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I.2.2 Bacillus megaterium Producing Recombinant Proteins Using a Xylose- ... The Gram positive bacterium Bacillus megaterium was first described by de Bary ...
Typology: Schemes and Mind Maps
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Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n
von Rebekka Katrin Johanna Biedendieck aus Berlin-Wilmersdorf
Malten, M., Biedendieck, R., Drews, A.-C., Gamer, M. and Jahn, D. (2004). Production and secretion of glycosyltransferases in Bacillus megaterium. Glycosciences 2004, Wageningen, 28/06 – 01/07 (Poster).
Malten, M., Biedendieck, R., Drews, A.-C., Hagemeister, J. and Jahn, D. (2004). Bacillus megaterium – a versatile tool for production and secretion of heterologous proteins. 3rd Recombinant Protein Production Meeting, Tavira, Portugal, 11 – 14/11 (Poster).
Biedendieck, R., Gamer, M., Stammen, S., Jahn, D. and Malten, M. (2005). High level production, export and purification of affinity tagged heterologous proteins using Bacillus megaterium. Systembiotechnologie für industrielle Prozesse, Dechema/GVA Vortrags- und Diskussionstagung, Braunschweig, Germany, 02 – 04/05 (Poster).
Malten, M., Biedendieck, R., Gamer, M. and Jahn, D. (2005). Bacillus megaterium – a versatile tool for production and secretion of heterologous proteins. 12th European Congress on Biotechnology, Kopenhagen, Denmark, 21 – 24/08 (Poster).
Biedendieck, R., Malten, M., Gamer, M. and Jahn, D. (2006). Bacillus megaterium : Tools for the intracellular protein production and purification of affinity tagged proteins. VAAM- Jahrestagung, Jena, Germany, 19 – 22/03 (Poster).
Biedendieck, R., Gamer, M., Malten, M. and Jahn, D. (2006). Tools for the intracellular production and purification of affinity tagged proteins using Bacillus megaterium. Society for Industrial Microbiology – Annual Meeting, Baltimore, MD, USA, 30/07 – 03/08 (Poster).
"Man sieht nur mit dem Herzen gut. Das Wesentliche ist für die Augen unsichtbar. Die Zeit, die Du für Deine Rose verloren hast, sie macht Deine Rose so wichtig. Die Menschen haben diese Wahrheit vergessen, aber Du darfst sie nicht vergessen. Du bist zeitlebens für das verantwortlich, was Du Dir vertraut gemacht hast. Du bist für deine Rose verantwortlich..." „Der kleine Prinz“, Antoine de Saint-Exupery, 1943
Table of Contents
II.6.11 Determination of Levansucrase Activity ............................................................... 72 II.6.11.1 Solution for Determination of Levansucrase Activity ................................................ 73 II.6.12 Determination of Penicillin G Acylase (PGA) Activity ........................................ 74 II.6.12.1 Solution for Determination of Penicillin G Acylase Activity ..................................... 74 II.6.13 Bioreactor Cultivation............................................................................................ 74 II.6.13.1 Analytical Procedures ................................................................................................. 75 II.6.14 Fluorescent Staining and Flow Cytometry............................................................. 75
III RESULTS AND DISCUSSION .................................................................................. 77 III.1 I NTRACELLULAR PRODUCTION OF RECOMBINANT PROTEINS IN B ACILLUS MEGATERIUM ............................................................................................................. 77 III.1.1 Vectors for the Intracellular Production of Recombinant Fusion Proteins Carrying Small Affinity Tags ................................................................................ 77 III.1.2 Production of Different Affinity Tag carrying GFP .............................................. 82 III.1.2.1 GFP as Model Protein for the Intracellular Recombinant Protein Production by Bacillus megaterium .................................................................................................... 82 III.1.2.2 Calculation of Intracellular GFP Amounts Using Purified GFP................................. 83 III.1.2.3 Production of GFP in Various Bacillus megaterium Strains at Different Growth Temperatures............................................................................................................... 83 III.1.3 Production of His 6 - and StrepII-Tagged GFP in Bacillus megaterium .................. 84 III.1.4 Purification of Affinity Tagged GFP from Cell-Free Extracts .............................. 87 III.1.4.1 Removal of Affinity Tags of Purified GFP Fusion Proteins ....................................... 90 III.1.5 Thioredoxin TrxA from Bacillus megaterium as Fusion Partner for GFP ............ 92 III.1.6 Employment of New Origins of Replication for Bacillus megaterium Vector Systems .................................................................................................................. 93 III.1.6.1 GFP Production Using Free Replicating Plasmids Based on Different Origins of Replication .................................................................................................................. 94 III.1.6.2 GFP Production Using a Two Vector System............................................................. 