# Patent

## Title
Method for the production of proteins and peptides

## Abstract

The invention relates to a method for producing monomeric or dimeric proteins or peptides containing internal or external disulfide bonds, comprising the following steps: a) a cell-free lysate, obtainable from eukaryotic cells, is provided, which contains functional microsomal vesicles, b) a nucleic acid coding the protein or peptide and additionally containing a signal sequence is added to the lysate, c) the lysate with the nucleic acid is held for a given time at a temperature in the range from 20 to 35° C., proteins or peptides formed with the nucleic acid being translocated into the microsomal vesicles, d) the microsomal vesicles are then dissolved, and the proteins or peptides obtained thereby are optionally separated from the lysate.

## Background
FIELD OF THE INVENTION
The invention relates to a method for producing monomeric or dimeric proteins or peptides containing internal or external disulfide bonds, comprising the following steps: a) a cell-free lysate is provided, b) a nucleic acid coding the protein or peptide is added to the lysate, c) the lysate with the nucleic acid is held for a given time at a temperature in the range from 20 to 35° C., d) the proteins or peptides obtained thereby are optionally separated from the lysate. The invention further relates to proteins or peptides obtainable by such a method and to a preparation obtainable by such a method.
PRIOR ART AND BACKGROUND OF THE INVENTION
Monoclonal antibodies are used in a wide field of applications also including the use as research tools and for diagnostic and therapeutic purposes.
Since scFv (single-chain variable fragment) and Fab (antigen-binding fragment) as parts of complete antibodies are small and expressible in Escherichia coli and permit a systematic survey and a selection for molecules binding with high affinity, the potential range of applications of these molecules quickly widens. One of the most important limitations of this widening is however the speed of the generation and the analysis of the efficiency of the bond and the effects of antibodies on their target molecules. An acceleration of the antibody production would permit a faster response to health risks caused by a pandemic spread of pathogenic viruses and microorganisms.
The conventional production of antibodies occurs by the culture of animal cells (Nilsang S, Nandakumar K S, Galaev I Y, Rakshit S K, Holmdahl R, Mattiasson B, Kumar A. (2007) Monoclonal antibody production using a new supermacroporous cryogel bioreactor. Biotechnol Prog 23:932-939; Rodrigues M E, Costa A R, Henriques M, Azeredo J, Oliveira R (2010) Technological progresses in monoclonal antibody production systems. Biotechnol Prog 2:332-51). A distinct acceleration and a more economic production of antibodies could be achieved in recent years by the expression thereof in Escherichia coli cells (Humphreys D P (2003) Production of antibodies and antibody fragments in Escherichia coli and a comparison of their functions, uses and modification. Curr Opin Drug Discov Devel 6:188-196). Cell-based methods of the antibody production require however a time and work-intensive cell culture execution. For a bacterial expression, furthermore additional efforts are necessary for cloning the expressible matrices coding for antibodies (Hoogenboom H R (2005) Selecting and screening recombinant antibody libraries. Nat Biotechnol 23:1105-1116).
Meanwhile, the cell-free protein expression is regarded as a promising alternative for overcoming the limitations of cell-based methods. One reason for this is the significant improvement of the productivity, economy and the activity of the synthesized proteins in cell-free prokaryotic systems achieved in recent years (Spirin A S, Baranov V I, Ryabova L A, Ovodov S Y, Alakhov Y B (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242:1162-4; Strey J, Merk H, Stiege W (2004) Verfahren zur präparativen in vitro Proteinbiosynthese. Patent DE 102004032460; Kim T W, Oh I S, Keum J W, Kwon Y C, Byun J Y, Lee K H, Choi C Y, Kim D M (2007) Prolonged cell-free protein synthesis using dual energy sources: combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate. Biotechnol Bioeng 97:1510-1515) and eukaryotic systems (Madin K, Sawasaki T, Ogasawara T, Endo Y (2000) A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. Proc Natl Acad Sci USA 97(2):559-564; Tsuboi T, Takeo S, Iriko H, Jin L, Tsuchimochi M, Matsuda S, Han E, Otsuk, H, Kaneko O, Sattabongkot J, Udomsangpetch R, Sawasaki T, Tori, M, Endo, Y (2008) Wheat germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates. Infection and Immunity 1702-1708; Tarui H, Murata M, Tani I, Imanishi S, Nishikawa S, Hara T (2001) Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment. Appl Microbiol Biotechnol 55:446-453; Kubick S, Schacherl J, Fleischer-Notter H, Royall E, Roberts L O, Stiege W (2003) In vitro Translation in an Insect-Based Cell-Free System. In: Swartz, J. R. (Ed.) Cell-Free Protein Expression. Springer, Berlin Heidelberg New York, 209-217; Mikami S, Masutani M, Sonenberg N, Yokoyama S, Imataka H (2006) An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr Purif 46(2):348-357). Modifications of some of these systems permitted the synthesis of disulfide-bonded proteins including scFv (single-chain antibody) (Ryabova L A, Desplancq D, Spirin A S, Pluckthun A (1997) Functional antibody production using cell-free translation: effects of protein disulfide isomerase and chaperones. Nat Biotechnol 15:79-84; Merk H, Stiege W, Tsumoto K, Kumagai I, Erdmann V A (1999) Cell-free Expression of two Single-Chain Monoclonal Antibodies against Lysozyme: Effect of Domain Arrangement on the Expression. J Biochem 125:328-333). Fab antibody fragments are however more attractive, due to their better binding properties, than scFv. Recently, the cell-free expression of a Fab in a system based on E. coli was shown (Oh I S, Lee J C, Lee M S, Chung J H, Kim D M (2010) Cell-free production of functional antibody fragments. Bioprocess Biosyst Eng 33:127-32).
The drawbacks of the cell-free systems based on E. coli are however a low specific activity of the produced Fab and the necessity of the removal von toxins of the expression system, before the synthesized Fab can be used for a cell-based quality test. E. coli systems meanwhile offer in their simple version (batch system) synthesis performances of up to 1 milligram newly synthesized protein per milliliter reaction solution. The highest value for the yield of functional Fab from a cell-free system is however only 30 μg/ml (Oh I S, Lee J C, Lee M S, Chung J H, Kim D M (2010) Cell-free production of functional antibody fragments. Bioprocess Biosyst Eng 33:127-32). In spite of the high productivity of the cell-free E. coli system, thus only a fraction of the synthesized protein can be used. Furthermore, in order to obtain precise measurement results, the active portion of the synthesized protein must be separated from the inactive portion, which means more efforts for this cleaning step.
Cell-free systems based on E. coli have high activities, which during the reaction will substantially vary the redox potential. This variation reduces the activity of the synthesized antibody fragments. A reduction of the variation of the redox potential was achieved by chemical pre-treatment of the cell lysate, Kim D M, Swartz J R (2004) Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli. Biotechnol Bioeng 85:122-9; Oh I S, Kim D M, Kim T W, Park C G, Chloi C Y (2006) Providing an oxidizing environment for the cell-free expression of disulfide-containing proteins by exhausting the reducing activity of Escherichia coli S30 extract. Biotechnol Prog 22:1225-89. This is however disadvantageous for the handling of the system, since another step is required, which furthermore can be automated with additional efforts only.
In order to be able to synthesize in E. coli-based cell-free systems noteworthy concentrations of soluble and active antibody fragment, they are supplemented with chaperones, for instance GroE, DnaK and protein disulfide isomerase, for instance PDI, DsbC (Ryabova L A, Desplancq D, Spirin A S, Pluckthun A (1997) Functional antibody production using cell-free translation: effects of protein disulfide isomerase and chaperones. Nat Biotechnol 15:79-84; Merk H, Stiege W, Tsumoto K, Kumagai I, Erdmann V A (1999) Cell-free Expression of two Single-Chain Monoclonal Antibodies against Lysozyme: Effect of Domain Arrangement on the Expression. J Biochem 125:328-333; Tsumoto K, Nakaoki Y, Ueda Y, Ogasahara K, Yutani K, Watanabe K, Kumagai I (1994) Effect of the order of antibody variable regions on the expression of the single-chain HyHEL10 Fv fragment in E. coli and the thermodynamic analysis of its antigen-binding properties. Biochem Biophys Res Commun 201:546-51; Oh I S, Lee J C, Lee M S, Chung J H, Kim D M (2010) Cell-free production of functional antibody fragments. Bioprocess Biosyst Eng 33:127-32; Kim D M, Swartz J R (2004) Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli. Biotechnol Bioeng 85:122-9; Oh I S, Kim D M, Kim T W, Park C G, Chloi C Y (2006) Providing an oxidizing environment for the cell-free expression of disulfide-containing proteins by exhausting the reducing activity of Escherichia coli S30 extract. Biotechnol Prog 22:1225-8).
TECHNICAL OBJECT OF THE INVENTION
It is therefore the technical object of the invention to propose a method for producing proteins and peptides, in particular antibodies or antibody fragments, wherein the obtained proteins or peptides have an improved highly specific activity and simultaneously the synthesis effort is reduced, in particular also with regard to the used nucleic acid and the post-treatment of the obtained proteins or peptides.



