MXPA01000224A - Expression of functional eukaryotic proteins. - Google Patents

Abstract

This invention relates to the improved expression of evolved polynucleotide and polypeptide sequences encoding for eukaryotic enzymes, particularly peroxidase enzymes, in conventional or facile expression systems. Various methods for directed evolution of polynucleotide sequences can be used to obtain the improved sequences. The improved characteristics of the polypeptides or proteins generated in this manner include improved folding, without formation of inclusion bodies, and retained functional activity. In a particular embodiment, the invention relates to improved expression of the horseradish peroxidase gene and horseradish peroxidase enzymes.

Description

EXPRESSION OF PROTEINS EU CARIOTICAS FU NCIONALES The government has certain rights in this invention in accordance with the concessions us. N0014-96-1 -0340 and N00014-98-1 -0657, adjudicated by the Navy of the United States. This application claims priority of the US application no. 60 / 094,403, filed July 28, 1,998 and No. 60/1 06,840 filed on November 3, 1998.

BACKGROUND OF THE INVENTION Field of the Invention This invention relates to methods for the selection and production of polynucleotides that encode functional polypeptides or proteins, especially eukaryotic proteins, and in particular, in easy host cell expression systems. Easy expression systems include robust prokaryotic cells (e.g., bacteria) and eukaryotic systems (e.g., yeast). In particular, the invention concerns the recombinant production of functional eukaryotic proteins resistant to expression by host cells, in high yield, and without deactivation, denaturation, inclusion bodies or other loss of structure or function. In preferred embodiments, the expressed proteins are secreted by the host cells. Preferred proteins of the invention include peroxidases and heme-containing proteins, such as horseradish peroxidase (H-RP) and cytochrome c-peroxidase (CCP). The polynucleotides which encode and express these proteins in expression systems of recombinant host cells also they are encompassed by the invention.

Description of the Related Art The publications and reference materials noted herein and listed in the appended Bibliography are each incorporated by reference in their entirety. They are referenced numerically in the text and the Bibliography later. Many proteins of interest are produced by organisms that have "eukaryotic" cells. These are cells that have a nucleus surrounded by their own membrane and that contain DNA in structures called chromosomes. All multicellular organisms, such as humans and animals, and many simple cell animals, have eukaryotic cells. Other simple cell organisms, such as bacteria, have "prokaryotic" cells. These cells have a primitive nucleus with DNA in a defined structure, but without chromosomes and a nuclear membrane that is characteristic of eukaryotes. Prokaryotic organisms are, in general, much easier and less expensive to grow, maintain and manipulate than eukaryotic cells. Genetic engineering and recombinant DNA and RNA technologies have made it possible to produce proteins, hormones and enzymes that are natural. for an organism, by using the cells of a different organism as "factories" or systems of host cell expression. In particular, it is often desirable to express a protein of eukaryotic origin in a prokaryotic host cell, because the prokaryotes can be grown in large numbers of identical cells, to produce large amounts of the desired foreign protein. For example, certain human proteins may be useful as medicaments if they can be supplied in sufficient quantity for patients having a protein deficiency. Such proteins can not be obtained easily or ethically by isolating them from human cells, nor can they be easily made by direct chemical synthesis or by growing them in isolated tissue cultures. Other proteins and enzymes are useful in the industry. For example, certain enzymes can decompose food products, and are useful in laundry detergents. However, commercial applications require large amounts of protein and a high degree of quality control. To solve some of these problems, recombinant genetic engineering techniques have been developed to use genetic machinery from other cells, such as bacteria and yeast, to produce human proteins or other proteins. Selected genetic material, such as a polynucleotide encoding a desired protein, is "recombined" with genetic material in a host cell, such that the host cell expresses the introduced foreign genetic material and produces the desired polypeptide or protein. Bacteria and yeasts can be suitable host cells because they are easy and inexpensive to grow and maintain in large quantities, and can be used reliably and repeatably to produce foreign proteins.

However, many proteins can not be easily expressed in foreign host cells, including bacteria and yeast. Such expression-resistant polypeptides or proteins may not be expressed at all or are expressed inefficiently, for example, in low yield. The protein can be expressed, but it can lose some or all of its functions. In some cases, the expressed protein may lose some or all of its active folded structure, and may even become denatured or become completely inactive. The expressed proteins can also be encapsulated within inclusion bodies within a host cell. These are discrete particles or globules inside and separated from the rest of the cell, and which contain expressed protein, perhaps in an agglomerated or inactive form. This makes it difficult to collect the protein produced from the host cells, since the isolation and purification techniques can be difficult, inefficient, slow and expensive. Efforts to produce expression-resistant polypeptides in active or functional form and in relatively high yields have spanned many years and have been markedly unfortunate. In particular, expression-resistant enzymes that are commercially important, such as horseradish peroxidase-like peroxidase enzymes, have not been functionally expressed in reasonably high yield or in convenient, economical or easy host cells. These enzymes are produced, in their place, in a non-functional or inactive form, for example, as insemination bodies and are laboriously manipulated and reconstituted to obtain active enzymes at relatively poor yields. Some proteins that are made by cells can be secreted or delivered outside the cell, which can improve the performance and efficiency of subsequent isolation and purification steps. However, many proteins are not secreted naturally and are difficult to secrete artificially, for example, because they contain chemical groups that do not easily cross the cell membrane. In particular, it is difficult to design a host cell and compatible protein system to secrete a protein that has a tendency to form inclusion bodies. Therefore, improved techniques are needed to express foreign proteins, particularly proteins of eukaryotic origin and particularly recombinant proteins that can be secreted by host cells in high yield and without loss of activity or function. As discussed, a particular challenge when producing foreign proteins in a host cell expression system is the inability of many foreign proteins to properly bend into functional proteins when common recominant hosts are used, such as. E. coli and yeast (1-4). As a result, polypeptide chains that are produced in a recombinant host cell system are often degraded on synthesis or accumulated in inclusion bodies. This is particularly true for eukaryotic proteins that contain disulfide bonds or are glycosylated in the natural form. The underlying reasons, which are not clearly understood and are probably multifactorial, may include the "unnatural" recombinant environments in which proteins accumulate (35) and the lack of appropriate folding cofactors, such as molecular chaperones in the E. coli host (3) Additionally, glycosylation has been implicated in protein doubling in eukaryotic organisms (36), such function is absent in bacteria. The problem of folding presents a challenging obstacle to the large-scale production of proteins for pharmaceutical or industrial applications. The lack of high efficiency functional expression systems has become one of the bottlenecks to apply directed evolution techniques to optimize the reaction conditions and proteins for desired uses. Using random mutagenesis and gene recombination followed by classification or selection, directed evolution has been successfully applied to improve a variety of enzymatic properties, such as substrate specificity, activity in organic solvents and stability at high temperatures, which are often critical for industrial applications (5). Eukaryotic enzymes have innumerable existing and potent applications, but the improvement of these proteins by directed evolution has been limited by the inability to express them functionally in an easy recombinant host. For example, the difficulty of expressing peroxidase enzymes in a host of easy expression has posed at least two technical challenges to realize the potential of peroxidases as biocatalysts. First, efforts to modify these enzymes for industrial applications have been hindered by engineering methods. protein Directed evolution, for example, exploits expression in a host, such as E. coli or S. cerevisiae, organisms in which large libraries of mutants or variants can be made. Second, the lack of efficient expression in an appropriate foreign (heterologous) host prevents the mass production of some of these proteins on an economic scale. One way to obtain the active form of recombinantly expressed proteins is to double them in vitro from inclusion bodies, but these processes are often laborious and inefficient (1 -3). Additionally, this is not a viable option for directed evolution in which the classification of tens of thousands of mutants is required. A more advantageous means to solve the problem "may be to identify mutations in an objective gene that may facilitate the redoubling in host environments." Evidence from a variety of studies increasingly suggests that certain residues of an amino acid sequence have a profound influence on Thus, it would be highly advantageous if the scientists could identify mutations in an objective gene that facilitate folding in the host environment, which can avoid the inclusion body's obstacle, but such techniques require the discovery, identification, and use of particular beneficial agents For example, a series of studies by King and coworkers has shown that several substitutions of simple amino acids interfered with the productive folding of the appendage protein of phage p22 at restriction in vivo, and which second site suppressor mutations were able to rescue the defective folding mutants (6). In another study, the replacement of tyrosine 35 with leucine in bovine pancreatic trypsin inhibitor (BPTI) eliminated kinetic traps in the in vitro folding pathway (7). Additionally, it was reported that several mutants of human ßterleucin 1 ß, created by u-cartridge mutagenesis of any selected residues, were expressed in E. coli in soluble form, while the wild-type was largely insoluble and bodies were formed. inclusion (8). In a separate study, a simple engineered site mutation was found to improve the performance of the folding of a recombinant antibody (9). It is difficult to predict which residues are critical for the function or stability of the protein, leaving only the fold. In this way, it would be advantageous if there were a method to systematically search for beneficial mechanisms that affect the folding and expression of the proteins, without compromising the biological activity. Targeted evolution techniques can prove useful in meeting this objective. This evolution approach uses intermixing of DNA, for simultaneous random mutagenesis and recombination, to generate a variant having an improved desirable property over the existing wild-type protein. Point mutations are generated due to the intrinsic infidelity of Taq-based polymerase reactions (PCR) associated with reassembly of n-ucleic acid sequences. In an exampleStem mer and collaborators applied this technique to the gene that encodes green fluorescence protein (GFP), which resulted in a protein that doubled better than the wild type in E. coli (10). A group of proteins of particular interest are heme proteins, that is, they have heme groups containing iron. These proteins have many biological and biochemical uses and include certain enzymes called peroxidases, which are enzymes that facilitate oxidation and reduction reactions in which a peroxide (for example, hydrogen peroxide) is one of the reactants. Peroxides are compounds, different from molecular 02, in which the oxygen atoms are united with each other. For example, the heme enzyme horseradish peroxidase (H RP) is widely used as a reporter in diagnostic tests. HRP catalyzes a reaction in which the starting materials or substrates are combined chemically in the presence of a peroxide, such as hydrogen peroxide (H2O2), with water (H2O) as a by-product. This reaction can be exploited to indicate if another reaction of interest has occurred, or if certain materials, such as H RP start materials, are present in a mixture or sample. It will be beneficial to provide a means to produce large quantities of H RP, and other heme or peroxidase enzymes, using efficient and cost-effective systems, such as prokaryotic expression systems. However, natural RP H contains four disulfides and is highly glilazylated (-21%), although the carbohydrate moiety has no apparent effect on activity or stability (11). As a consequence, previous attempts to express H RP in bacteria have produced inclusion bodies without any functional expression (1 2-14). Successful expression in yeast has not been achieved before this invention. Accordingly, there is a need to develop new and improved methods for expressing proteins, which ordinarily have been difficult to express in order to obviate the need for laborious in vitro doubling protocols. In particular, there is a need for protein expression methods that are well suited for use in connection with directed evolution techniques. In particular, this invention describes methods for classifying libraries of HRP mutants produced by error-prone PCR and intermixing of DNA to identify mutations that facilitate functional expression in bacteria (E. coli, B. subtilis) and yeast (S. cerevisiae ). In an exemplary embodiment, the variant of the invention is a working horseradish peroxidase (H RP), which is expressed in E. coli without inclusion bodies at levels of approximately 10 μg / l. This is comparable with quantities previously obtained from much more expensive, slow and laborious in vitro re-blending techniques used to recover other HRP enzymes from inclusion bodies.