94 III.1.6.3 Influence of Antibiotics on Bacillus megaterium Growth Profiles and GFP Production ................................................................................................................... 97 III.1.7 High Cell Density Cultivations (HCDCs).............................................................. 98 III.1.7.1 HCDC of Bacillus megaterium WH323 Carrying pRBBm56 (GFP-Strep)................ 98 III.1.7.1.1 HCDC of Bacillus megaterium with Early Induction of Recombinant Gene Expression....................................................................................................................... 98 III.1.7.1.2 HCDC of Bacillus megaterium with Late Induction of Recombinant Gene Expression....................................................................................................................... 99 III.1.7.1.3 Comparison of HCDC with Early and Late Induction of Recombinant Gene Expression..................................................................................................................... 100 III.1.7.2 Fluorescent Activated Cell Sorting (FACS) Analysis of GFP-Production in Bacillus megaterium Cells ........................................................................................ 101
Table of Contents
III.2 EXTRACELLULAR PRODUCTION OF RECOMBINANT PROTEINS ............................ 103 III.2.1 Construction of the Protein Secretion Vector pMM1525..................................... 103 III.2.2 Cloning of Lactobacillus reuteri Levansucrase Gene into Bacillus megaterium Secretion Vectors.................................................................................................. 105 III.2.3 Recombinant Protein Production and Secretion by Bacillus megaterium ............ 106 III.2.3.1 Production and Secretion of Lactobacillus reuteri Levansucrase by Bacillus megaterium ............................................................................................................... 106 III.2.3.2 Improved Protein Secretion by Bacillus megaterium in Nutrient Rich Medium...... 110 III.2.3.3 Influence of Fusion to Myc-Epitope and His 6 -Tag on the Secretion of Lactobacillus reuteri Levansucrase by Bacillus megaterium ................................... 110 III.2.4 Production and Secretion of Recombinant Affinity Tag Carrying Fusion Proteins ................................................................................................................. 111 III.2.4.1 Bacillus megaterium Expression Vectors for the Extracellular Production of Fusion Proteins with Small Affinity Tags................................................................. 111 III.2.4.2 Export of Affinity Tagged Forms of Lactobacillus reuteri Levansucrase by Bacillus megaterium ................................................................................................. 113 III.2.5 Approaches to Increase the Levansucrase Production and Export by Bacillus megaterium ........................................................................................................... 116 III.2.5.1 Utilisation of a Terminator for Target Gene Transcription....................................... 116 III.2.5.2 Coexpression of the Extracellular Chaperon Gene prsA ........................................... 116 III.2.5.3 Coexpression of Signal Peptidase Gene sipM ........................................................... 117 III.2.5.4 Extended Culture Volume and Time ........................................................................ 118 III.2.6 Affinity Chromatography Purification of Recombinant Secreted Proteins.......... 119 III.2.6.1 Affinity Chromatographic Purification of StrepII-Tagged LevΔ773 from the Cell-Free Growth Medium........................................................................................ 119 III.2.6.2 Affinity Chromatographic Purification of His 6 -Tagged LevΔ773 from Concentrated Cell-Free Growth Medium ................................................................. 120 III.2.6.3 Affinity Chromatographic Purification of His 6 -Tagged LevΔ773 from the Cell-Free Growth Medium by Nickel Charged Magnetic Beads.............................. 120 III.2.6.4 Purification of His 6 -Tagged LevΔ773 from the Cell-Free Growth Medium by Ni-NTA Sepharose in a Batch Process ..................................................................... 121 III.2.6.5 Comparison of the Purification Procedures for Secreted Fusion Proteins by Ni-NTA or Strep-Tactin Sepharose .......................................................................... 122 III.2.6.6 Comparison of the Production and Purification of His 6 -Tagged LevΔ733 from Bacillus megaterium and Escherichia coli ................................................................ 122 III.2.7 Comparison of Different Signal Peptides for Secretion of Lactobacillus reuteri Levansucrase LevΔ773 by Bacillus megaterium ................................................. 125 III.2.8 Production and Secretion of Additional Recombinant Proteins by Bacillus megaterium ........................................................................................................... 128 III.2.8.1 Production and Secretion of the Penicillin G Acylase PGA by Bacillus megaterium ............................................................................................................... 128
Abbreviations