DESCRIPTION OF THE DRAWINGS
FIG. 1 shows in detail the dimerization of Fab chains by disulfide bonding.
FIG. 2 shows the result of the comparison of tests of anti-hen egg white lysozyme Fab antibody fragment according to the invention versus anti-hen egg white lysozyme Fab antibody fragment according to prior art.
FIG. 3 shows the determination of the synthesis yield of Fab synthesized according to the invention and in comparison with vesicle-depleted lysate.
FIG. 4 shows the determination of activity of Fab synthesized according to the invention and with vesicle-depleted lysate.
FIG. 5 shows the plasmid pIX5.0-Mel-LaLys (plasmid DNA) having SEQ.ID.NO: 1.
FIG. 6 shows the plasmid pIX5.0-Mel-HaLys (plasmid DNA) having SEQ.ID.NO: 2.
FIG. 7 shows the plasmid pIX3.0-LaLys (plasmid DNA) having SEQ.ID.NO: 3.
FIG. 8 shows the plasmid pIX3.0a-HaLys (plasmid DNA) having SEQ.ID.NO: 4.
FIG. 9 shows the employed matrix Mel-VLCL-SII, linear, PCR-generated DNA having SEQ.ID.NO: 5.
FIG. 10 shows the matrix Mel-VHCH1, linear, PCR-generated DNA having SEQ.ID.NO: 6.