BRIEF DESCRI PCI OF THE I NVE NTION It is thought that the restrictions observed in the use of natural proteins are a consequence of evolution. Porteins have evolved in the context and ambience of a living organism, to perform specific biological functions under conditions that lead to life - not in the laboratory or under industrial conditions. In some cases, evolution may favor or even require less than optimally efficient enzymes. It is not thought that the production, efficiency, working conditions, stability and other properties of known expression systems are unalterable, nor that they are limitations, which should be seen as intrinsic to the nature of cellular expression systems. It is possible that the proteins used in these systems can be developed in vitro, or that they can be developed in another way analogous proteins, to alter or intensify the properties of the protein, for example, to obtain a much more efficient expression, bending and secretion, while maintaining the activity of the protein. Improved proteins can also be obtained by classifying cultures of natural organisms or gene libraries of expressed genes (3). Many proteins, when expressed using easy expression systems (e.g., E. coli) result in inclusion bodies or are inactive due to an inability to properly bend. The invention takes advantage of directed evolution techniques to create novel polynucleotides that encode functional proteins with mutation, which have an increased capacity to be produced in an expression system, without inactivation or inclusion bodies. In preferred embodiments, the protein is secreted out of the cell. There are several advantages to secreting protein from bacteria in the culture medium, because in many cases, desired substrates can not easily pass through the E. coli membranes. Secretion can facilitate classification in directed evolution studies, because, by allowing the secreted enzyme to catalyze a reaction in the culture medium, substrates can be used that can not enter the cells. It can also significantly simplify the production of recombinant proteins, since the supernatant of the culture is largely free of contaminating substances, if the level of secretion is sufficiently high. However, the secretion of bacterial proteins in culture media remains a difficult task, particularly for enzymes containing bulky prosthetic groups, such as heme. This problem can be solved by using a suitable signal peptide, such as the signal from pectate lyase B (PelB) from Erwinia carotovora (27), to efficiently direct the secretion of a peroxidase, such as H RP or CCP in the culture medium This signal peptide is generally also applicable to other proteins containing heme prosthetic groups, such as cytochrome P450 enzymes and other peroxidases. According to one embodiment of the invention, directed or random mutagenesis is used to produce in vitro proteins, which readily bend after expression, even in yeasts and in prokaryotic expression systems, such as E. coii, and are readily secreted out of the host cell in expected quantities for proteins produced by such expression systems. Additionably, the activity of these proteins is not compromised by the mutagenic step after the appropriate selection is made.

Thus, the invention provides a method for improving the expression of a polynucleotide encoding peroxidase enzymes by using directed evolution, and polynucleotides encoding variant horseradish peroxidase, which have improved expression in conventional expression systems. The above features and many other concomitant advantages of the invention will be better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic map of an E. coli HRP expression vector pETHRP, the pETpelBHRP plasmid. The HRP gene (with an extra methionine residue at the N-terminus) was inserted into pET-22b (+), immediately downstream of the signal sequence of pectate lyase B (PelB) from Erwinia carotovora for periplasmic localization. The expression is under the control of the T7 promoter. FIG. 2 shows the nucleic acid and amino acid sequences of the pelB signal peptide [SEQ. ID. NO .: 1 and SEQ. ID. NO: 2]. FIG. 3 shows a nucleotide and amino acid sequence encoding a HRP enzyme designated HRP1A6 ([SEQ ID NO.3 and SEQ ID NO.4]). FIG. 4 is a map of expression vector pETpelBHRPI A6. FIG. 5A shows the relative activities of natural type HRP and HRP1A6. FIG 5B shows a representative overview of the first generation HRP mutants classified by activity in descending order. The activities are normalized with those of the natural type FIG. 6 shows activity levels of HRP 1A6 at various concentrations of ITPG. FIG. 7 is a representation of the structure of HRP. This figure was generated from published HRP coordinates (34), using the computer program Insight II (Molecular Biosystems). FIG. 8 is a map of the expression vector pYEXS1-HRP containing a coding sequence for HRP cloned in the secretion plasmid pYEX-S1. FIG. 9 shows the activity levels of HRP1A6 and three mutants obtained by directed evolution in S. cerevisiae HRP1-77E2, HRP1-117G4 and HRP2-28D6. In this example, HRP1A6 was the father of HRP1-77E2 and HRP1-117G4, while HRP1-117G4 was the father of HRP2-28D6. FIG. 10 shows the residual activity of several HRP mutants as a function of temperature, in a thermal inactivation curve indicating the relative thermostability of the mutants. FIG. 11 shows the residual activity of several HRP mutants as a function of concentration of hydrogen peroxide, in a titration curve indicating the relative ability of the mutants to resist degradation in the presence of hydrogen peroxide FIG. 12 shows a sequence of nucleotides and amino acids encoding a variant HRP enzyme designated HRP1-77E2 ([SEQ ID NO.5 and SEQ ID NO.6]). FIG. 13 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant designated HRP1-4B6 ([SEQ ID NO.7 and SEQ ID NO.8]). FIG. 14 shows a nucleotide and amino acid sequence encoding a variant HRP enzyme designated HRP1-28B11 ([SEQ ID NO.9 and SEQ ID NO: 10]). FIG. 15 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant designated HRP1-24D11 ([SEQ ID NO: 11 and SEQ ID NO: 12]). FIG. 16 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant designated HRP1-117G4 ([SEQ ID NO: 12 and SEQ ID NO: 13]). FIG. 17 shows a nucleotide and amino acid sequence encoding a variant HRP enzyme designated HRP1-80C12 ([SEQ ID NO: 17 and SEQ ID NO: 18]). FIG. 18 shows a nucleotide and amino acid sequence encoding a variant HRP enzyme designated HRP2-28D6 ([SEQ ID NO: 19 and SEQ ID NO: 20]). FIG. 19 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant designated HRP2-13A10 ([SEQ ID NO 21 and SEQ ID NO: 22]) FIG. 20 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant designated HRP3-17E12 ([SEQ ID NO.23 and SEQ ID NO.24]). FIG. 21 shows the activities of natural type HRP, father (HRP1A6) and mutants of HRP developed in S. cerevisiae strain BJ5465. The values were obtained with the ABTS test. The cells were grown in flasks with shaking at 30 ° C for 64 h. FIG. 22 shows A) The correlation between reactivity and stability (Ares? IA,) of HRP mutants. FIG. 23 shows the reactivity of HRP mutants in organic solvent / water systems. FIG. 24 shows the lineage of the mutants. Nucleotide substitutions are shown in parentheses following the corresponding amino acid substitutions, and synonymous mutations in italics. For each generation new mutations with "*" were donated. FIG 25 shows the accumulation of secreted HRP activity of Pichia for the HRP3-17E2 variant. FIG. 26 is a schematic map of yeast cytochrome c peroxidase expression vector pETCCP.

DETAILED DESCRIPTION OF THE INVENTION This invention concerns methods for improving the expression of proteins using conventional expression systems, said proteins would ordinarily result in inclusion bodies or be degraded upon synthesis due to an inability to properly bend in the environment of the expression system.