2D two dimensional Aλ absorption at wavelength λ in nm aa amino acid Amp ampicillin AP alkaline phosphatase APB alkaline phosphatase buffer APS ammonium peroxodisulfate ATCC American Type Culture Collection ATP Adenosine 5’-triphosphate bp base pair BSA bovine serum albumin BCIP 5-Brom-4-chlor-3-indolyl phosphate CAI codon adaptation index CDW cell dry weight CHAPS 3-[(3-cholamidopropyl)-dimethyalmino] CIP calf intestinal alkaline phosphatase Cml chloramphenicol CV column volume Da dalton DNA deoxyribonucleic acid D.N.S. dinitrosalicylic acid (d)dNTP (di)deoxyribonucleotide triphosphate dsDNA double stranded DNA DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen DTT 1,4-dithio-D,L-threitol EDTA ethylenediaminetetraacetic acid e.g. exempli gratia (for example) et al. et alteri (and others) FACS fluorescent activated cell sorting Fig. figure for forward FSC frontal scatter
Abbreviations
g Æ centrifugation : earth gravity Æ weight : gram GFP green fluorescent protein h hour HCDC high cell density cultivation HPLC high pressure liquid chromatography H 2 Odeion deionised water IEF isoelectric focussing IPTG isopropyl-β-D-thiogalactopyranoside kb kilo base pair KD dissociation constant kDa kilo Dalton λ wave length LB Luria Bertani m milli M molar [mol l-1^ ] μ micro μset growth rate MALDI matrix assisted laser desorption/ionisation Mb mega base pairs MCS multiple cloning site min minute MOPS 3-(N-morpholino)-propan sulfonacid MOPSO 3-(N-morpholino)-2-hydroxy propan sulfonacid M (^) r relative molecular mass MS mass spectrometry n nano NBT nitroblue tetrazolium NIPAB 6-Nitro-3-phenylacetamido-benzoic acid NTA nitrilotriacetic acid ODλ optical density at wavelength λ in nm ORF open reading frame ori origin of replication
Abbreviations
UV ultraviolet vs. versus v/v volume per volume w/v weight per volume
Introduction
The project outlined in the following thesis was part of the Collaborative Research Centre SFB 578 (Sonderforschungsbereich 578) “Development of Biotechnological Processes by Integrating Genetic and Engineering Methods – From Gene to Product”. It is based on the collaboration of different institutes from the Technical University of Braunschweig, the Helmholtz Centre for Infection Research (formerly called “Gesellschaft für Biotechnologische Forschung”), and the University of Applied Sciences of Magdeburg. One major aim of this collaboration is the investigation of a complete biotechnological production process starting with the first cloning step to the final downstream processing of the target product by using the prokaryotic host organism Bacillus megaterium. For this purpose, the collaboration was divided into five main project areas (Hempel, 2006):
[A] Molecular Biology of Product Formation [B] Systems Biotechnology of Product Formation [C] Process Technology [D] Application Technology [Z] Genome Project: Bacillus megaterium
Each of the main project areas consists of two to five sub-projects. This thesis was incorporated into the sub-project A1 “Production of recombined glycosyltransferases in Aspergillus niger and Bacillus megaterium ”. Aim of our sub-project was the establishment and investigation of genetic tools for B. megaterium. This includes vector construction for recombinant protein production, the production of the recombinant proteins in shaking flask scale as well as the purification of recombinant proteins for further applications within the Collaborative Research Centre SFB578.
The Gram positive bacterium Bacillus megaterium was first described by de Bary more than one century ago (De Bary, 1884). With its eponymous size „megat(h)erium“ (Greek: “big animal”) of 1.5 x 4 μm, this microorganism belongs to the larger bacteria. Due to the
Introduction
those plasmids reaches from only 5.4 kb to over 165 kb (Kieselburg et al. , 1984). The two smallest ones replicate by the rolling circle mechanism whereas the other five are theta replication plasmids with cross-hybridisation replicons (Stevenson et al. , 1998). The rolling circle replicons show similarities to sequences of plasmids known from Bacillus thuringiensis and Bacillus anthracis (Scholle et al. , 2003). The theta replicons appear to be unique. They may form a new class of compatible replicons (Kunnimalaiyaan and Vary, 2005). Beside these different DNA sequences, the plasmids of B. megaterium QM B1551 carry several interesting genes, e.g. genes coding for proteins involved in cell division, in germination, in heavy metal resistance, in cell wall hydrolysis, or in rifampin resistance. Even a complete rRNA operon is located on one of the plasmids (Kunnimalaiyaan and Vary, 2005; Scholle et al. , 2003). B. megaterium strain PV361, a derivative of strain QM B1551 (Sussman et al. , 1988), lacks all seven plasmids. Surprisingly, this plasmidless strain is able to grow on rich medium and shows no differences in sporulation compared to QM B1551. Therefore, the plasmids of QM B1551 may play a role in its adaptation to various environmental conditions. For industrial applications and research, plasmidless strains are used as hosts for efficient plasmid encoded production of foreign proteins. In comparison to B. subtilis , B. megaterium is known for its ability to stably replicate and maintain recombinant plasmids (Rygus and Hillen, 1991; Vary, 1994). Moreover, an efficient protoplast transformation system does exist (Barg et al. , 2005; von Tersch and Robbins, 1990).
