction. The amount of secreted scFv protein continuously increased until 24 hours after induction. A longer cultivation time resulted in a slight reduction of functional scFv with the total amount of antibody fragments still increasing. From 12 h on, degradation products of the antibody fragment were detected.

The anti-lysozyme D1.3 scFv was purified from culture supernatant of B. megaterium or E. coli periplasm and supernatant. The material was purified by immobilized metal affinity chromatography (IMAC). A yield of 410 μg/L scFv after purification was obtained from B. megaterium, whereas 290 μg/L were obtained from E. coli.

Serial dilutions of the purified scFv D1.3 produced by either E. coli or B. megaterium were analyzed by antigen ELISA on lysozyme (Fig. 5). The scFvs produced in B. megaterium showed higher specific antigen binding compared to the scFvs produced in E. coli, when same amounts of antibody fragments were applied to ELISA.

Both the higher concentration and the better specific activity indicate an improved enrichment from B. megaterium culture supernatant by a single IMAC purification step, when compared to E. coli material.

Standard cloning procedures were performed according to Sambrook and Russell 45. The pEJBmD1.3scFv vector was derived from the B. megaterium expression vector pHIS1525 25. The vector pHIS1525 was modified for the cloning and expression of antibody fragments. For this purpose, the terminator T4 46 and a sequence encoding the 6xHis-tag were introduced using the two hybridised oligonucleotide primer EJoligoHisTermf and EJoligoHisTermr with the restriction sites SphI and AgeI. A residual part of the E. coli tetracycline resistence gene was deleted by digestion with AflII and religation. Furthermore, the signal peptide sp_lipA 25 was optimized regarding the codon usage and a NheI restriction site was introduced for further cloning steps using the overlapping oligonucleotide primer EJopSplipAf and EJopSplipAr. The signal peptide gene fragment was cloned into the BglII/BsrGI restriction site, resulting in the vector pEJBmopSplipA. These cloning steps were performed in E. coli DH5α (Invitrogen, Karlsruhe, Germany). The DNA encoding the D1.3 scFv was amplified from the plasmid pHAL1-D1.3scFv, an scFv variant of pHAL1-D1.3 32, using the oligonucleotide primer EJD1.3NheIf and EJD1.3r and cloned into the NheI/NotI site to yield pEJBmD1.3scFv. This cloning step was performed in E. coli DH10B containing the plasmid pMMEc4, encoding the xylose repressor protein to ensure the repression of the Xylose promoter during the cloning step in E. coli. The plasmid pMMEc4 was derived from pBAD33 (ATCC 87402) by cloning the amplified xylose repressor gene using the oligonucleotide primer XylRfor and XylRrev from B. megaterium DSM319 into the SacI/SphI site. Transformation of E. coli DH10B [pMMEc4] with pEJBmD1.3scFv was performed as outlined before 45. All affected regions of the constructs were confirmed by DNA sequencing using an ABI Prism 310 sequencer. All oligonucleotide primer sequences are given in Table 1.

Transformations of non-sporulating B. megaterium strain MS941 47 were performed as described by Barg et al. 48.

The D1.3 scFv was produced in shaking flasks. 100 mL medium + 10 μg/mL tetracyline were inoculated with 1 mL overnight culture at 37°C and 250 rpm. The media used were A5 pH 6.8 (30 g/L glucose, 2 g/L (NH₄)₂SO₄, 0.3 g/L MgSO₄, 0.5 g/L yeast extract, 3.5 g/L KH₂PO₄, 7.3 g/L NaHPO₄ × 2H₂O, 40 mg/L MnCl × 2H₂O, 53 mg/L CaCl₂ × 2H₂O, 2.5 mg/L FeSO₄ × 7H₂O, 2.5 mg/L (NH₄)₆Mo₇O₂₄ × H₂O, 2.5 mg/L CoCl₂ × 6H₂O), M9, LB, 2xTY and TB 45. The induction was started by adding 0.5 % xylose at O.D._{600 nm} = 0.3 – 0.4. The culture was shaken at 250 rpm and temperatures from 23°C to 45°C for up to 48 h. The supernatant was directly used for ELISA. For SDS-PAGE analysis and protein purification, the proteins of the supernatant were precipitated using 440 g/L ammonium sulfate.