Definitions As used herein, "about" or "about" should mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. The term "substrate" means any substance or compound that is converted or intended to be converted into another compound by the action of an enzymatic catalyst. The term includes aromatic and aliphatic compounds, and includes not only a simple compound, but also combinations of compounds, such as solutions, mixtures and other materials, which contain at least one substrate. An "oxidation reaction" or "oxygenation reaction", as used herein, is a chemical or biochemical reaction that involves the addition of oxygen to a substrate, to form a substrate or oxygenated product. rusty. An oxidation reaction is usually accompanied by a reduction reaction (hence the term "redox" reaction, for oxidation and reduction). A compound is "oxidized" when it receives oxygen or loses electrons. A com ponent is "reduced" if it loses oxygen or gains electrons. The term "enzyme" means any substance com- pletely or substantially composed of proteins or polypeptides that cata- lyze or promote, more or less specifically, one or more chemical or biochemical reactions. A "polypeptide" (one or more peptides) is a chain of chemical building blocks called amino acids that are linked by chemical bonds called peptide bonds. A protein or polypeptide, including an enzyme, can be "natural" or "wild type," meaning that it occurs in nature; or it can be a "mutant", "variant" or "modified", meaning that it has been made, altered, derivatized or in some way is different or changed from a natural protein, or from another mutant A polypeptide or "parent" enzyme is any polypeptide or enzyme from which another polypeptide or enzyme is derived or made, using any method, tool or technique, and whether or not the parent is itself a natural or mutant polypeptide or enzyme. A parent ucleotide polynucleotide is one that encodes a polypeptide A "test enzyme" is a protein-containing substance that is tested to determine if it has properties of an enzyme The term "enzyme" can also refer to a catalytic polynucleotide (eg, RNA or DNA). The "activity" of an enzyme is a measure of its ability to catalyze a reaction and can be expressed as the ratio to which the product of the reaction is produced, for example, the enzyme activity can be represented as the product quantity produced per unit time, per unit (eg, concentration or weight) of enzyme. The "stability" of an enzi means its ability to function, over time, in a particular environment or under particular conditions.A way to assess stability is to assess its ability to withstand a loss of activity over time. , under given conditions.The stability of the enzyme can also be evaluated in other ways, for example, by determining the relative degree to which the enzyme is in a doubled or unfolded state.Thus, one enzyme is more stable than another, or has Improved stability, when it is more resistant than the other enzyme to a loss of activity under the same conditions, is more resistant to unfolding, or is more durable by any suitable measure.For example, a more "thermally stable" or "thermostable" enzyme "is one that is more resistant to structure loss (splitting) or function (enzymatic activity) when exposed to heat or an elevated temperature." One way to evaluate this is to determine the "melting temperature" or Tm of the protein. The melting temperature, also called a central point, is the temperature at which half of the protein is split from its fully folded state. This central point is usually determined by calculating the center point of a titration curve that graphs the unfolding of the protein as a temperature function. In this way, a protein with a higher Tm requires more heat to cause splitting and is more stable or more thermostable, in other words, a protein with a higher Tm indicates that fewer molecules of that protein they are split at the same temperature as a protein with a lower Tm, again meaning that the protein that is more resistant to unfolding is more stable (it has less "splitting at the same temperature). Another measure of stability is T1 / 2, which is the central point of transition of the inactivation curve of the protein as a function of temperature. T1 / 2 is the temperature at which the protein loses half of its activity. Thus, a protein with a higher T1 2 indicates that fewer molecules of that protein are inactive at the same temperature as a protein with a lower T1 / 2, again meaning that the protein that is more resistant to deactivation is more stable (it has more activity at the same time). These tests are also called "thermal displacement" tests, because the curve of inactivation or splitting, plotted against temperature, is "displaced" at higher or lower temperatures when the stability increases or decreases. Thermostability can also be measured in other ways. For example, a longer half-life (-1/2) for enzyme activity at elevated temperature is an indication of thermostability. An "oxidation enzyme" is an enzyme that catalyzes one or more oxidation reactions, usually by adding, inserting, contributing or transferring oxygen from a source or donor to a substrate. Such enzymes are also called oxido-reductases or redox enzymes, and include oxygenases, hydrogenases or reductases, oxidases and peroxidases. The terms "oxygen donor", "oxidizing agent" and "oxidant" mean a substance, molecule or compound that donates oxygen to a substrate in an oxidation reaction. Normally, the oxygen donor is reduced (accepts electrons) Exemplary oxygen donors, which are not (imitating, they include molecular oxygen or dioxygen (02) and peroxides, including alkali peroxides, such as, peroxide of t- Butyl, and most preferably hydrogen peroxide (H202) A peroxide is any compound that has two oxygen atoms attached to each other.A "luminescent" substance means any substance that produces detectable electromagnetic radiation, or a change in electromagnetic radiation. , most notably visible light, by any mechanism, including color change, UV absorbance, fluorescence and phosphorescence.Preferably, a luminescent substance according to the invention produces a "detectable" UV color, fluorescence or absorbance. chemiluminescent "means any substance that enhances the detectability of a luminescent signal (eg, fluo rescente), for example, by increasing the strength or life time of the signal. An exemplary and preferred in vivo chemiluminating agent is 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) and the like. Other chemiluminescent agents include 1,2-dioxetanes, such as, tetramethyl-1,2-dioxetane (TMD), 1,2-dioxetanones and 1,2-dioxeta nodiones. "The term" polymer "means any substance or compound that It consists of two or more form blocks ("groupers") that are repetitively linked to one another, for example, a "dimer" is a compound in which two building blocks have been joined together. non-protein substance that is necessary or beneficial for the activity of an enzyme A "coenzyme" means a cofactor that interacts directly with ys to promote a reaction catalyzed by an enzyme.Many coenzymes serve as carriers., NAD + and NADP "carry hydrogen atoms from one enzyme to another An" auxiliary protein "means a protein substance that is necessary or beneficial for the activity of an enzyme The term" host cell "means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any form, for the production of a substance by the cell, for example, the expression by the cell of a gene, a DNA or RNA sequence, a "protein" or an enzyme. "DNA" (deoxyribonucleic acid) means any chain or sequence of chemical forming blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, which are They link together in a deoxyribose sugar skeleton DNA can have a strand of nucleotide bases, or two complementary strands, which can form a double helix structure. "RNA" (ribonucleic acid) means any chain or sequence of the chemical forming blocks adenine (A), g uani na (G), cytosine (C) and uracil (U), called n-nucleotide bases, which are bound together in a sugar skeleton of ribose. Normally, RNA has a filament of nucleotide bases. A "polynucleotide" or "nucleotide sequence" is a series of nucleotide bases (also called "nucleotides") in DNA and RNA, and means any chain of two or more nucleotides.A nucleotide sequence normally carries genetic information, including the information used by the cellular machinery to make proteins and enzymes.These terms include DAN double or single strand, genomic and cDNA, RNA, any polynucleotide or genetically and genetically manipulated, and polynucleotides both sense as anti-sense (although they are only represented here as meaning.) This includes double or single strand molecules, ie DNA-DNA, RNA-RNA and RNA-RNA bridges, as well as "protein nucleic acids". (PNA) formed by conjugating bases to an amino acid skeleton This also includes nucleic acids containing modified bases, for example, thio-uracil, thio-guanine and fluoro-uracil The polynucleotides in the present they may be flanked by natural regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5 'and 3' non-coding regions, and the like. The nucleic acids can also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more naturally occurring nucleotides with an analog, and modifications of internucleotides, such as, for example, those with uncharged bonds (eg, methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged bonds (for example, phosphorothioates, phosphorodithioates, etc.). The polynucleotides may contain one or more covalently adiutional linked portions, such as, for example, proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalcors ( for example, acridine, psora len, etc.), chelators (for example, metals, radioactive metals, iron, oxidative metals, etc.) and alkylating agents. The polynucleotides c_-3 may be derivatives peormation of u? methyl or ethyl phosphorous = ter e ~ 'Acetone of phosphoramidate z alkyl. Additionally, the po < In addition to this, they can also be modified with a brand name or can be converted to a direct or direct signal. Many examples include: • Radioisotopes, fluorescent molecules. is, bi and similar. Proteins and e-zymes are made in a host cell. -sirucciones in DNA. < = NA, according to the gere code:? Co. -añera general, one se:. -ency of DNA that has instructions oara '-otein or enzyme z. "Cular is 4transcite" in a secu -.- respondent of RNA _a sequence of RNA is "translated" to SJ see = aminca sequence: DOS that form the protein c enz ~ a. "Amino acid sequence" is any chain of DNA or syna- chine. Each am-: acid is represented in either DNA or RNA DOG: ~ the triplets of? LG e-olidos. . Each ~ piété form c "-respondiente a a ~ ~ acid By ejemp 'amino acid sa: -¡ede be co.dificado PZ ~ triplet -suelee, two or coden AA.- - op c: . Aon AAG (CoC =: genetic has algjna ta redundancy - • mada degeneration 5; ~ ificando the r ~ ost 'os a ~ - Noah: SNEN more than one Codor, "espondiente" > OD to nuc e ottid =. = DNA sequences _. ~ '\\ is read er grupes of three for z: -odu - proteins is imc: ~ ante begins "reading the secuer: a e - • ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~ ~ añera en q "e una se: _. - ia of nucleotides is grouped in z don . "na el 'mar: 3 de! ec: _'; The term "gene", also called a "structural gene", means a DNA sequence that encodes or corresponds to a particular amino acid sequence, which comprises all or part of one or more proteins or enzymes, and may or may not include Regulatory DNAs, such as promoter sequences, which determine, for example, the conditions under which the gene is expressed. Some genes, which are not structural genes, can be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. A "coding sequence" or a sequence "encoding" a polypeptide, protein or enzyme, is a nucleotide sequence that, when expressed, results in the production of that polypeptide, protein or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence is "under the control" control sequences transcription or translation in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is trans-RNA spliced and translated into the protein encoded by the sequence coding. Preferably, the coding sequence is a double-stranded DNA sequence, which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The limits of the sequence of. coding are determined by a start codon at the 5 'end (amino) and a stop codon of translation at the 3' end (carboxyl). A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eukaryotic (eg ífero mam) and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, the polyadenylation signal and transcription termination sequence will usually be located at the 3 'end of the coding sequence. The transcription and translation control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators and the like, which provide for the expression of a coding sequence in a host cell. In eukaryotic cells, the polyadenylation signals are control sequences. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating the transcription of a downstream coding sequence (3 'direction). For purposes of defining this invention, the promoter sequence is linked at its 3 'end by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate the transcription initiation site. transcription at detectable levels above the support. A transcription initiation site (conveniently defined) will be found within the promoter sequence., for example, by mapping with nuclease S 1), as well as protein nion domains (consensus sequences) responsible for the binding of RNA polymerase. As described above, the promoter DNA is a DNA sequence that initiates, regulates, mediates or otherwise controls the expression of the coding DNA. A promoter can be "inducible", singling out that it is influenced by the presence or amount of another compound (an "inducer"). For example, an inducible promoter includes those that initiate or increase the expression of a downstream coding sequence in the presence of a particular inducing compound. A "bored" inducible promoter is a promoter that provides a high level of expression in the presence of an inductive compound and a comparatively very low level of expression, and a detectable level of expression in a minimum, in the absence of an inducer. . A "signal sequence" is included at the beginning of the coding sequence of a protein to be expressed in the periplasmic space or outside the cell. This sequence encodes a signal peptide, N-terminal for the mature polypeptide, which directs the host cell to transubject the polypeptide. The term "translocation signal sequence" is also used to refer to a signal sequence. Translocation signal sequences may be associated with a variety of natural proteins for eukaryotes and prokaryotes, and are frequently functional in both types of organisms. The proteins of the invention can also be modified and improved upon the addition of a sequence, which directs the secretion of the protein out of the host cell. The addition of the signal sequence does not interfere with the folding of the secreted protein, and evidence of it is easily proven to use known techniques in the area and that depend on the protein (for example, tests for activity of a protein). protein given after the modification).