I.2.2 Bacillus megaterium Producing Recombinant Proteins Using a Xylose-Inducible Promoter System
Rygus and Hillen identified a xylose-inducible promoter P xylA with the according repressor protein XylR in the genome of B. megaterium strain DSM319 (Rygus et al. , 1991). This promoter is located upstream of an operon coding for the xylose isomerase XylA, the xylulokinase XylB and the xylose permease XylT. XylA and XylB are necessary for the biochemical phosphorylation of xylose to xylose-5-phosphate while XylT is responsible for the active transport of xylose into the cell. The gene encoding the repressor protein XylR is located divergently oriented upstream of this operon ( Fig. 1 ) while the promoter regions of xylR and of the xyl -operon are overlapping. The regulation of the xyl -operon expression occurs on transcriptional level. In the absence of xylose, XylR binds to the two tandem overlapping operator sequences located in P xylA and prevents transcription of the xyl -operon (Dahl et al. , 1994; Gärtner et al. , 1988). In the presence of xylose, the sugar binds to the repressor XylR. This results in a conformational change of XylR and its release from the
Introduction
operators. In this case, the RNA-polymerase is able to recognise the promoter and initiates gene expression. An additional level of regulation is mediated by glucose (Gärtner et al. , 1988). A so called catabolite response element ( cre ) sequence is situated in the xylA open reading frame from base 23 to 200 (Rygus and Hillen, 1992). This cis -active cre element and the trans -active catabolite controlled protein (CcpA) are essential for catabolite regulation in B. megaterium. In the presence of glucose HPr, a phospho-carrier protein of the phosphoenolpyruvate:glycose phosphotransferase system (PTS), is phosphorylated at Ser- which enhanced CcpA-binding to the cre sequence (Deutscher et al. , 1995). Xylose induced gene expression is repressed 14-fold. In the presence of xylose and glucose as sole energy sources, B. megaterium shows diauxic growth. As long as glucose is consumed, expression of the xyl -operon is inhibited by CcpA binding to the cre sequence. When glucose becomes exhausted, the organism is able to switch from glucose to xylose consumption and enters a second log phase (Rygus and Hillen, 1992). Beside glucose, also other carbon-sources like fructose and mannitol are known to induce catabolite regulation. Based on this xylose-inducible promoter, Rygus and Hillen developed a xylose-dependent plasmid-borne system for the overproduction of recombinant proteins. This plasmid encoded system includes the coding sequences for XylR, the promoter P xylA and the first 195 bp of the xylA ( xylA’’ ) followed by a multiple cloning site (MCS). The designed plasmid called pWH1520 ( Fig. 1B ) was successfully used for the recombinant intracellular production of prokaryotic and eukaryotic proteins including E. coli β-galactosidase, B. megaterium glucose dehydrogenase, Acinetobacter calcoaceticus mutarostase, human urokinase-like plasminogen activator (Rygus and Hillen, 1991) and Clostridium difficile toxin A (Burger et al. , 2003). Toxin A, with a high molecular mass of 308,000, was produced even in a higher amount compared to recombinant production in E. coli. This was probably due to a higher similarity in codon usage of toxA to the host B. megaterium. In further studies in our laboratory, the promoter region of xylA in pWH1520 was modified and optimised for recombinant gene expression. The cre sequence was eliminated and an enhanced MCS was inserted (Malten, 2002; Malten et al. , 2005a). The resulting plasmid pMM1520 ( Fig. 1C ) allows simple cloning of target genes by the use of 15 different DNA restriction enzyme cleavage sites located in the new designed MCS. Moreover, glucose and other sugars present in nutrient rich medium do not inhibit recombinant gene expression by catabolite control anymore. To further optimise this system, a derivative of the plasmidless B. megaterium strain DSM319, WH323, was constructed. This strain has a deletion in the