The D1.3 scFv was produced in shaking flasks according to Dübel et al. 37 using the vector pOPE101 49 and the E. coli strain XL1-Blue MRF' (Stratagene, Amsterdam, Netherland). Briefly, 300 mL 2 × TY + 100 mM glucose + 100 μg/mL ampicillin were inoculated with an overnight culture yield to O.D._{600 nm} = 0.1 and cultured at 37°C and 250 rpm. The scFv production was induced by adjusting to 50 μM IPTG at O.D._{600 nm} = 0.5 and shaking for 3 h at 30°C. Bacteria were harvested by 10 min at 4400 × g and 4°C. The supernatant was used for ammonium sulfate precipitation (see below). Bacteria pellets were resuspended in 30 mL ice cold PE buffer, pH 8 (20 % sucrose, 50 mM Tris, 1 mM EDTA) and incubated for 20 min on ice, interrupted by short vortexing every 2 min. Subsequently the bacteria were pelleted for 30 min at 30,000 × g and 4°C. The supernatant (periplasmic fraction) was stored at -20°C. The remaining supernatant from the first bacteria centrifugation was precipated using 132 g/300 mL ammonium sulfate, stirred 1 h at 4°C and centrifuged for 20 min at 7700 × g and 4°C. The protein pellet was dissolved in 10 mL PBS. The periplasmic fraction and the precipitated supernatant were combined and dialysed over night against PBS at 4°C.

Antibody fragments were purified from E. coli or B. megaterium derived material by affinity chromatography using IMAC. Chromatography using 1 mL Chelating Sepharose Fast Flow (Amersham Biosciences, Freiburg, Germany) was performed according to the manufacturers' instruction. The protein solution was adjusted to 10 mM imidazol containing buffer (20 mM Na₂HPO₄, 500 mM NaCl, 10 mM imidazol) for loading The column was washed one time with 10 mM imidazol, twice with 50 mM imidazol buffer and once with PBS. Two times 50 mM NaH₂PO₄, 300 mM NaCl and 250 mM imidazol and finaly with phosphate buffered saline (PBS) 45, pH 7.4, 100 mM EDTA were used for the elution.

Maxisorb MTPs (Nunc, Wiesbaden, Germany) were coated with 1 μg hen egg white lysozyme or 1 μg BSA in 100 μL PBS per well overnight at 4°C. Coated wells were washed three times with PBST (PBS + 0,1% (v/v) Tween 20) and blocked with 2% (w/v) skim milk powder in PBST for 1.5 h at RT, followed by three times washing with PBST. Soluble antibody fragments were diluted in 100 μL blocking solution and incubated for 1.5 h, followed by three times washing with PBST. Soluble antibody fragments were detected with mAb mouse anti-penta His-tag (1:10000) (Qiagen, Hilden, Germany) and polyclonal goat anti-mouse IgG conjugated with horse radish peroxidase (HRP) (Fab specific) (1:10000) (Sigma, Taufkirchen, Germany) and visualised with 100 μL TMB (3,3',5,5 '-tetramethylbenzidine) substrate. The staining reaction was stopped by adding 100 μL 1 N sulphuric acid. The absorbances at 450 nm and scattered light at 620 nm were measured using a microtitre plate reader SUNRISE (Tecan, Crailsheim, Germany). The absorbance at 620 nm was subtracted.

Soluble antibody fragments were separated by SDS-PAGE 50 and blotted onto polyvinyl fluoride (PVDF) membrane (millipore, Schwalbach, Germany). The membrane was blocked with 3% (w/v) skim milk powder in PBS for 1 h at RT. For the detection of soluble antibody fragments the mAb mouse anti-penta His-tag (1:2000) (Qiagen, Hilden, Germany) was used as first antibody and goat anti-mouse IgG (Fab specific) conjugated with alkaline phosphatase (AP) (Sigma, Taufkirchen, Germany) (1:5000) was used as second antibody. The visualisation was done by NBT/BCIP.