Preferred signal sequences of the invention include the pelB signal sequence, which normally directs a protein to the periplasmic space between the inner and outer membranes of the bacteria. Other signal sequences include, for example, ompA and ompT (52) The signal sequence is linked upstream of the nucleotide sequence encoding the protein, such that the sequence is present at the N-terminus of the protein after The expression. Conventional cloning techniques can be used as described. Some routine experimentation within the scope of one skilled in the art may be necessary to optimize the addition of the signal sequence for any given protein. The terms "expresses" and "expression" mean that it allows or causes the information in a gene or DNA sequence to become manifest, for example, by producing a protein by activating the cellular functions involved in the transcription and translation of a gene or sequence. of corresponding DNA. A DNA sequence is expressed in or by a cell to form an "expression product", such as a protein. The expression product itself, for example, the resulting protein, can also be said to be "expressed" by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or natural promoter, or in a natural host cell under the control of a promoter. strange. A polynucleotide or polypeptide is 'overexpressed' when it is expressed or produced in an amount or yield that is substantially greater than a given baseline yield, for example, a performance that occurs in nature. a polypeptide is overexpressed when the yield is substantially greater than the normal yield, base line average of the atural polypeptide in 5 natural host cells under given conditions, for example, conditions suitable for the life cycle of natural host cells. Overexpression of a polypeptide can be obtained, for example, by altering one or more of: (a) the growth or living conditions of the host cells; (b) the polynucleotide encoding the polypeptide to be overexpressed; (c) the promoter used to control the expression of the polynucleotide; and (d) the host cell by itself. This is relative, and thus, "over-expression" can also be used to compare or distinguish the level of expression of one polypeptide with another, without considering whether any polypeptide is a nautral polypeptide or is encoded by a 15 natural nucleotide poly. Normally, overexpression means a yield that is at least about twice a normal, average or baseline yield given. In this way, a polypeptide is overexpressed when it is produced in an amount or yield that is substantially greater than the amount or yield 20 of a parent polypeptide or under the conditions of the father. Likewise, a polypeptide is "under-expressed" when it is produced in an amount or yield that is substantially less than the amount or yield of a parent polypeptide or under the conditions of the parent, for example, at least half. of the performance of the base line. In this In the context, the level of expression or performance refers to the amount or concentration of polynucleotide that is expressed or the polypeptide that is produced (ie, expression product), whether or not it is in active or functional form. As an example, it can be said that a polynucleotide or polypeptide is sub-expressed when it is expressed in detectable amounts under the control of an inducible promoter, but without induction, ie, in the absence of an inducing compound. A product of expression can be characterized as intracellular, extracellular or secreted. The term "intracellular" means something that is inside the cell. The term "extracellular" means something that is outside of a cell. A substance is "secreted" by a cell if it is delivered to the periplasm or outside the cell, from somewhere on or inside the cell. As used herein, the terms "expression resistant polypeptide" and "functional resistant expression" are synonymous and refer to a polypeptide which is difficult to express functionally in selected host cells. For example, an expression-resistant polypeptide is not produced, or is produced in a very low yield or in non-functional form, when a polynucleotide encoding that polypeptide is transformed or introduced into host cells, for example , in a simple host cell expression system. These polypeptides include, for example, those which have disulfide bridges, which are composed of multiple subunits, or which require glycosylation. Expression resistant polypeptides also include those which are sensitive to bending conditions and splitting, splitting intracellular conditions (within the cell), such as temperature, pH, protein concentration, and the presence or absence of certain cofactors, coenzymes, auxiliary proteins, etc. Expression resistant polypeptides also include polypeptides that are encoded by polynucleotides that are sensitive to particular promoters or signal sequences in particular expression systems. In addition, expression-resistant polypeptides include those that tend to agglomerate, form inclusion bodies, or that are produced in a non-active or unfolded form. Polypeptides that are particularly suitable for use as parent polypeptides resistant to expression in the invention are those that are inactive (e.g., agglomerate, etc.) when they are produced in a high yield (e.g., when they are overexpressed). , but which are active (for example, they do not agglomerate, etc.) when they are produced in a very low yield (for example, when they are sub-expressed). These include, for example, polypeptides that: (a) tend to agglomerate, form inclusion bodies, or are inactive or unfolded, when expressed in the presence of an inducer, by a polynucleotide that is under the control of an inducible promoter; and (b) they tend not to be crowded, etc. , and are active, when expressed without inducer, by a nucleotide polynucleotide that is under the control of the inducible promoter. Such promoters are known and can be called "bulk" promoters. Polypeptides that include, incorporate, or are associated with heme groups are also examples of expression-resistant polypeptides. The polypeptides resistant to the expression of particles of the invention are peroxidase enzymes, such as horseradish peroxidase enzymes. An "expression-resistant polynucleotide" is a polynucleotide that encodes an expression-resistant polypeptide. A gene encoding a protein of the invention for use in an expression system, be it genomic DNA or cDNA, can be isolated from any source, particularly from a human cDNA or genomic library. Methods for obtaining genes are well known in the art, for example, Sambrook et al. (1 9). Accordingly, any animal cell can potentially serve as the source of nucleic acid for the molecular cloning of the gene of interest. The DNA can be obtained by standard procedures known in the art, such as, from cloned DNA (eg, a "library" of DNA), from cDNA library prepared from tissues with high level of protein expression, by chemical synthesis, by cloning NA cD, or by cloning genomic DNA, or fragments of the same, purified from the desired cell (1 9, 51). Clones derived from genomic DNA may contain regulatory and introns DNA regions in addition to coding regions; the clones derived from cD NA will not contain sequences of introns. In the molecular cloning of the genomic DNA gene, fragments of DNA are generated, some of which will encode the desired gene. The DNA can be cut at specific sites using several restriction enzymes. Alternately, one can use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically trimmed, as for example, by sonication. The linear DNA fragments can be separated according to size by standard techniques, including but not limited to, polyacrylamide and agarose flb gel electrophoresis and column chromatography. 5 The term "transformation" means the introduction of a gene "foreign" (ie, extrinsic or extracellular), DNA or RNA sequence to a host cell, such that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the gene or introduced sequence. He The introduced gene or sequence can also be called a "cloned" or "strange" gene or sequence, it can include regulatory or control sequences, such as start, stop, promoter, signal, secretion or other sequences used by a genetic machinery of the cell. The gene or sequence may include non-functional sequences or sequences without 15 no known function. A host cell that receives and expresses introduced DNA or RNA has been "transformed" and is a "transformant" or a "clone". The DNA or RNA introduced into a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species. The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a r sequence of DNA or RNA (eg, a foreign gene) can be introduced into a host cell, with in order to transform the host and promote the expression (eg, transcription and translation) of the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which the foreign DNA is inserted. A common way to insert a segment of DNA into another DNA segment involves the use of enzymes called restriction enzymes that cut DNA at specific sites (specific groups of nucleotides) called restriction sites. Generally, the foreign DNA is inserted into one or more restriction sites of the vector DNA, and then it is carried by the vector to a host cell together with the DNA of the transmissible vector. A segment or DNA sequence having inserted or added DNA, such as an expression vector, can also be called a "DNA construct". A common type of vector is a "plasmid", which is generally a self-contained double-stranded DNA molecule, which can readily accept additional (foreign) DNA and which can be easily introduced into a suitable host cell. A plasmid vector frequently contains coding DNA and promoter DNA and has one or more suitable restriction sites for inserting the foreign DNA. The promoter DNA and the coding DNA can be from the same gene or from different genes, and can be from the same organism or from different organisms. A large number of vectors, including fungal vectors and plasmids, have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK (Clonetech) plasmids, pU C plasmids, pET plasmids (Novagen, I nc, Madison, Wl), pRSÉT or pREP plasmids (I nvitrogen, San Díego Ca), or pMAL plasmids (New England) Biolabs, Beverly, MA) and many appropriate host cells, using methods described or cited herein or otherwise known to those of skill in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, eg, antibiotic resistance, and one or more expression cartridges. Preferred vectors are described in the Examples, and include without limitations pcWori, pET-26b (+), pXTD 14, pYEX-S 1, pMAL and pET22-b (+). Other vectors may be employed as desired by one skilled in the art. Routine experimentation in biotechnology can be used to determine which vectors are most suitable for use with the invention, if they are different from those described in the Examples. In general, the choice of vector depends on the size of the polynucleotide sequence and the host cell to be employed in the methods of this invention. A "cartridge" refers to a segment of D NA that can be inserted into a vector at specific restriction sites. The DNA segment encodes a polypeptide of interest, and the cartridge and restriction sites are designed to ensure insertion of the cartridge into the appropriate reading frame for transcription and translation. The term "expression system" refers to a compatible host cell and vector under suitable conditions, for example, for the expression of a protein encoded by foreign DNA carried by the vector and introduced into the host cell. The common expression systems include vectors of plasmids and cells of host bacteria (eg, E. coli and B. subtilis) or yeast (eg, S. cerevisiae), and insect host cells and Baculovirus vectors. As used herein, an "easy expression system" means any expression system that is foreign or heterologous to a selected polynucleotide or polypeptide, and which employs host cells that can be grown or maintained in a more advantageous manner than cell cultures. which are natural or heterologous to the selected polynucleotide or polypeptide, or which can produce the polypeptide more efficiently or in greater yield. For example, the use of robust prokaryotic cells to express a protein of eukaryotic origin would be an easy expression system. Preferred easy expression systems include host cells of E. coli, B. subtilis and S. cerevisiae and any suitable vector.

The terms "mutant" and "mutation" mean any detectable change in genetic material, for example, DNA, or any process, mechanism or result of such change. This includes gene mutations, in which the structure (eg, DNA sequence) of a gene, any gene or DNA that arises from any process of m utation, and any expression product (e.g. or enzyme) expressed by a modified gene or DNA sequence. The term "variant" can also be used to indicate a modified or altered gene, DNA sequence, enzyme, cell, etc. , that is, any kind of mutant. "Sequence-preserving variants" of a polynucleotide sequence are those in which a change of one or more nucleotides at a given codon position does not result in alteration in the amino acids encoded at that position. "Functional conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, acid, basic, hydrophobic and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are basic hydrophilic amino acids and can be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, can be replaced with leucine, methionine or valine. Other amino acids other than those indicated as conserved may differ in a protein or enzyme, so that the percentage of similarity of amino acid sequence or protein between two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme, such as, by the Cluster Method, where the sim ility is based on the MEGALI GN algorithm. A "function-conserving vanant" also includes a polypeptide or enzyme, having at least 60% amino acid identity as determined by the BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%, and even more preferably at least 90%, and which has the same or substantially similar properties or functions as the natural protein or enzyme or parent to which it is com pared. The term "DNA reassembly" is used when the recombination between identical sequences. The term "intermixed DNA" indicates the recombination between sequences that are substantially homologous but not identical. "Isolation" or "purification" of a polypeptide or enzyme refers to the derivation of the polypeptide by removing it from its original environment (e.g., from its natural environment if it occurs naturally, or from the host cell if it is produced by methods of recombinant DNÁ *). Methods for polypeptide purification are well known in the art, including without limitation, preparative gel-disk electrophoresis, isoelectric focusing, H PLC, reverse phase HPLC, gel filtration, partition chromatography and ion exchange, and distribution to against the current For some purposes, it is preferable to produce the polypeptide in a recombinant system, in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid phase matrix. Alternatively, antibodies raised against the protein or against peptides derived therefrom can be used as purification reagents. Other methods of purification are possible. A purified polynucleotide or polypeptide may contain less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cell components with which it was originally associated. A "substantially bistantial" enzyme indicates the highest degree of purity, which can be achieved using conventional purification techniques known in the art. The polynucleotides are "hybridizable" to one another when at least one strand of a polynucleotide can anneal to another polynucleotide under conditions of defined severity. The severity of the hybridization is determined, for example, by a) the temperature at which the hybridization and / or washing is performed, and b) the ionic strength and polarity (eg, formamide) of the hybridization and washing solutions, as well as as other parameters. Hybridization requires that the two polynucleotides contain substantially complementary sequences; depending on the severity of the hybridization, however, inequalities can be tolerated. Normally, hybridization of two sequences at high severity (such as, for example, in an aqueous solution of 0.5X SSC at 65 ° C), requires that the sequences exhibit some high degree of complementarity over their entire sequence. Intermediate severity conditions (such as, for example, an aqueous solution of 2X SSC at 65 ° C) and low severity (such as, for example, an aqueous solution of 2X SSC at 55 ° C), require correspondingly less global complementation between the hybridization sequences. (1X SSC is NaCl 0.15M, 0.015 M sodium citrate). The polynucleotides that "hybridize" to the polynucleotides herein can be of any length, in one embodiment, such polynucleotides are at least 10, preferably at least 15 and most preferably at least 20 nucleotides long. In another embodiment, the hybridizing polmucleotides are of approximately the same length. In another embodiment, the hybridizing polynucleotides include those which employ under conditions of appropriate stringency and which encode polypeptides or enzymes having the same function, such as, the ability to catalyze an oxidation, oxygenate or coupling reaction of the invention. The general genetic engineering tools and techniques discussed here, including transformation and expression, the use of host cells, vectors, expression systems, etc. , they are well known in the art.

Mutagenesis and directed evolution of proteins To improve the expression of proteins using conventional expression systems, the invention makes e! Unexpected discovery that directed evolution can be used to generate libraries of polynucleotide mutants which, when expressed using conventional or easy expression systems, result in functional proteins having normal or even greater activity than the native protein. Exposure bodies, which are commonly formed when proteins are expressed having disulfide bonds, and laborious in vitro duplication procedures by directed evolution can also be avoided. According to the invention, proteins that are expressed more readily in systems of easy gene expression can be obtained by using directed evolution to generate mutant polynucleotides in a library format for selection General methods for generating libraries and isolating and identifying improved proteins (also described as "variants") in accordance with the invention using directed evolution are briefly described below and more extensively, for example, in US Pat. 5,741,691 and 5,811,238. It should be understood that any method for generating mutations in polynucleotide sequences can be employed to provide a polynucleotide developed for use in expression systems. Proteins produced by directed evolution methods can then be classified for improved expression, doubling, secretion and function according to conventional methods. Any source of nucleic acid, in purified form, can be used as the starting nucleic acid. Thus, the process can employ DNA or RNA, including messenger RNA, said DNA or RNA can be single or double filament. In addition, a DNA-RNA hybrid containing one strand of each can be used. The nucleic acid sequence can be of various lengths depending on the size of the nucleic acid sequence to be mutated. Preferably, the specific nucleic acid sequence is 50 to 50,000 base pairs. It is contemplated that whole vectors containing the nucleic acid encoding the protein of interest can be used in the methods of this invention. Any specific nucleic acid sequence can be used to produce the population of mutants by the present process. An initial population of the specific nucleic acid sequences having mutations can be created by a variety of different known methods, some of which are discussed below.

