Production of recombinant antibody fragments in Bacillus megaterium
© Jordan et al; licensee BioMed Central Ltd. 2007
Received: 08 December 2006
Accepted: 15 January 2007
Published: 15 January 2007
Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments.
The lysozyme specific single chain Fv (scFv) fragment D1.3 was succesfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41°C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 μg of recombinant His6-tagged antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli.
High yields of functional scFv antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli.
Recombinant antibodies and antibody fragments are important tools for research, diagnostics  and therapy . Further, the generation of monoclonal antibodies for proteome research, ideally against each human protein, is a massive challenge . Here, phage display has evolved into a valuable method for the selection of antibody fragments for this purpose . Recombinant antibody fragments can be selected from huge antibody gene libraries against any target by an in vitro panning procedure [5, 6]. Furthermore, the panning procedure can be integrated into high throughput technologies . For the production of large numbers of selected antibodies the established systems like mammalian cell culture , Pichia pastoris  or plants  are expensive, complicated and time consuming. The recombinant production of antibody fragments requires the folding and association of at least two different domains (VH and VL) and includes the formation of disulphide bonds. This results in lower yields compared to single domain enzymes. Overall, the production rate is strongly dependent on the individual sequence of the antibody. However, using appropriate bacterial signal peptides, antigen binding Fv fragments consisting of the VH and VL domain of an antibody have been produced in E. coli . Improved yields of functional material were achieved by the linking of VH and VL by a 15 – 25 amino acid linker, resulting in the production of a single polypeptide, the scFv (single chain fragment variable) [12, 13]. However, Gram-negative bacteria like E. coli secrete proteins mostly into the periplasm . Only in rare cases can the scFv be isolated in higher amounts from the supernatant . The use of a Gram-positive bacterium could facilitate the scFv production due to the lack of an outer membrane allowing direct secretion of proteins into the growth medium. The Gram-positive bacteria Bacillus brevis [16, 17] and Bacillus subtilis [18, 19] have already been succesfully used for the production of antibody fragments. In contrast to B. subtilis, B. megaterium does not produce alkaline proteases. Another advantage of this bacterium is the high stability of plasmids during growth , which allows a stable gene expression in long term cultivations and bioreactors. B. megaterium has been used for the production of several recombinant proteins, e.g. dextransucrase , glucanase  and Clostridium difficile toxin A . Recently, a set of free replication vectors and genetically optimized B. megaterium strains for the intra- and extracellular production of affinity tagged recombinant proteins were developed. They were succesfully employed for the production and purification of dextransucrase , levansucrase [24, 25], penicillin amidase  and a hydrolase .
Construction of the pEJBmD1.3scFv vector
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.
Comparison of B. megaterium and E. coli
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.
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.
For many applications of antibodies, full length IgG material is not mandatory. For example, for research and diagnostics, correctly glycosylated IgG produced in mammalian cell lines are not necessary. Antigen binding antibody fragments, like Fab or scFv, are sufficient for many immunological standard assays e.g. immunoblot, ELISA, immunohistochemistry or immunoprecipitation. In contrast to full IgG molecules, these antigen binding fragments can conveniently be produced in E. coli. Although this production system is widely used, corresponding yields are somehow limited and in particular hampered by the inefficient secretion, leaving more than 90 % of the produced scFv e inside of the cell. Moreover, the majority of protein is found denatured in form of insoluble inclusion bodies . These observations stimultated the search for alternative production systems.