BASICS OF THE INVENTION AND PREFERRED EMBODIMENTS
For achieving this technical object, the invention teaches a method for producing monomeric or dimeric proteins or peptides containing internal or external disulfide bonds, comprising the following steps: a) a cell-free lysate, obtainable from eukaryotic cells, is provided, which contains functional microsomal vesicles, b) a nucleic acid coding the protein or peptide and additionally containing a signal sequence is added to the lysate, c) the lysate with the nucleic acid is held for a given time at a temperature in the range from 20 to 35° C., proteins or peptides formed with the nucleic acid being translocated into the microsomal vesicles, d) the microsomal vesicles are then dissolved, and the proteins or peptides obtained thereby are optionally separated from the lysate.
The method according to the invention differs in several aspects from prior art. Instead of the prokaryotic E. coli cells used up to now for the cell-free synthesis of Fab antibody fragments, eukaryotic cells are used here for the production of the expression system. Heretofore, the cell-free synthesis of Fab took place in a medium that corresponds to the cytosol of a living cell. In the invention described here, the matrices coding for antibody fragments contain signal peptide sequences. By means of these signal peptide sequences, the antibody fragments are cotranslationally translocated by natural mechanisms into microsomal vesicles, which originate from the endoplasmic reticulum. According to prior art for the cell-free protein synthesis in a system of insect cells (TNT® T7 Insect Cell Extract Protein, Promega #L1101, L1102 and EasyXpress Insect Kit II, Qiagen #32561, 32562), the protein synthesis occurs at 28 to 30° C. for one to four hours. In the system described here, the synthesis occurs at a lower incubation temperature of for instance 25° C. and an incubation time of four to five hours or less. Following the synthesis reaction, the microsomal vesicles, in which synthesized Fab is present, are dissolved by means of a detergent, and Fab is released in this way. Heretofore, pre-fabricated mRNA or circular DNA coding for Fab were used as matrices for the protein synthesis. In the method described here, linear DNA generated by PCR (polymerase chain reaction) can also be used as a matrix directly, without prior purification. The PCR-generated matrix used here can be employed as a matrix in a prokaryotic system as well as in eukaryotic systems based on insect cells (for instance Spodoptera frugiperda) and on mammal cells (Chinese Hamster Ovary). In the system described here the lysate used for the protein synthesis is produced from insect cells in a non-reducing medium, in contrast to prior art. The synthesis of Fab in this insect cell lysate occurs, different from prior art, not in a reducing medium, but in a defined redox medium. Different from prior art, the cell lysate preferably is not chemically pre-treated, in order to suppress activities that strongly modify the redox potential in the course of the reaction. In contrast to prior art, the system described here preferably is not supplemented with chaperones nor with protein disulfide isomerase.
Based on these differences, various advantages are achieved. The specific activity of the proteins or antibodies or antibody fragments produced according to this invention is substantially higher compared to prior art. Whereas a prokaryotic cell-free system, compared to a typical protein synthesis performance of several hundred microgram up to one milligram per milliliter reaction solution, achieves only about 30 microgram per milliliter reaction solution, and thus a very low percentage of active Fab, proteins or antibodies or antibody fragments of the invention described here are mainly produced in an active form. The consequence in prior is that for an application, where the inactive portion of the antibody fragments is disturbing, this portion has to be separated before the application by additional work steps. Such additional work steps make an automation of an antibody fragment production difficult. A disturbing effect of inactive antibody fragment may for instance be the quantification of synthesized, active antibody fragment by immunodetection (Western Blot or Enzyme Linked Immuno Sorbant Assay), since this method does normally not allow a distinction of active and inactive protein. Compared to prior art with respect to the cell-free protein synthesis system used here and based on insect cells, the advantage of the system described here is that only therein active Fab antibody fragments can be produced. The method described here permits the synthesis of antibody fragment in a completely cell-free medium, since the generation of the matrix coding for antibody fragment can also take place by the cell-free method of the PCR, in contrast to prior art. This has several advantages. The time and labor needed for the generation of the matrices and the antibody fragments is reduced from about one to two weeks to one to two days. Since for the production of antibody fragments described here, no living cells are needed in any of the steps, and no recombinant plasmid DNA needs to be produced, risks and legal limitations in dealing with genetically modified organisms do not apply. According to prior art, antibody fragments are produced in a cell-free manner up to now in systems based on E. coli cells. Such systems contain endotoxins. Antibody fragments produced in such systems cannot be used directly for assays based on eukaryotic cells, for instance human cells, since endotoxins normally act cytotoxically on these cells, and the measurement of the specific effect of antibody fragment on these cells is overlaid by the cytotoxic effect. In order to be able to use antibody fragments from E. coli-based systems in a reasonable manner in such assays, the antibody fragments must be cleaned before in additional steps. This is disadvantageous for the working time and for an automation of the production of antibody fragments. Since the system used in this invention does not contain any endotoxins, these problems do not exist here. The cell lysate of the system described here needs not be chemically pre-treated to suppress activities that would strongly modify the redox potential in the course of the reaction. Reducing chemicals need not be added to the lysate. This simplifies the handling of the system generally and particularly with regard to the automatation of the protein synthesis reaction. The system described here does not require the production and supplementation of chaperones and protein disulfide isomerase. Thus, it has a simpler structure and is more economic with respect to these components than systems according to prior art.
For the purpose of the invention, various improvements are possible. Preferably, the proteins or peptides are dimeric monoclonal antibodies or physiologically effective preferably dimeric antibody fragments. Because of the disulfide bonds within such structures, the advantages of the invention have a particularly strong effect.
Eukaryotic cells may in particular be insect cells, but HeLa cells, CHO (Chinese Hamster Ovary) cells, HEK (Human Embryonic Kidney) cells, wheat germ cells, rabbit reticulocyte cells, yeast cells (e.g. Saccharomyces cerevisiae), protozoa (e.g. Leishmania) cells or green algae may also be used. From the insect cells, for instance cells of the species Spodoptera frugiperda, Drosophila melanogaster or Trichoplusia ni may be used.
As signal peptides can be used in the case of the insect cells, besides melittin from honey bees, PPD1, diphosphonucleotide phosphatase/phosphodiesterase from Lupinus luteolus, azurocidin from Homo sapiens or gp67 from Autographa californica. Typical signal peptides for other cell species are: a) protein synthesis systems from mammal cells (e.g. HeLa, CHO, HEK, rabbit reticulocyte) the sequences from: luciferase from Gaussia, luciferase from Metridia, luciferase from Vargula, chymotrypsinogen from Homo sapiens, human Interleukin-2 from Homo sapiens, human Trypsinogen-2 from Homo sapiens, oikosin 1 from Oikopleura dioica, b) for protein synthesis systems from yeasts (e.g. Saccharomyces cerevisiae): inulinase from Kluyveromyces marxianus, xylanase from Aureobasidium pullulans, c) for protein synthesis systems from algae the sequences from: calreticulin from Bigelowiella natans, protein disulfide isomerase Bigelowiella natans, d) for protein synthesis systems from protozoa (e.g. Leishmania) the sequences from: gp63 from Leishmania, IFN-7 from Mus musculus. The nucleic acid to be used in the method then contains, besides the sequence coding for the protein or peptide, an additional sequence coding for the signal peptide. The latter is in most cases disposed N-terminally of the sequence coding for the protein/peptide, may however in principle also be located within it or C-terminally of it.
In step d), all conventional substances, in particular detergents may be used. Besides Brij-35, examples are: Triton X-100, NP40 (Nonidet P-40), Tween 20, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), DDM (n-dodecyl-beta-D-maltoside), digitonin or glucopyranoside.
Apart from that, all conventional methods and means, for instance for separating proteins or peptides from lysates, can be applied, if desired.