The error-prone polymerase chain reaction (20, 45, 46) and cartridge mutagenesis (38-44), in which the optimized specific region is replaced with a synthetically mutagenized oligonucleotide, can be employed in the invention. Error-prone PCR can be used to mutagenize a mixture of fragments of unknown sequences. These techniques can also be employed under low fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence, or to mutagenize a mixture of fragments of unknown sequence. Oligonucleotide-directed mutagenesis, which replaces a short sequence with a synthetically mutagenized oligonucleotide, can also be employed to generate developed polynucleotides having improved expression. Alternatively, the intermixing of DNA or nucleic acid, which uses a homologous recombination method in vitro or in vivo of nucleic acid fragment deposits or polynucleotides, can be used to generate polynucleotide molecules having variant sequences of the invention. "Parallel PCR is another method that can be used to develop polynucleotides for enhanced expression in conventional expression systems, which uses a large number of different PCR reactions that occur in parallel in the same vessel, so that the product of one reaction initiates the product of another reaction. The sequences can be randomly mutagenized at several levels by random fragmentation and reassembly of the fragments by mutual initiation. Site-specific mutations can be introduced into long sequences by random fragmentation of the template followed by reassembly of the fragments in the presence of mutagenic oligonucleotides. A particularly useful parallel PCR application, which can be used in the invention, is called sexual PCR. In sexual PCR, also known as DNA intermixing, parallel PCR is used to perform in vitro recombination in a repository of DNA sequences. Sexual PCR can also be used to build gene chimera libraries of different species. The polynucleotide sequences for use in the invention can also be altered by chemical mutagenesis. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. Other agents that are analogs of nucleotide precursors include nitrosoguanidine, 5-bromuracil, 2-aminopurine or acridine. Generally, these agents are added to the PCR reaction in place of the nucleotide precursor, thereby mutating the sequence. Intercalation agents, such as proflavine, achalavinine, quinacrine and the like can also be used. Random mutagenesis of the polynucleotide sequence can also be achieved by radiation with X-rays or ultraviolet light, or by subjecting the polynucleotide to propagation in a host (such as, E. coli) that is deficient in the repair function of normal DNA damage. In general, the plasmid DNA or DNA fragments thus mutagenized are introduced into E. coli and propagated as a pool or library of mutant plasmids. Alternatively, a mixed population of specific nucleic acids may be found in nature, since they may consist of different alleles of the same gene or the same gene of different related species (ie, consanguineous genes.). are related DNA sequences found within a 'species, for example, the peroxidase class of genes Once the mixed population of the specific nucleic acid sequences is generated, the polynucleotides can be used directly or can be inserted into a appropriate cloning vector, using techniques well known in the art Once the developed polynucleotide molecules are generated, they can be cloned into a suitable vector selected by the skilled artisan according to methods well known in the art. the specific nucleic acid sequence is cloned into a vector, can be clonally amplified insert each vector into a cell and allow the host cell to amplify the vector.

The mixed population can be tested to identify the desired recombinant nucleic acid fragment. The method of selection will depend on the desired DNA fragment. For example, in this invention, a fragment of DNA that encodes a protein with improved folding properties can be determined by tests for functional activity of the protein and absence of the formation of inclusion bodies. Such tests are well known. In the technique Using the methods of directed evolution, the invention provides a novel means to produce solos proteins, properly folded, functional and soluble in conventional or easy expression systems, such as E. coli or yeasts. Conventional tests can be used to determine whether a protein of interest produced from an expression system has expression, folding and / or improved functional properties. For example, to determine whether a polynucleotide subject to directed evolution and expressed in a foreign host cell produces a protein with improved folding, one skilled in the art can perform experiments designed to test the functional activity of the protein. Briefly, the developed protein can be classified quickly, and is easily isolated and purified from the expression system or medium if it is secreted. Then it can be subjected to tests designed to test the functional activity of the particular protein in a natural way. Such experiments of various proteins are well known in the art and are discussed in the Examples below. In one embodiment, the invention contemplates the use of polynucleotides that encode protein variants containing heme. In this manner, the invention employs directed evolution to generate novel peroxidase enzymes, such as, H-RP, which are properly folded into host cells (e.g., E. coli) used in the expression system, retain the functional activity and avoid the problems associated with the formation of inclusion bodies.

The invention can also be applied to select or optimize an expression system, including the selection of host cells, promoters and signal sequences. The expression conditions can also be optimized according to the invention. The Examples below describe the methods of the invention and, in particular, show the use of directed evolution to generate variants of HRP, which when expressed using conventional expression systems, do not form inclusion bodies and retain functional activity. Ordinarily, the corresponding natural proteins form inclusion bodies and show little functional activity retained after expression in conventional expression systems. Examples of practicing the invention are provided, and it is understood that they are only examples and that they do not limit the scope of the invention or the appended claims. A person of ordinary skill in the art will appreciate that the invention can be practiced in many ways in accordance with the claims and descriptions herein.

EJ EM PLO 1 Functional expression of horseradish peroxidase in E. coli and yeast There is a growing interest in exploiting eukaryotic peroxidases for use as industrial biocatalysts. However, protein engineering and evolution aimed at improving specific properties are complicated by the lack of easy recommending expression systems. In an effort to develop a functional bacterial expression system suitable for high volume classification of horseradish peroxidase mutants (H RP), the present Example describes the development of a bacterial expression system for hemo-associated proteins, such as, horseradish peroxidase (HRP), by inserting a corresponding gene as a fusion to the PelB signal peptide. further, by subjecting these genes to directed evolution, the hemo-associated proteins bend more efficiently in E. coli and become more resistant to heat (thermostable) and more resistant to inactivation by H202. This Example provides an approach to greatly facilitate efforts to "tune" many enzymes that are promising industrial biocatalysts, but for which adequate bacterial or yeast expression systems are currently lacking because the proteins form inclusion bodies or are secreted inefficiently by the cell.

Cloning of HRP The H RP gene (with an extra methionine residue at the N-terminus) was cloned from the plasmid pBBG IO (British Biotechnologies, Ltd., Oxford, UK) by PCR techniques to introduce Aat II site into the start codon and a Hi nd lll site immediately downstream from the stop codon. This plasmid contains the synthetic horseradish peroxidase (HRP) gene described in Smith et al. (1 3), whose DNA sequence is based on an amino acid sequence published for the H protein RP - (49) pBBG IO was made by inserting the H RP sequence between the Hindlll and EcoR1 sites of the polylinker in the Well known plasmid pUC19. The PCR product obtained from this plasmid was digested with Aat II first, blunt-ended with t4 DNA polymerase, and then further restricted with Hind III. The digested product was ligated into pET-22b (+) (purchased from Novagen) treated with Mcsl and Hind III, to produce the pETpelBHRP vector. A map of this expression vector is shown in FIG. 1. In this construct, the HRP gene was placed under the control of the T7 promoter and fused in frame to the pelB signal sequence (See [SEQ ID NO.1 and SEQ ID NO.2] and FIG.2) , which theoretically directs the transport of proteins in the periplasmic space, that is, for delivery outside the cellular cytoplasm (27). The ligation product was transformed into E. coli strain BL21 (DE3) for protein expression in cells with or without induction by 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG). In cells that were induced with IPTG, peroxidase activity was not detected above the support, for BL21 (DE3) cells or BL21 cells (DE3 harboring pET-22b (+), even though the level of HRP polypeptides responds above 20% of the total cellular proteins This was consistent with previous observations (12-14) .In the cells that were not induced with IPTG, clones were found that showed weak but measurable activity against azino-di- (ethylbenzthiazoline) sulfonate ( ABTS) The T7 promoter in the vector pET-22b (+) is known to have leakage (31) and it is theoretically possible, therefore, that some of the HRP polypeptide chains produced at this basal level are capable of bending into the natural form Adversely, the addition of IPTG leads to high-level HRP synthesis, which favors chain aggregating instead and prevents proper folding. Subsequently, random mutagenesis and classification were used to identify m utations that lead to a greater expression of H RP activity. Thus, one aspect of the invention includes the use of a promoter which can regulate the production of small amounts of polypeptide under some conditions and larger amounts under other conditions. For example, "leaky" inductible promoters can be used. This type of promoter produces high levels of a particular protein or proteins in the presence of an inducing compound, and much lower levels in the absence of the inducer. In some embodiments, a polypeptide can be overexpressed under certain conditions (e.g., in the presence of inducer) and under-expressed under other conditions (e.g., without inducer). Polypeptides that are inactive when expressed at normal levels or when they are overexpressed, but are active when they are b-expressed, are particularly suitable for use as parent polypeptides of the invention. Such expression-resistant polypeptides can be improved, using the methods of the invention, to provide functional and active expression, at suitably high yields and activity levels Generation of random library and classification One of the HRP clones that showed detectable peroxidase activity was used in the first generation of prone mutagenesis PCR to error. The aleatoric libraries were generated by a modification of the error-prone PCR protocol described above (20, 21, 22), in which 0.15 mM of MnCl2 was used instead of 0.5 mM MnCI2 This protocol incorporates both manganese ions and unbalanced nucleotides, and has been shown to generate both transitions and transversions, and therefore a spectrum broader amino acid changes (50). Briefly, the PCR reaction solution contained a template of 20 fmoles, 30 pmoles of each of two primers, 27 mM MgCl, 50 mM KCI, 10 mM Tris-HCl (pH 8.3), 0.01% gelatin, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 0.15 mM MnCl2 and 5 units of Taq polymer in a volume of 100 μl. The PCR reactions were performed on an MJ PTC-200 cycler (MJ Research, MA) for 30 cycles with the following parameters: 94 ° C for 1 min, 50 ° C for 1 min and 72 ° C for 1 min. The primers used were: 5'-TTATTGCTCAGCGGTGGCAGCAGC [SEQ. ID NO. 15], and 5'-AAGCGCTCATGAGCCCGAAGTGGC [SEQ. ID NO. 16]. The PCR products were purified with a Promega set Wizard PCR and were digested with Nde I and Hind III. The digestion products were subjected to gel purification with a QIAEX II gel extraction set and the HRP fragments were ligated back into the pET-22b (+) vector digested and gel purified in a similar manner. The ligation mixtures were transformed in BL21 cells (DE3) by electroporation with a Gene Pulser II (Bio-Rad). Cell growth and expression was performed either in 96-well microplates or 384 wells in LB medium at 30 ° C. Peroxidase activity tests were performed with H202 and ABTS (26). For each generation, normally 12,000-15,000 colonies were collected and classified into 96-well plates. This number represents an exhaustive search of all accessible single mutants, with a 95% probability for any mutant to be sampled at least once (25). Colonies were collected either manually or using an automated colony collector at Caltech, Q-bot (Genetix, UK). Of the 12,000 colonies that were classified (no IPTG added), a clone designated HRP1A6 showed peroxidase activity 10-14 times higher than the parent clone. FIG.5A and 5B. This clone also showed markedly decreased activity when as little as 5 μM of IPTG was added. FIG. 6. Sigma reports that 1 mg of highly purified HRP of horseradish has a total activity of 1,000 units, as determined by the ABTS assay. Other workers reported similar results (13). Based on these data, the concentration of active HRP was estimated to be -100 ug / l. HRP1A6 shows a total activity of more than 100 units / l. This compares favorably with the yield obtained from the redoubling of added HRP chains in vitro (13). This level of expression for clone HRP1A6 is also similar to that for bovine pancreatic trypsin inhibitor (BPTl) in E. coli (32), a non-glycosylated protein with three disulfide bonds. More than 95% of H RP activity was found in the LB culture medium as judged by the activity of ABTS. Clone HRP1 A6 remained stable for up to one week at 4 ° C. I PTG was omitted in all HRP expression experiments, unless otherwise specified. Peroxidase activity tests for HRP were performed with a classic peroxidase assay, ABTS and hydrogen peroxide (26). Fifteen μl of cell suspension was mixed with 140 μl of ABTS / H202 (2.9 mM ABTS, 0.5 mM H202, pH 4.5) in microplates, and the activity was determined with a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA) at 25 ° C. ° C. One unit of HRP is defined as the amount of enzyme that oxidizes 1 μmol of ABTS per minute under the test conditions. The sequence of H RP 1 A6 is shown in FIG. 3 [SEQ ID NO. 3]. A map of a plasmid gone pETpelBHRP I A6 containing the gene H RP 1 A6 is shown in the FI G. 4. A representation of the structure of this HRP clone is shown in FI G. 7.