Novel production systems could enhance the yield of functional antibody fragments or allow cheaper and easier production and purification. Various microorganism beside E. coli, like B. subtilis [18, 19], B. brevis [16, 17], Proteus mirabilis  or Lactobacillus zeae  have been used for the production of recombinant antibody fragments. Among these, Gram-positive bacteria in particular offer the opportunity to avoid the periplasm preparation step by secreting directly into the medium. In this study, the parameters for the scFv production in B. megaterium were established using the anti-lysozyme scFv D1.3 [30, 31], an antibody fragment that has already been widely used to evaluate selection a production methods. Remarkably, the optimal yields of functional scFv fragments were obtained at 41°C, 24 h production time and in TB medium. These conditions differ significantly from those established for the production of this protein class in E. coli, Bacillus subtilis or Bacillus brevis. When using E. coli, a broad temperature range using 22°C, 25°C, 30°C or 37°C was successfully used for the expression of different antibody fragments with a slight advantage of lower temperatures [31, 32, 36–40]. For B. subtilis 37°C, 6 – 8 h expression time and rich medium was used for the expression of scFvs , wheras for Bacillus brevis 30°C, 3 days and THB medium was preferable . For the production of other recombinant proteins in B. megaterium, like dextransucrase, 37°C was found to be optimal . As it is known that folding is usually the limiting step in antibody production in various organisms [37, 41, 42], a possible heat shock effect, leading to improved chaperone expression, may explain these differences.
The production of the D1.3 scFv in B. megaterium led to a higher yield of functional antibody fragments compared to our standard E. coli production system, whereas a fraction of degraded scFvs were detected in the B. megaterium production. The production of this particular scFv can yield up to of 0.8 – 1 mg/L using a different E. coli expression system after extensive optimization . The B. megaterium production strains have not been optimized compared to the long lasting optimization of E. coli and other Bacillus strains. When using E. coli, a yield of 1 – 2 g/L were obtained when producing a human breast tumor marker protein p185HER2 specific Fab fragment . When using Bacillus strains, e.g. using B. subtilis 10 – 15 mg/L of a fibrin specific scFv were produced  or when using B. brevis Fab fragments were produced at a level of 100 mg/L . Furthermore, the yield is strongly depend on the individual sequence of the produced antibody fragment.
This study showed, that after a single purification step using a recombinant His6-tag, both the concentration of the obtained material and its functional activity are superior to E. coli scFv prepared in parallel.
B. megaterium allows the production of functional scFv fragments by secretion into the culture medium. The specific activity of material obtained from B. megaterium was higher than that obtained from E. coli. Therefore, B. megaterium can be added to the list of microbial production hosts able to handle the demanding folding and secretion of antibody fragments. The B. megaterium production system offers a serious alternative to E. coli for the production of recombinant antibody fragments, in particular for research and diagnostic purposes.
5' cg ccg ctc atc acc atc acc atc act aaa aag ccc tca atg aag agg gct ttt ttt aa 3'
5' ccg gtt aaa aaa agc cct ctt cat tga ggg ctt ttt agt gat ggt gat ggt gat gag cgg ccg cat g 3'
5' cgc agt gta caa tga aaa aag tat taa tgg ctt tca tta ttt gtt tat cat taa ttt tat cag tat tag c 3'
5' cgc aga gat ctt caa tgc ggc gct agc acc tga tgg tgg agc agc taa tac tga taa aat taa tga taa 3'
5' cgc agg cta gcg ccg aag tta aac tgc agg agt cag gac ct 3'
5' cgc agg cgg ccg cct tca gct cga 3'
5' ata aag agc tca agg aga caa agg aat ggt tat tat tca aat tgc 3'
5' agg ctg cat gcc gtt cac tta act aac tta tag g 3'
Transformation of B. megaterium
Production and export of scFvs using B. megaterium
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 . 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.
Production of scFvs in E. coli
The D1.3 scFv was produced in shaking flasks according to Dübel et al.  using the vector pOPE101  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.
Immobilized metal affinity chromatography (IMAC) purification of antibody fragments
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) , pH 7.4, 100 mM EDTA were used for the elution.
Antigen binding ELISA
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.
SDS-PAGE and Immunoblot
Soluble antibody fragments were separated by SDS-PAGE  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.
We would like to thank Bronwyn M. Kenny for corrections on the manuscript. We gratefully acknowledge the financial support by the German Research Foundation (DFG, SFB 578) and the German ministry of education and research (BMBF, SMP "Antibody Factory" in the NGFN2 program).
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