The invention furthermore relates to proteins or peptides or preparation containing proteins or peptides, obtainable by a method according to the invention, as well as a preparation (for instance obtained in step c) or d)) containing proteins or peptides and a cell-free lysate, obtainable from eukaryotic cells, the lysate containing functional microsomal vesicles or products of a dissolution of such microsomal vesicles, and the preparation not containing any (endogenous and/or added) chaperones and/or protein disulfide isomerases. The explanations with respect to the method apply in an analogous manner.
In the following, the invention is explained in more detail with reference to examples of execution.
Example 1
In the examples of execution, cell lysate is produced from Spodoptera frugiperda insect cells, as described in the document Kubick S, Schacherl J, Fleischer-Notter H, Royall E, Roberts L O, Stiege W (2003) In vitro Translation in an Insect-Based Cell-Free System. In: Swartz, J. R. (Ed.) Cell-Free Protein Expression. Springer, Berlin Heidelberg New York, 209-217, with the difference that the elution buffer used for the chromatography of the centrifugation supernatant of the cell lysate does not contain a reduction agent.
Thereafter, the lysate with a final concentration of 6.9 U S7 micrococcal nuclease (Roche) per milliliter lysate at a final concentration of 1 mM CaCl2 is incubated for 20 minutes at 20° C., and the digestion is stopped by adjustment of a final concentration of 5 mM EGTA and cooling on ice. Coupled transcription/translation reactions contain 35 vol-% nuclease-treated cell lysate and further 30 mM HEPES/KOH pH 7.6, 2.9 mM magnesium acetate, 75 mM potassium acetate, 0.25 mM spermidine, 20 mg/ml creatine phosphate, 1.75 mM ATP, 0.3 mM CTP, 0.3 mM GTP, 0.3 mM UTP, 0.33 mM P1,P3-di-(guanosine-5′)-triphosphate, 2.5 mM glutathione in oxidized form, 0.5 mM glutathione in reduced form, 100 U/ml ribonuclease inhibitor RNasin (Promega), 50 U/μl T7 RNA polymerase (Roche), 17.5 μg/ml tRNA from baker's yeast (Roche), 0.1 mM each of all 20 natural amino acids, 0.1 mg/ml creatine kinase (Roche) and respectively 7.5 μg/ml plasmid DNA coding for light and heavy chain of a Fab antibody fragment. As needed, L-[U-14C]leucine (GE Healthcare) is added to the reaction for adjusting a molar activity of 40 dpm/pmol. The reaction batches are incubated for 4 hours at 25° C. under agitation at 700 rpm in the thermomixer (Eppendorf).
Following the synthesis reaction, the reaction batch is reacted for the lysis of the microsomal vesicles containing Fab with Brij-35 solution in a final concentration of 0.05% and incubated for 5 min at room temperature.
Example 2
Anti-hen egg white lysozyme Fab antibody fragment and anti-human CD4 Fab antibody fragment are synthesized as described in Example 1. As matrices are used the plasmid pIX5.0-Mel-LaLys and pIX5.0-HaLys (anti-lysozyme) and the linear DNA matrices Mel-VLCL-SII and Mel-VHCH1 (anti-CD4) generated with the EasyXpress Linear Template Kit plus (Qiagen), which code according to the invention described here for a signal peptide at the N-terminus of the Fab chains.
After the reaction, two 5 μl aliquots of each synthesis reaction were precipitated with acetone. Of the respectively two resulting pellets, one was dissolved in SDS sample buffer with reduction agent, and disulfide bonds were cleaved by heating to 90° C. for 3 minutes. The respectively second pellet was dissolved in SDS sample buffer without reduction agent by incubation for 30 minutes at 37° C., without cleaving the generated disulfide bonds. FIG. 1 shows the result of the analysis as an autoradiograph after SDS PAGE.
According thereto, in the autoradiograph with the samples treated with reduction agent, the co-expressed light and heavy chains can be seen as bands with the expected molecular weight, whereas under non-reducing conditions, bands are detected having a molecular weight as it is expected for Fab dimer. The monomeric chains were reacted in the protein synthesis system mainly to dimers.
FIG. 1 shows in detail the dimerization of Fab chains by disulfide bonding. Anti-hen egg white lysozyme Fab antibody fragment and anti-human CD4 Fab antibody fragment were synthesized in a cell-free manner according to the invention described here and the linkage of the individual light and heavy immunoglobulin chains to the disulfide-bonded dimer was analyzed by gel electrophoresis. Tracks 1-3: The disulfide bonds of the Fab chains in the reaction batches were cleaved by heating in reduction agent-containing sample buffer. Tracks 5-7: The disulfide bonds of the Fab chains in the reaction batches were maintained by treating in sample buffer without reduction agent. Tracks 1 and 5: co-expression of light and heavy chain of anti-hen egg white lysozyme Fab antibody fragment, Tracks 2 and 6: control batch with anti-lysozyme single-stranded antibody without signal peptide synthesized in presence of 0.5 μM protein disulfide isomerase (Takara), Tracks 3 and 7: co-expression of light and heavy chain of anti-human CD4 Fab antibody fragment, Track 4: empty.
Example 3
An anti-hen egg white lysozyme Fab antibody fragment is synthesized as described in Example 1. As matrices are used the plasmid pIX5.0-Mel-LaLys and pIX5.0-HaLys coding according to the invention described here for a signal peptide at the N-terminus of the Fab chains. The plasmids used for carrying out prior art, coding for the same proteins as the pIX5.0 plasmid variants, not having however a sequence coding for signal peptide, are pIX3.0-LaLys and pIX3.0a-HaLys. For the determination of the Fab activity, hen egg white lysozyme (Sigma-Aldrich) with a specific activity of 56,400 U/mg is incubated with an aliquot of the reaction batch with the synthesized Fab and parallelly with an aliquot from a control batch, and the specific inhibition of the lysozyme-dependent lysis of Micrococcus lysodeicticus cells is determined as described in the document Merk H, Stiege W, Tsumoto K, Kumagai I, Erdmann V A (1999) Cell-free Expression of two Single-Chain Monoclonal Antibodies against Lysozyme: Effect of Domain Arrangement on the Expression. J Biochem 125:328-333. By way of comparison, anti-hen egg white lysozyme Fab antibody fragment according to prior art is parallelly synthesized with the EasyXpress Insect Kit II (Qiagen) according to manufacturer's instructions and is also used for the determination of the activity. The result of the comparison tests is shown in FIG. 2. Anti-hen egg white lysozyme Fab antibody fragment was synthesized in a cell-free manner a) according to prior art and b) according to the invention described here. 5, 15 and 30 μl reaction batch each with synthesized anti-hen egg white lysozyme Fab antibody fragment from the different synthesis methods and 30 μl control reaction batch with synthesized erythropoietin were incubated with 20 ng hen egg white lysozyme, and the specific inhibition of lysozyme was detected. The values for unspecific inhibition due to the effect of the respective control reaction batch were deducted from the measurement values.
Fab that is synthesized according to the method described here has an activity being an order of magnitude higher compared to a prior art batch.
Example 4
An anti-hen egg white lysozyme Fab antibody fragment is synthesized as described in Example 1, with the difference that before the synthesis reaction, microsomal vesicles originating from the endoplasmic reticulum were to a large extent removed from the nuclease-treated lysate. For this purpose, the lysate is centrifuged for 20 minutes at 16,000×g and 4° C. The centrifugation supernatant of the nuclease-treated lysate liberated from the pelletized vesicles is then used for the synthesis reaction of Fab. In parallel, the synthesis of Fab is carried out according to the invention with lysate containing vesicles as described in Example 2. The yield and activity of the synthesized proteins are shown in FIGS. 3 and 4.
FIG. 3 shows the determination of the synthesis yield of Fab synthesized according to the invention and in comparison with vesicle-depleted lysate. The yields of the newly synthesized, radioactively marked proteins were determined by scintillation measurement of the material insoluble in hot trichloroacetic acid in 5 μl aliquots of the reaction batches. FIG. 4 shows the determination of activity of Fab synthesized according to the invention and with vesicle-depleted lysate. The activity of anti-hen egg white lysozyme Fab antibody fragment was determined analogously to Example 3. There is shown the respective extent of the lysozyme inhibition as a function of the employed Fab quantity, which was determined according to FIG. 3 by a radioactivity measurement.
Fab that is synthesized according to the method described here has an activity being approx. one order of magnitude higher compared to a batch that does not include the translocation of Fab in microsomal vesicles and thus represents part only of the method of this invention.
Example 5
In the following, the vector maps and sequences of the DNA matrices used for the cell-free protein synthesis are shown and described.
FIG. 5 shows the plasmid pIX5.0-Mel-LaLys (plasmid DNA) with the following sequence details:















Type
Start
End
Name





Region
 622
 638
T7 transcription promoter


 


Gene
 701
1408
Mel-LaLys, light chain





anti-lysozyme Fab with





melittin signal peptide


 


Region
1475
1513
T7 transcription





terminator


 


Region
2569
1896
complementary, replica-





tion origin (ori)


 


Gene
3574
2714
complementary, ampicillin





resistance gene





(β-lactamase)










(SEQ. ID. NO: 1)


TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCT


 


CCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAG


 


ACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGG


 


CTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCAC


 


CATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATAC


 


CGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAG


 


GGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG


 


GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCC


 


CAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTGCATGC


 


AAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACC


 


ATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCC


 


CGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCG


 


CACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGA


 


GGATCGAGATCGATCTCGATCCCGCGAAATTAATACGACTCACTAT


 


AGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTT


 


TAAGAAGGAGATATACCATGAAATTCTTAGTCAACGTTGCCCTGGT


 


TTTTATGGTGGTGTATATTAGCTATATTTATGCCGATATTGTGCTGA


 


CCCAGAGTCCGGCAACCCTGAGCGTTACACCGGGTAATAGCGTTAG


 


CCTGAGCTGTCGTGCAAGCCAGAGCATTGGTAATAATCTGCATTGG


 


TATCAGCAGAAAAGCCATGAAAGTCCGCGTCTGCTGATTAAATATG


 


CAAGCCAGTCAATTAGCGGTATTCCGAGCCGTTTTAGCGGTAGCGG


 


TAGTGGCACCGATTTTACCCTGAGCATTAATAGCGTTGAAACCGAA


 


GATTTTGGCATGTATTTTTGCCAGCAGAGCAATAGCTGGCCGTATA


 


CCTTTGGTGGTGGCACCAAACTGGAAATTAAACGTGCAGATGCAGC


 


ACCGACCGTTAGCATTTTTCCGCCGAGCAGCGAACAGCTGACCAGC


 


GGTGGTGCAAGCGTTGTTTGTTTTCTGAATAACTTTTATCCGAAAG


 


ATATCAATGTGAAATGGAAAATTGATGGCAGCGAACGTCAGAATG


 


GTGTTCTGAATAGCTGGACCGATCAGGATAGCAAAGATAGCACCT


 


ATAGCATGAGCAGCACCCTGACCCTGACCAAAGATGAATATGAAC


 


GCCATAATAGCTATACCTGTGAAGCAACCCATAAAACCAGCACCA


 


GCCCGATTGTTAAAAGCTTTAATCGCAATGAATGCTAATAACTCGA


 


GCGGCCGCCTGCAGATCTAAATAATAAGTAATTAACTAGTGAGCA


 


ATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGT


 


TTTTTGATATCCCGGGATTTAAATAGGCCTGAATTCGTAATCATGG


 


TCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACA


 


CAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTA


 


ATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCT


 


TTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCC


 


AACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTC


 


CTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCG


 


GTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAG


 


GGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAG


 


GCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGC


 


TCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGA


 


GGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCC


 


CTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC


 


GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC


 


ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTC


 


CAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGC


 


GCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACG


 


ACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG


 


CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA


 


CTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTG


 


AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA


 


AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCA


 


GATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTT


 


TCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG


 


ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTT


 


TAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA


 


AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATC


 


TCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGT


 


CGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGT


 


GCTGCAATGATACCGCGAGATCCACGCTCACCGGCTCCAGATTTAT


 


CAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTC


 


CTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGA


 


AGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT


 


GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGG


 


CTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATC


 


CCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATC


 


GTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGG


 


CAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT


 


TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTA


 


TGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATAC


 


CGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGT


 


TCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCA


 


GTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTT


 


ACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT


 


GCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC


 


ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTG


 


TCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAA


 


ATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCT


 


AAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTAT


 


CACGAGGCCCTTTCGTC









FIG. 6 shows the plasmid pIX5.0-Mel-HaLys (plasmid DNA) with the following sequence details:















Type
Start
End
Name





Region
 622
 638
T7 transcription promoter


 


Gene
 701
1411
Mel-HaLys, light chain





anti-lysozyme Fab with





melittin signal peptide


 


Region
1478
1516
T7 transcription





terminator


 


Region
2572
1899
complementary, replica-





tion origin (ori)


 


Gene
3577
2717
complementary, ampicillin





resistance gene (β-





lactamase)










(SEQ. ID. NO: 2)


TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCT


 


CCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAG


 


ACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGG


 


CTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCAC


 


CATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATAC


 


CGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAG


 


GGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG


 


GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCC


 


CAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTGCATGC


 


AAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACC


 


ATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCC


 


CGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCG


 


CACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGA


 


GGATCGAGATCGATCTCGATCCCGCGAAATTAATACGACTCACTAT


 


AGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTT


 


TAAGAAGGAGATATACCATGAAATTCTTAGTCAACGTTGCCCTGGT


 


TTTTATGGTGGTGTATATTAGCTATATTTATGCCGATGTGCAGCTGC


 


AGGAAAGCGGTCCGAGCCTGGTTAAACCGAGCCAGACCCTGAGCC


 


TGACCTGTAGCGTTACCGGTGATAGCATTACCAGCGATTATTGGAG


 


CTGGATTCGTAAATTTCCGGGTAATCGTCTGGAATATATGGGTTAT


 


GTTAGCTATAGCGGCAGCACCTATTATAATCCGAGCCTGAAAAGCC


 


GTATTAGCATTACCCGTGATACCAGCAAAAATCAGTATTATCTGGA


 


TCTGAATAGCGTGACCACCGAAGATACCGCAACCTATTACTGTGCA


 


AATTGGGATGGTGATTATTGGGGTCAGGGCACCCTGGTTACCGTTA


 


GCGCAGCAAAAACCACACCGCCGAGCGTTTATCCGCTGGCACCGG


 


GTAGCGCAGCACAGACCAATAGCATGGTTACCCTGGGTTGTCTGGT


 


GAAAGGTTATTTTCCGGAACCGGTTACCGTTACCTGGAATAGCGGT


 


AGCCTGAGCAGCGGTGTTCATACCTTTCCGGCAGTTCTGCAGAGCG


 


ATCTGTATACCCTGAGCAGCAGCGTTACCGTTCCGAGCAGTCCGCG


 


TCCGAGCGAAACCGTTACCTGTAATGTTGCACATCCGGCAAGCAGC


 


ACCAAAGTTGATAAAAAAATTGTTCCGCGTGATTGCTAATAACTCG


 


AGCGGCCGCCTGCAGATCTAAATAATAAGTAATTAACTAGTGAGC


 


AATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGG


 


TTTTTTGATATCCCGGGATTTAAATAGGCCTGAATTCGTAATCATG


 


GTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCAC


 


ACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCT


 


AATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGC


 


TTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGC


 


CAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTT


 


CCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGC


 


GGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCA


 


GGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAA


 


GGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGG


 


CTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG


 


AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCC


 


CCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTAC


 


CGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC


 


ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTC


 


CAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGC


 


GCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACG


 


ACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG


 


CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA


 


CTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTG


 


AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA


 


AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCA


 


GATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTT


 


TCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG


 


ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTT


 


TAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA


 


AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATC


 


TCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGT


 


CGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGT


 


GCTGCAATGATACCGCGAGATCCACGCTCACCGGCTCCAGATTTAT


 


CAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTC


 


CTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGA


 


AGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT


 


GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGG


 


CTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATC


 


CCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATC


 


GTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGG


 


CAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT


 


TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTA


 


TGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATAC


 


CGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGT


 


TCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCA


 


GTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTT


 


ACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT


 


GCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC


 


ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTG


 


TCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAA


 


ATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCT


 


AAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTAT


 


CACGAGGCCCTTTCGTC









FIG. 7 shows the plasmid pIX3.0-LaLys (plasmid DNA) with the following sequence details:















Type
Start
End
Name





Region 
 423
 439
T7 transcription promoter


 


Gene
 502
1149
HaLys, light chain anti-





lysozyme Fab


 


Region 
1173
1211
T7 transcription





terminator


 


Region 
2260
1627
complementary, replica-





tion origin (ori)


 


Gene
3265
2405
complementary, ampicillin





resistance gene





(β-lactamase)










(SEQ. ID. NO: 3)


TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCT


 


CCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAG


 


ACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGG


 


CTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCAC


 


CATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATAC


 


CGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAG


 


GGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG


 


GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCC


 


CAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTCGAG


 


CGGCCGCTAGCTAATACGACTCACTATAGGGAGACCACAACGGTTT


 


CCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATAAACAAT


 


GGATATTGTGCTGACCCAGAGTCCGGCAACCCTGAGCGTTACACCG


 


GGTAATAGCGTTAGCCTGAGCTGTCGTGCAAGCCAGAGCATTGGTA


 


ATAATCTGCATTGGTATCAGCAGAAAAGCCATGAAAGTCCGCGTCT


 