Functional expression of HRP in yeast The natural HRP protein contains four disulfide bonds and E. coli has only a limited capacity to support the formation of disulfides. In theory, these well-preserved isoflurates in HRP (and other plant peroxidases) are likely to be important for the structural integrity of the protein and may not be replaceable by mutations elsewhere. Yeast has a much greater capacity to support the formation of disulfide bonds. In this way, the yeast can be used as a suitable expression host, instead of E. coli, particularly if it is desired to relieve the apparent limitation in the doubling of HRP imposed by any restriction on the formation of disulfides in E. coli. For example, S. cerevisiae can be used as a host for the expression of mutant proteins and HRP genes. This HRP (HRP1A6 gene) was cloned into the secretion vector pYEX-S1 obtained from Clontech (Palo Alto.

, CA) (35), producing pYEXS1-HRP (FIG 8). This vector uses the constitutive phosphoglycerate kinase promoter and a Kluveromyces lactis secretion signal peptide. The plasmid was first propagated in E. coli, and then transformed into S. cerevisiae strain BJ5465, obtained from the Yeast Genetic Stock Center (YGSC), University of California, Berkeley using the LiAc method as described (36). BJ5465 is deficient in protease and has been found to be generally suitable for secretion. A first generation of PCR prone to error of HRP in yeast was performed. Among the first 7,400 classified mutants, four variants showed 400% more HRP1A6 activity in yeast. Additional details and results are given in Example 2.

EXAMPLE 2 Functional expression of HRP in yeast through direct evolution This example describes the use of directed evolution to further improve the functional expression of HRP. As explained in the Example 1 Horseradish peroxidase was isolated (HRP1A6 gene).

Because HRP contains four well-preserved disulfides, and E. coli has only limited capacity to support the formation of disulfide bonds, further improvement in bacterial expression of HRP in E. coli can be restricted by forming correct pairs of disulfide-containing cysteines . Yeast cells, for example, S. cerevisiae, have a much greater ability to support the formation of disulfide bonds and may be more able to accommodate the disulfide bonds in peroxidase enzymes. In theory, these well-preserved disulfides in HRP (and other plant peroxidases) are probably important for the structural integrity of the protein and may not be replaceable by mutations elsewhere. Thus, the yeast can be used as a suitable expression host, instead of E. coli, particularly if it is desired to relieve the apparent limitation in the H-fold of RP imposed by any restriction on disulfide formation in E. col ?. According to this, S was chosen. cerevisiae as an alternative host for the expression of HRP. S. cerevisiae is both a microorganism and a eukaryote, and has much of the secretion and post-translational machinery of eukaryotic protein, such as ER and Golgi, which catalyze the formation of disulfide bonds and glycosylated polypeptides. Genetic manipulation techniques (in particular, gene transformation) are also readily available. One disadvantage is that the yeast naturally secretes few proteins. Moreover, the glycosylation of yeast differs significantly from that in larger eukaryotic organism, which could present problems for the secretion of glycoproteins (4). However, several proteins have been efficiently secreted from yeast (4). Strategically, the experiments in this example take advantage of the ability of the yeast to catalyze the formation of disulfide bonds while tuning to the glycosylation factor through the process of directed evolution.

Construction of the yeast expression system for HRP The HRP1 A6 HRP mutant of Example 1 was cloned into the yeast secretion vector pYEX-S 1 obtained from Clontech (Palo Alto, CA) (35), yielding pYEXS 1 -HRP ( FIG 8). This vector uses the constitutive phosphoglycerate kinase promoter and a K. lactis secretion signal peptide. PYEX-S 1 was digested with Sacl and then terminated blunt-ended with T4 DNA polymerase. The H RP 1 A6 mature gene was cloned from pETpelBH RP I A6 by PCR techniques using the pfu reading test polymerase (Stratagene, CA) that generate blunt-ended products. The used forward and backward primers were 5'-CAGTTAACCCCTACATTC-3 '[S EQ. I D NO. 25] and 5'- TCATTAAGAGTTGCTGTTGAC-3 '[SEQ I D NO. 26], respectively. The PCR fragments were then ligated into the restricted and blunt pYEX-S 1, and transformed into E. coli DH5a cells. A variety of colonies was collected and classified by the presence of the HRP gene by PCR reactions of colonies 1 8 with these two primers: 5'-CGTAGTTTTTCAAGTTCTTAG-3 [SEQ I D NO. 27] and 5'-TCCTTACCTTCCAATAATTC-3 '[SEQ IDN O. 28] The correct orientation of the HRP gene was further confirmed by sequencing This yeast expression vector is generally referred to hereafter as pYEXS 1 -HRP (FIG 8). In this construct, the HRP gene was placed directly downstream of the K. lactis secretion signal peptide and the expression is under the control of the constitutive phosphog licerate kinase promoter. The vector also carries the Amp resistance gene of E. coli, as well as the selectable yeast markers Ieu2-d and U RA3 (47). For expression experiments, the plasmid was first propagated in E. coli strain DH5a, and then transformed into S. cerevisiae strain BJ5465, obtained from the Yeast Genetic Stock Center (YGSC; University of California, Berkeley), using a LiAc method that uses single filament DNA as described by Gietz et al. (48). BJ5465 is deficient in protease and is generally suitable for secretion (4). Following the transformation, the cells were platinized in YN B selective medium supplemented with 20 μg / μl of leucine, 20 μg / μl of histidine, 20 μg / ml of adenine and 20 μg / ml of tryptophan. Colonies were harvested and grown in 96-well microplates in YEPD medium at 30 ° C in a circulating air incubator for 2 days and 1 6 hours. HRP activity tests were performed with a classic peroxidase assay, ABTS and hydrogen peroxide (26). The activity obtained from yeast for H RP 1 A6 was only about 1/10 of that of E coli, and in fact, slightly less than that obtained by the wild type in this construct Generation and classification of HRP mutants They were constructed libraries of HRP mutants by error-prone PCR (20) as described (53), except that the following two flanking primers of the HRP gene were used in the mutagenic PCR reactions: 5'-CAGTTAACCCCTACATTC-3 '[SEQ ID NO . 25] and 5'-TGATGCTGTCGCCGAAGAAG-3 '[SEQ ID NO. 29]. In addition, the parameters of thermal cyclization were: 95 ° C for 2 min, (94 ° C for 1 min, 50 ° C for 1 min and 72 ° C for 1 min, 30 cycles). The PCR products were purified with a Promega Wizard PCR set (Madison, Wl), digested with Sac I and Bam Hl (the first 27 amino acid residues of HRP were left unmodified). The digestion products were then subjected to gel purification with a QIAEX II gel extraction set (QIAGEN, Valencia, CA) and the HRP fragments were ligated again into pYEXS1-HRP1 A6. The ligation mixtures were transformed into HB101 cells of E. coli by electroporation with a Gene Pluser II (Bio-Rad), and selected in LB medium supplemented with 100 mg / ml of ampicillin. The colonies were collected directly from the LB plates. This plasmid DNA was subsequently used for transformation in yeast BJ5465 as described above. Single colonies were harvested from yeast nitrogen base plates (YNB), and grown at 30 ° C for 64 h in 96 well microplates containing YEPD medium (1% yeast extract, 1% peptone, 2 % glucose) in an incubator. The microplates were then centrifuged at 1,500 g for 10 min and 10 ml of the supernatant in each well was transferred to a new microplate with a Beckman 96-channel pipetting station (Mu lti mek, Beckman, Fulerton, CA) and tested for a total H RP activity. The standard global deviations of this measurement (including pipetting errors, which were approximately 2%) did not exceed 10%. The improved mutants (showing the highest total HRP activity) were recovered directly from the microplates, washed three times with sterile H202 and returned to grow in YNB selective medium. The plasmids containing the HRP mutants were first extracted from the yeast cells with a mini-preparation set of Zymo yeast plasmid (Zymo Research, Orange, CA) and then returned to E. coli X1 0-Gold for further propagation and isolation preparative. Where indicated, a pre-sorting of levant clones was performed expressing H RP as follows. Colonies were replicated on YNB plates on pure nitrocellulose membranes supported on MSI (Micron Separations I nc, Westboro, MA), which were grown on fresh YEP D agar at 30 ° C for 34 h. The membranes were then immersed in 1 00 ml of membrane substrate TM B (TM B 0.8 mM, H202 2.9 mM and 0 1 2% (w / v) of dextran sulfate as enhancer) for 5 min to allow A colored product was developed. Those yeast clones that exhibited a bright green color were again screened to the master YNB selective plates, and harvested and grown in YEPD for further classification as described above.

Mutagenesis of first-generation HRP in yeast to improve expression A first generation of HRP1A6 error-prone PCR in yeast was aimed at improving the level of expression. An error-prone PCR protocol was used that incorporates both unbalanced nucleotide and manganese ion concentrations as previously described (20, 21). This protocol showed to generate scarcely random mutations, allowing a sampling of a broader spectrum of changes of amino acid residues. The concentration of manganese ions used was 100 μM, which generated an error ratio of approximately 1-2 mutations per gene on average (22). The PCR products were purified with a Promega Wizard PCR set, digested with Sac I and Bam Hl (in this manner, the first 27 amino acid residues of HRP were left unmodified). The digestion products were then subjected to gel purification with a QIAEX II gel extraction set, and the HRP fragments were ligated back into the digested and gel purified pEXS1-HRP1A6 in a similar manner. The ligation mixtures were transformed into HB101 cells by electroporation with a Gene Pluser II (Bio-Rad). The colonies were scraped from the E. coli plates and resuspended in LB medium, from which the plasmids were prepared. The plasmids were then transformed into yeast and yeast colonies were obtained and grown as described above.

A total of approximately 14,000 colonies were collected and classified for this generation, which represented an exhaustive search for all accessible single mutants and a 95% probability for any mutant to be sampled at least once (25). Of these colonies, a number of mutants showed significantly greater activity than the parent (HRP1A6) in yeast. Two exemplary improved mutants are designated HRP1-117G4 [SEQ ID NO. 12 and SEQ ID NO.13] and HRP1-77E2 [SEQ ID NO. 5 and SEQ ID NO. 6]. HRP1-117G4 gave an activity 16 times greater than the parent or a total activity of approximately 220 units / l (FIG 9). HRP1-77E2 showed a total activity of approximately 147 units / l. Both were greater than the highest level obtained from E. coli. See also FIG. 12 (HRP1-77E2) and FIG. 16 (HRP1 -117G4).

Second generation mutagenesis of HRP in yeast to improve expression The second generation of error-prone PCR used HRP1-117G4 as the father. For this generation, a higher concentration of manganese ions was used to increase the mutation ratio. This change was made based on the following considerations. Because the classification can only handle a library of approximately 104 to 105 mutants at the present time, the mutagenesis ratio has been conservatively limited to create predominantly simple mutants in the past (15). In this example, the fraction of clones that is more active than the parent for a given generation remains relatively constant with an error ratio of up to 6 mutations per gene. The advantage of using higher error ratios is that it would allow neutral mutations to exist along with isolated beneficial mutations through classification. These accumulated neutral mutations may become useful in subsequent generations, either by providing a bridge to generate new types of mutations, or by synergistic interactions with newly created mutations. The concentration of manganese ions used in this generation was 350 μM, which generated an error ratio of approximately 4-5 mutations per gene on average (22). Additionally, a pre-classification of the colonies was performed using nitrocellulose membranes. This was possible because the higher error ratio significantly reduces the number of colonies that exhibit similar or greater activity than that of the father. The procedures were as follows. First, colonies of the master plates were replicated on nitrocellulose membranes and grown on YEPD plates at 30 ° C for one day and 6 hours. The membranes were then recovered from the plates and immersed in a mixture of TMB (tetramethyl benzidine) and H202. The colonies with the brightest color were identified and the corresponding mother colonies of the remaining plates were harvested and grown. For this generation, approximately 1 20,000 colonies were sorted (approximately 5,000 were harvested and fully grown) and the mutant HRP2-28 D6 was obtained. This showed an activity of 85% greater than his father, H RP 1 - 1 1 7G4, or a total activity of 41 0 a idades / l (FI G.9) Third generation of HRP mutagenesis in yeast to improve expression The third round of random mutagenesis was performed under similar conditions with HRP2-28D6 as the father. For this generation, a total of 90,000 colonies were pre-sorted and 3,000 were collected and grown. The best mutant, HRP3-17E12, gives an expression level of 1080 units / l, an increase of 160% over the HRP2-28D6 parent, or 85 times over the initial mutant, HRP1A6.