GCTGATTAAATATGCAAGCCAGTCAATTAGCGGTATTCCGAGCCGT


 


TTTAGCGGTAGCGGTAGTGGCACCGATTTTACCCTGAGCATTAATA


 


GCGTTGAAACCGAAGATTTTGGCATGTATTTTTGCCAGCAGAGCAA


 


TAGCTGGCCGTATACCTTTGGTGGTGGCACCAAACTGGAAATTAAA


 


CGTGCAGATGCAGCACCGACCGTTAGCATTTTTCCGCCGAGCAGCG


 


AACAGCTGACCAGCGGTGGTGCAAGCGTTGTTTGTTTTCTGAATAA


 


CTTTTATCCGAAAGATATCAATGTGAAATGGAAAATTGATGGCAGC


 


GAACGTCAGAATGGTGTTCTGAATAGCTGGACCGATCAGGATAGC


 


AAAGATAGCACCTATAGCATGAGCAGCACCCTGACCCTGACCAAA


 


GATGAATATGAACGCCATAATAGCTATACCTGTGAAGCAACCCATA


 


AAACCAGCACCAGCCCGATTGTTAAAAGCTTTAATCGCAATGAATG


 


CTAATAACTAACTAACCAAGATCTGTACCCCTTGGGGCCTCTAAAC


 


GGGTCTTGAGGGGTTTTTTGGATCCGAATTCACCGGTGCAATTCGT


 


AATCATGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACA


 


ATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGG


 


GGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCAC


 


TGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATG


 


AATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTC


 


TTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGC


 


GGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCAC


 


AGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCC


 


AGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTT


 


TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTC


 


AAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC


 


GTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC


 


CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC


 


GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC


 


GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCG


 


ACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT


 


AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGAT


 


TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTG


 


GTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGC


 


GCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTT


 


GATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTG


 


CAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCC


 


TTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCA


 


CGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCT


 


AGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTAT


 


ATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG


 


GCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTG


 


ACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCT


 


GGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTC


 


CAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCA


 


GAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGT


 


TGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCA


 


ACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTT


 


TGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTT


 


ACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTC


 


CTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCAT


 


GGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTA


 


AGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAG


 


AATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACG


 


GGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATT


 


GGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGT


 


TGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTC


 


AGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGA


 


AGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATG


 


TTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC


 


AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAA


 


AAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCC


 


ACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAA


 


AATAGGCGTATCACGAGGCCCTTTCGTC









FIG. 8 shows the plasmid pIX3.0a-HaLys (plasmid DNA) with the following sequence details:















Type
Start
End
Name





Region
 423
 439
T7 transcription promoter


 


Gene
 502
1152
HaLys, light chain anti-





lysozyme Fab


 


Region
1219
1257
T7 transcription





terminator


 


Region
2313
1640
complementary, replica-





tion origin (ori)


 


Gene
3318
2458
complementary, ampicillin





resistance gene





(β-lactamase)










(SEQ. ID. NO: 4)


TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCT


 


CCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAG


 


ACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGG


 


CTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCAC


 


CATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATAC


 


CGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAG


 


GGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG


 


GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCC


 


CAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTCGAG


 


CGGCCGCTAGCTAATACGACTCACTATAGGGAGACCACAACGGTTT


 


CCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATAAACAAT


 


GGATGTGCAGCTGCAGGAAAGCGGTCCGAGCCTGGTTAAACCGAG


 


CCAGACCCTGAGCCTGACCTGTAGCGTTACCGGTGATAGCATTACC


 


AGCGATTATTGGAGCTGGATTCGTAAATTTCCGGGTAATCGTCTGG


 


AATATATGGGTTATGTTAGCTATAGCGGCAGCACCTATTATAATCC


 


GAGCCTGAAAAGCCGTATTAGCATTACCCGTGATACCAGCAAAAA


 


TCAGTATTATCTGGATCTGAATAGCGTGACCACCGAAGATACCGCA


 


ACCTATTACTGTGCAAATTGGGATGGTGATTATTGGGGTCAGGGCA


 


CCCTGGTTACCGTTAGCGCAGCAAAAACCACACCGCCGAGCGTTTA


 


TCCGCTGGCACCGGGTAGCGCAGCACAGACCAATAGCATGGTTAC


 


CCTGGGTTGTCTGGTGAAAGGTTATTTTCCGGAACCGGTTACCGTT


 


ACCTGGAATAGCGGTAGCCTGAGCAGCGGTGTTCATACCTTTCCGG


 


CAGTTCTGCAGAGCGATCTGTATACCCTGAGCAGCAGCGTTACCGT


 


TCCGAGCAGTCCGCGTCCGAGCGAAACCGTTACCTGTAATGTTGCA


 


CATCCGGCAAGCAGCACCAAAGTTGATAAAAAAATTGTTCCGCGT


 


GATTGCTAATAACTCGAGCGGCCGCCTGCAGATCTAAATAATAAGT


 


AATTAACTAGTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAA


 


ACGGGTCTTGAGGGGTTTTTTGATATCCCGGGATTTAAATAGGCCT


 


GAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATC


 


CGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTA


 


AAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTT


 


GCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTG


 


CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATT


 


GGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGT


 


TCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG


 


GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGC


 


AAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT


 


GGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAAT


 


CGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGA


 


TACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCC


 


GACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA


 


AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGG


 


TGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGT


 


TCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC


 


AACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGT


 


AACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTC


 


TTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTG


 


GTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG


 


TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTT


 


TTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA


 


GAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACG


 


AAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGAT


 


CTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATC


 


TAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA


 


TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATA


 


GTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCT


 


TACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGATCCACGCTC


 


ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGC


 


CGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT


 


ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATA


 


GTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACG


 


CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCA


 


AGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCT


 


CCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTT


 


ATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATG


 


CCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGT


 


CATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC


 


GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT


 


GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATC


 


TTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCA


 


ACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCA


 


AAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGAC


 


ACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAA


 


GCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG


 


TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGA


 


AAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAA


 


CCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC









FIG. 9 shows the employed matrix Mel-VLCL-SII, linear, PCR-generated DNA with the following sequence details:















Type
Start
End
Name





Region
 25
 41
T7 transcription promoter


 


Gen
104
844
Mel-LaLys, light chain





anti-lysozyme Fab with





melittin signal peptide,





C-terminal strep-tag


 


Region
868
906
T7 transcription





terminator










(SEQ. ID. NO: 5)


ATGATATCTCGAGCGGCCGCTAGCTAATACGACTCACTATAGGGAG


 


ACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAA


 


GGAGATAAACAATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTAT


 


GGTCGTATACATTTCTTACATCTATGCGGACGATATCCAGATGACC


 


CAGTCTCCGGCTTCTCTGTCTGCTTCTGTTGGTGAAACCGTTACCTT


 


CACCTGCCGTGCTTCTGAAATGATCTACTCTTACCTGGCTTGGTATC


 


AGCAGAAACAGGGTAAATCTCCGCAACTGCTGGTTCACGACGCTA


 


AAACCCTGGCTGAAGGTGTTCCGTCCCGTTTCTCTGGTGGTGGTTC


 


TGGCACCCAGTTCTCTCTGAAAATCAACACCCTCCAGCCGGAAGAC


 


TTCGGTACCTACTACTGCCAGCACCACTACGGTAACCCGCCGACCT


 


TCGGTGGTGGCACCAAACTCGAGATCAAACGGGGGATCGCTGCAC


 


CATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGG


 


AACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAG


 


GCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC


 


TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC


 


AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAA


 


CACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGC


 


CCGTCACAAAGAGCTTCAACCGCGGAGAGTGTTCTGCCTGGTCTCA


 


TCCGCAATTCGAAAAATAATAACTAACTAACCAAGATCTGTACCCC


 


TTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGGATCCGAATTC


 


ACCGGTGATATCAT









FIG. 10 shows the matrix Mel-VHCH1, linear, PCR-generated DNA, with the following sequence details:















Type
Start
End
Name





Region
 25
 41
T7 transcription promoter


 


Gene
104
832
Mel-HaLys, heavy chain





anti-lysozyme Fab with





melittin signal peptide


 


Region
856
894
T7 transcription





terminator










(SEQ. ID. NO: 6)


ATGATATCTCGAGCGGCCGCTAGCTAATACGACTCACTATAGGGAG


 


ACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAA


 


GGAGATAAACAATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTAT


 


GGTCGTATACATTTCTTACATCTATGCGGACCAGGTTCAGCTGAAA


 


CAGTCTGGTCCGGGTCTTGTACAGCCGTCCCAGTCTCTGTCTATCA


 


CCTGCACCGTTTCCGGATTCTCTCTGACCACCTTCGGTGTTCACTGG


 


GTTCGTCAGTCCCCGGGTAAAGGTCTGGAATGGCTGGGTGTTATCT


 


GGCGTTCTGGTATCACCGACTACAACGTTCCGTTCATGTCTCGTCT


 


GTCTATCACCAAAGACAACTCTAAATCTCAGGTTTTCTTCAAACTG


 


AACTCTCTGCAACCGGACGACACCGCTATCTACTACTGCGCTAAAA


 


ACGATCCGGGTACCGGTTTCGCTTACTGGGGTCAGGGCACCCTGGT


 


CACCGTTTCTGCAGGGAGCACCAAGGGCCCATCGGTCTTCCCCCTG


 


GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT


 


GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAA


 


CTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTA


 


CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCT


 


CCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTTAATCACAA


 


ACCCAGCAACACCAAGGTCGACAAGAAAGTTGAGCCCAAATCTTG


 


CTAATAACTAACTAACCAAGATCTGTACCCCTTGGGGCCTCTAAAC


 


GGGTCTTGAGGGGTTTTTTGGATCCGAATTCACCGGTGATATCAT

## Claims

1. A method for producing a dimeric monoclonal antibody or physiologically effective antibody fragment containing internal or external disulfide bonds, comprising the following steps:
a) providing a cell-free lysate, obtained from eukaryotic cells selected from the group consisting of insect cells, HeLa cells, CHO cells, HEK cells, wheat germ cells, rabbit reticulocyte cells, yeast cells, protozoa cells and green alga, which contains functional microsomal vesicles,
b) adding a nucleic acid coding the dimeric monoclonal antibody or antibody fragment to the lysate wherein said nucleic acid comprises a signal sequence capable of assisting the translocation of said antibody or antibody fragment formed with the nucleic acid into the microsomal vesicles,
c) holding the lysate with the nucleic acid for a given time at a temperature in the range from 20° C. to 35° C., to allow the signal sequence to assist in the translocation of said antibody or said antibody fragment formed with the nucleic acid into the microsomal vesicles, and
d) digesting the microsomal vesicles, whereby said antibodies or antibody fragments the proteins or peptides obtained thereby are optionally separated from the lysate.

2. The method according to claim 1, wherein the lysate does not contain any chaperones and/or protein disulfide isomerases, or wherein no chaperones and/or protein disulfide isomerases are added to the lysate.

3. The method according to claim 1, wherein the lysate is not subjected to any chemical pre-treatment for stabilizing the redox potential.

4. The method according to claim 1, wherein said cells are insect cells.

5. The method according to claim 1, wherein step c) is carried out below a temperature of 30° C., in particular below 26° C.

6. The method according to claim 1, wherein step c) is carried out for a time of 0.5 to 5 hours.

7. The method according to claim 1, wherein the nucleic acid is a linear DNA.

8. A preparation containing dimeric monoclonal antibodies or physiologically effective antibody fragments and a cell-free lysate, obtained from eukaryotic cells selected from the group consisting of insect cells, HeLa cells, CHO cells, HEK cells, wheat germ cells, rabbit reticulocyte cells, yeast cells, protozoa cells and green alga, wherein the lysate contains functional microsomal vesicles or a digest of such microsomal vesicles, and wherein the preparation does not contain any endogenous and/or added chaperones and/or protein disulfide isomerases.