First generation mutagenesis of HRP in yeast to improve stability A generation of random mutagenesis of HRP was performed to improve thermostability and resistance to H202 using HRP1-77E2 as the parent. Random mutagenesis (with 100 μM manganese) and cell growth was performed essentially as described above (without any pre-sorting). Thermostability tests were performed with a MJ PTC-200 cyclizer (MJ Research, MA) at 73 ° C with an incubation time of 10 min. H202 resistance tests were performed separately on 25 mM H202 at room temperature and a pre-incubation time of 30 min, followed by ABTS classification at H20225 mM. Mutants that were more thermostable or chemically stable (resistant to H202) than the parent were further characterized at various temperatures (by thermostability) or H202 concentrations (for stability to H202).

Of 3,000 colonies classified, a thermostable mutant (HRP1-4B6) showed a T1 2 of about 6 ° C higher than that of the parent (T1 2 is the midpoint of transition of the inactivation curve of HRP as a function of temperature) ( FIG 10). Another mutant, HRP1-28B11 also showed some improvement in thermostability. The mutant HRP1-24D11 was not significantly more thermostable than its parent HRP1-77E2, but was more resistant to degradation of H202. (A common feedback mechanism to HRP enzymes is that they are degraded by H202, which is a reagent in the enzymatic reactions that facilitates HRP.) The mutant HRP1-24D11 retained approximately 60% > activity after incubation with H20225 mM for 30 min, while the father exhibited a residual activity of 42% under the same conditions (FIG 11).

Construction of vector for HRP expression in Pichia pastoris Enhanced HRP mutants were additionally cloned into the Pichia expression vector pPIZaB (Invitrogen Corp., Carlsbad, CA) to facilitate production of the mutants for biochemical characterization (54). This vector contains the signal peptide of factor a including a four-residue separator sequence Glu-Ala-GIu-Ala at the C-terminus of the secretion signal, and the PAOX1 promoter inducible with methanol. pPIZaB was restricted with Pst I first, terminated in blunt end with T4 DNA polymerase and then further digested with EcoR I. The sample was purified with a purification set of DNA Promega. The coding sequences for the HRP variants were obtained from the corresponding pYEXS1-HRP plasmids by PCR techniques using the Pfu test reading polymerase. The following two primers were used in the PCR reactions: 5'-TCAGTTAACCCCTACATTC-3 '(forward) [SEQ ID. DO NOT. 30] and 5'-CCACCACCAGTAGAGACATGG-3 '(backward) [SEQ ID NO. 31]. The PCR products were restricted with Eco RI, and ligated into pPIZaB digested and purified, yielding pPIZaB-HRP (Fig. 1b), in which the HRP genes were placed immediately downstream of the a-factor signal. The ligation products were first transformed into E. coli strain XL10-Gold and selected in low LB medium in salt (1% tryptophan, 0.5% yeast extract, 0.5% NaCl, pH adjusted to 7.5) supplemented with 25 mg / ml Zeocin (Cayla, Toulouse cedex, France). The colonies were classified by the presence of the HRP genes by colony PCR reactions (55) with these two primers: 5'-GAGAAAAGAGAGGCTGAAGCTC-3 '(forward) [SEQ ID NO. 32] and 5'-TCCTTACCTTCCAATAATTC-3 '(backward) [SEQ ID NO. 33]. The forward primer contained the last three nucleotides of the signal sequence and the first nucleotide of the HRP sequence (as underlined), which ensured that the positive colonies carried the full-length HRP genes in the correct orientation. The plasmids were isolated with a QIAgen mini-preparation set of liquid cultures of positive transformants, and were used for further transformation in Pichia for the expression of HRP The Pichia transformation was performed with electroporation according to "the instructions of the manufacturer (Invitrogen). Before the transformation, the plasmids were linearized with Pme I, purified with a Promega DNA purification set and were further treated with Princeton Centri-Sep columns balanced in dd H20 to remove any residual impurities Linearized vectors were integrated into the Pichia genome on the transformation via homologous recombination between the transforming DNA and the Pichia genome The transformed cells were platinized in YPDS medium (1% yeast extract, 2% peptone, 2% glucose, 1M sorbitol) supplemented with 100 mg / ml Zeocin For each construct containing a HRP mutant different, normally 4-6 were collected 10 transformants, and purified on fresh YPDS plates (supplemented with 100 mg / ml Zeocin) to isolate single colonies, which were then classified to identify the clones that conferred the highest expression levels. The Pichia strain X-33 was used in all expression experiments. It was determined in tests 15 initials that X-33 (Mut +) provided an expression of HRP significantly better than KM17 (MutS).

Expression of HRP in Pichia pastoris Pichia cell growth was performed at 30 ° C in a shaker. HE 20 cells were first grown to harbor pPIZaB-HRP overnight in BMGY (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1 34% YNB, 4X10-5% biotin, 1% glycerol) supplemented with 1% casamino acids at an OD 600 of 1.2-1 6. The cells were then pelleted and resuspended to an OD600 of -.- > 1.0 in BMMY medium (identical to BMGY, except 0.5% methanol instead of 1% or glycerol) supplemented with 1% casamino acids. Growth was continued for another 54-72 h. Sterile methanol was added every 24 h to maintain the induction conditions. H RP levels in the supernatants peaked around 54-60 h post-induction (time at which the OD60o reached approximately 8.0-10.0). Where applicable, at the induction point, vitamin B 1 1.0mM, 1.0mM d-ALA and 0.5ml / l trace element mixture were added (0.5g / l MgCl2, 30g / l of FeCI2.6H20, 1 g / I of ZnCI2.4H20, 0.2 g / l of CoCI2.6H20, 1 g / l of Na2Mo04.2H20, 0.5 g / l of CaCl2.2H20, 1 g / l of CuCI2 and 0.2 g / l of H2B03) were added to the growth medium.

Peroxidase activity assay Peroxidase activity tests were performed for H RP with a classical peroxidase assay, ABTS and hydrogen peroxide (26). 1 μl (or 1 5 μl) of cell suspension was mixed with 1 40 μl (or 1 50 μl) of ABTS / H202 (the concentrations of ABTS and H202 are 0.5 μM and 2.9 μM respectively, pH 4.5) in a microplate , and the increase in absorbance at 405 nm (e of oxidized ABTS is 34 700 cm "1 M" 1) was determined with a SpectraMax plate reader (Molecu lar Devices, Sunnyvale, CA) at 25 ° C. U nity of H RP is defined as the amount of enzyme that oxidizes 1 μ mol of ABTS per min under the test conditions Ensai de Guajacol The assay is performed with 1 mM H202 and 5 mM Guajacol in 50 mM phosphate buffer pH 7.0 and an increase in absorbance is followed at 470 nm (e of oxidized product at 470 nm is 26,000 cm ~ 1M "1) after addition of the yeast supernatant The stability of mutants is assessed using tests for initial activity (A,) and residual activity (AreS |), performed as described above with ABTS as substrate.AsS? d is measured after incubation of HRP mutants in NaOAc buffer pH 4.5, without containing H202 or containing H2021mM and incubating at 50 ° C for 10 min The assay for stability in organic solvent / buffer mixture (50 mM NaOAc buffer pH 4.5) was made with 1 mM H202 and 2 mM ABTS using supernatant of HRP mutants expressed in yeast (10 ul) in dioxane / buffer (20/80).

Production of HRP mutants in Pichia To obtain sufficient quantities of purified enzymes, Pichia was used in an additional effort to increase the production of HRP mutants. HRP-C (wild type), HRP2-13A10 (FIG.19, [SEQ ID No. 21 and SEQ ID No. 22]) and HRP3-17E12 (FIG. 20, [SEQ ID No. 23 and SEQ ID NO. 24]) were cloned into the Pichia pPICZaB secretion vector. In this construct (pPICZaB-HRP, FIG 1b), HRP was fused to the signal peptide of factor a and the expression was induced with methanol A normal expression curve is shown in FIG 25. For HRP3-17E12, after 55 h In culture, approximately 6,500 units / l of HRP activity were detected in the supernatant (FIG 25, empty squares) or 6.5 times of that obtained from yeast. The work of others as well as our laboratory found that the addition of trace metal elements, aminolevulinic acid, heme synthesis intermediate, and vitamin supplements to the growth medium (such as thiamin), results in a substantial improvement in holoenzyme yields. of proteins containing heme in E. coli (59-62). The addition of these additives to the growth medium of Pichia in our experiments led to an increase of 32% >; in the activity of HRP3-17E12 detected in the supernatant (FIG.25, filled squares).

Sequencing data The sequencing revealed that HRP1-77E2, the parent used for studies of thermostability and stability to H202 carries a reverted D255 to N255 (GAC to AAC) and a second mutation L37I (TTA to ATA). This residue is part of the helix 2, and is close to the heme pocket (34). See, FIG. 12, [SEQ. ID. NO.5] and [SEQ. ID. NO 6]. The HRP1-4B6 mutant carries K232M (AAG to ATG) in addition to L37I. This residue is part of the helix 14 and is exposed to solvent on the surface. See, FIG. 13, [SEQ. ID NO.7] and [SEQ ID NO. 8]. HRP1-28B11, the mutant with thermostability between HRP1-77E2 and HRP1-4B6 has the mutation F221L (TTT to TTA) in addition to L37I. This residue is in a structural loop and part of the substrate access channel (34). See, FIG. 14 [SEQ. ID. NO.9] and [SEQ ID NO 10] The mutant HRP1-24D11 contains the L131P (CTA to CCA) mutation in addition to L37I. This residue is at the tip of the helix 7 and is on the surface. See, FIG. 15, [SEQ. ID NO. 11] and [SEQ. ID. DO NOT. 12]. The mutant HRP1-117G4, a preferred mutant of the first generation in terms of total activity, contains five mutations with respect to its parent: (1) a reversion of D255 to N255 (GAC to AAC) (the wild-type sequence); (2) L131P (CTA to CCÁ); (3) L223Q (CTG to CAG); with silent mutations (4) in N135 (AAC to AAT) and (5) T257 (ACT to ACÁ). For the L223Q mutation, this amino acid residue is in a loop and is exposed to solvent. See, FIG. 16, [SEQ. ID. DO NOT. 13] and [SEQ. ID NO. 14]. Impressively, the improved HRP mutants, HRP1-80C12 (FIG.17, [SEQ ID NO: 17] and [SEQ ID NO.18]) and HRP1-77E2 (FIG. 20, [SEQ. NO. 23] and [SEQ ID NO. 24]) also carry the reverse D255 to N255 (GAC to AAC). In addition, HRP1-80C12 contains L131P (CTA-> CCA), found in HRP1-77G4. On the other hand, HRP-77E2 has a second mutation L37I (TTA-> ATA), which is part of the B helix and is in the heme pocket, presumably accessible to solvent as well. HRP2-28D6 (FIG.18, [SEQ ID NO: 19] and [SEQ ID NO: 20]) contains two additional mutations with respect to HRP1-117G4: T102A (ACT-> GCT) and P226Q ( CCA -> CAA). T102A is part of the D helix, and it is the only mutation found buried within the structure. P226Q is located in the same loop as L223Q. HRP2-13A10, on the other hand, contains four more mutations with respect to HRP1-117G4: R93L (CGA -> CTA); T102A (ACT -> GCT), K241T (AAA -> ACA); and V303E (GTG -> GAG). R93L, which is accessible to solvent, is in the structure loop that connects the helices C and D. K241T is in the structural loop that connects the helices G and H. This residue is exposed again to the solvent. Finally, V303E is part of the long filament that extends from the J helix to the C-terminus of the protein. These three mutations seem to contribute to the increased stability of this mutant compared to others. HRP3-17E12 contains three more mutations with respect to the parent HRP2-28D6: N47S (AAT -> AGT); K241T (AAA -> ACA), and a silent mutation in G121 (GGT - > GGC). It is worth noting that K241T was also found in HRP2-13A10. N47S is located in a structural loop that connects helix B and a 3-helix, and is also accessible to the solvent.

Mutation analysis The three improved mutant forms of the first generation of evolution carry the reverse D255N with respect to the father HRP1A6. This seems to suggest that the glycosylation sites in HRP are beneficial for doubling and expression. The function of glycosylation in proteins has been an intriguing subject, but its role in the doubling, processing and secretion of proteins is being gradually recognized (56-58). The three synonymous mutations can not easily be explained by changes in codon usage (63) Two of them, N135 (AAC -> ATT) and T257 (ACT -> ACA), resulted in few changes in the frequencies of codons used, whereas G121 (GGT -> GGC), a codon used more frequently (GCT, 61%) was replaced by a less frequent codon (GGC, 16%). However, it is not clear how this substitution would significantly affect the translation of HRP mRNA.

Characterization of mutants with respect to reactivity and stability In addition, the ABTS assay also uses the guajacol system as a second assay of independent activity for HRP mutants (FIG 21). Both tests show a good correlation with respect to the activity of the mutants. Figures 22a and 22b show the correlation between reactivity and stability after incubation at 50 ° C without H202 (a) and with 1 mM H202 (b). In both cases HRP 2-1 3A10 mutant shows the highest stability in combination with good reactivity. As revealed by sequencing, it appears that three amino acid changes are responsible for this stability. A similar pattern is observed in the organic solvent system (Figure 23) where HRP2-1 3A1 0 mutant shows the best proportions of activity in dioxane / buffer system versus that in buffer only.

EX EMPLO 3 Expression and secretion of CCP in E. coli Construction of expression vector for CCP _ The cytochrome c peroxidase gene (CC P) of S. cerevisiae from pT7CCP (16, 17), donated by Dr. Dave Goodin, The Scppps Research Institute, La Jolla, CA) it was recloned by PCR techniques to introduce an Msc I site at the start codon and a Hind III site immediately downstream from the stop codon. The PCR product was restricted with Msc I and Hind I 11, and then ligated into pET-22b (+) deferred in a similar manner, yielding pETCCP (Fig. 26). PT7CCP carries a gene for CCP, in which the N-terminal sequence has been modified to encode Met-Lys-Thr amino acids, as described in Goodin et al. (17) and Fitzgerald et al. (16) Thus, in this construct, the CCP gene was placed under the control of the T7 promoter, and fused in frame within the pelB signal sequence for periplasmic localization.

Expression of CCP The expression experiments of CCP in E. coli BL21 (DE3) were performed in LB medium containing 1 00 μg / ml of ampicillin. The cells were grown at 37 ° C at an A6oo of 0.7-0.8, at which time 1 PTG was added to a final concentration of 1 μM to induce the synthesis of CCP from the T7 promoter. Growth was continued at 30 ° C for an additional 20 hours and cells and supernatant were collected by centrifugation. It is known that CCP is correctly folded into E. coli. Surprisingly, more than 95% of the CCP protein was found in the LB culture medium at high levels (approximately 1000 mg / liter, as assessed by SDS-PAGE). The protein was active towards ABTS, showing that the secreted CCP is bent and contains the required ferric heme.

Having thus described exemplary embodiments of the invention, it should be noted by those skilled in the art that the descriptions are only exemplary and that various other alternatives, adaptations and modifications may be made within the scope of the invention. For example, practitioners will understand that the steps of any method of the invention can be performed in a general manner in any order, including simultaneously or contemporaneously, unless a particular order is expressly required, or is necessarily inherent or implicit with the purpose of practicing the invention. Accordingly, the invention is not limited to any specific embodiment or illustration herein. The invention is defined according to the appended claims and is limited only in accordance with the claims.

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SEQUENCE LISTING < 1 1 0 > Caltech Arnold, F. H. Lin, Z. < 1 20 > EUCARI OTI CAS PROTEIN VARIANTS THAT ARE EXPRESSLY EXPRESSED IN E. COLI < 1 30 > 9373 / 2E804 < 140 > To be assigned < 1 51 > 1 999-07-28 < 160 > 3"3 <170> FastSEQ for Windows Version 3.0 <21 0> 1 <21 1> 66 <212> DNA <21 3> Erwinia carotovora <400> 1 atgaaatacc tattgcctac ggcaggcgct ggattgttat tactcgctgc ccaaccagcc 60 atggcc 66 < 210 > 2 < 21 1 > 22 < 212 > PRT < 213 > Erwínia carotovora < 400 > 2 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala 20 < 210 > 3 < 211 > 927 < 212 > DNA < 213 > Escheric: hia coli < 400 > 3 atgcagttaa cccctacatt ctacgacaat agctgtccca acgtgtccaa catcgttcgc 60 gacacaatcg tcaacgagct cagatccgat cccaggatcg ctgcttcaat attacgtctg 120 cacttccatg actgcttcgt gaatggttgc gacgctagca tattactgga caacaccacc 180 agtttccgca ctgaaaagga tgcattcggg aacgctaaca gcgccagggg ctttccagtg 240 atcgatcgca tgaaggctgc cgttgagtca gcatgcccac gaacagtcag ttgtgcagac 300 tagctgcgca ctgctgacta acagagcgtg actcttgcag gcggaccgtc ctggagagtg 360 ccgctcggtc gacgtgactc cctacaggca ttcctagatc tggccaacgc caacttgcct 420 gctccattct tcaccctgcc ccagctgaag gatagcttta gaaacgtggg tctgaatcgc 480 ncgagtgacc ttgtggctct gtccggagga cacacatttg gaaagaacca gtgtaggttc 540 ggctctacaa atcatggata tttcagcaac actgggttac ctgáccccac gctgaacact 600 acgtatctcc agacactgag aggcttgtgc ccactgaatg gcaacctcag tgcactagtg 660 gactti-GATC tgcggacccc aaccatcttc gataacaagt actatgtgaa tctagaggag 720 cagaaaggcc tgatacagag tgatcaagaa ctgtttagca gtccagacgc cactgacacc 780 atcccactgg tgagaagttt tgctaactct actcaaacct tctttaacgc cttcgtggaa 840 gccatggacc gtatgg gtaa cattacccct ctgacgggta cccaaggcca gattcgtctg 900 aactgcagag tggtcaacag caactct 927 < 210 > 4 < 211 > 305 < 212 > PRT < 213 > Escherichia coli < 400 > 4 Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Se 1 5 10 15 Asn He Val Arg Asp Thr He Val Asn Glu Leu Arg Ser Asp Pro Ar «20 25 30 He Ala Ala Ser He Phe His Asp Cys Phe Val Asn Gly Cys Asp Al, 35 40 45 Be He Leu Leu Asp Asn Thr Thr Be Phe Arg Thr Glu Lys Asp Al. 50 55 60 Phe Gly Asn Wing Asn Be Wing Arg Gly Phe Pro Val He Asp Arg Me 65 70 75 80 Lys Wing Wing Val Glu Be Wing Cys Pro Arg Thr Val Ser Cys Wing As] 85 90 95 Leu Leu Thr He Wing Wing Gln Gln Ser Val Thr Leu Wing Gly Gly Pp 100 105 110 Ser Trp Arg Val Pro Leu Gly Arg Arg Asp Be Leu Gln Ala Phe Read 115 120 125 Asp Leu Ala Asn Ala Asn Leu Pro Ala Pro Phe Phe Thr Leu Pro Gil 130 135 1. 0 Leu I-ys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg Ser Ser Asp Read 145 150 155 16 'Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn Gln Cys Arg Phi 165 170 175 He Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu Pro Asp Pn ISO 185 190 Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly Leu Cys Pro Read 195 200 205 Asn Gly Asn Leu Be Ala Leu Val Asp Phe Asp Leu Arg Thr Pro Th: 210 215 220 He Phe Asp Asn Lys Tyr Tyr Val Asn Leu Glu Glu Gln Lys Gly Read 225 230 23ET 24? He Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asp Wing Thr Asp Th: 245 250 255 He Pro 'Leu Val Arg Ser Phe Wing Asn Ser Thr Gln Thr Phe Phe Asi 260 265 270 Wing Phe Val Glu Wing Met Asp Arg Met Gly Asn He Thr Pro Leu Th: 275 280 285 Gly Thr Gln Gly Gln He Arg Leu Asn Cys Arg Val Val Asn Ser AS 290 295 300 Ser 305 < 210 > 5 < 211 > 927 < 212 > DNA < 213 > Escherichia coli < 400 ^ 5 atgcagttaa cccctacatt ct; acgacaat agctgtccca acgtgtccaa catcgttcgc 60 gacacaatcg tcaacgagct cagatccgat cccaggatcg ctgcttcaat aatacgtctg 120 cacttccatg * actgcttcgt gaatggttgc gacgctagca tattactgga caacaccacc 180 agtttccgca ctgaaaagga aacgctaaca tgcattcggg gcgccagggg ctttccagtg 240 atcgatcgca tgaaggctgc cgttgagtca gcatgcccac gaacagtcag ttgtgcagac 300 tagctgcgca ctgctgacta acagagcgtg actcttgcag gcggaccgtc ctggagagtg 360 ccgctcggtc gacgtgactc cctacaggca ttcctagatc tggccaacgc caactitgcct 420 gctccattct tcaccctgcc ccagctgaag gatagcttta gaaacgtggg tctgaatcgc 480 tcgagtgacc ttgtggctct gtccggagga. cacacatttg gaaagaacca gtgtaggttc 540 ggctctacaa atcatggata tttcagcaac: actgggttac ctgaccccac gctgaacact 600 acgtatctcc agacactgag aggcttgtgc ccactgaatg gcaacctcag tgcactagtg 660 gactttgatc tgcggacccc aaccatcttc gataacaagt actatgtgaa tctagaggag 720 tgatacagag cagaaaggcc tgatcaagaa. ctgtttagca gtccaaacgc cactgacacc 780 atcccactgg tgagaagttt tgctaactct actcaaacct tctttaacgc cttcgtggaa 840 gccatggacc gtatgggtaa cattacccct ctgacgggta cccaaggcca gattcgtctg 900 aactgcagag tggtcaacaca caactct 927 < 210: > 6 < 211: > 307 < 212: > PRT < 213: > Escherichia coli < 400 > . 6 Met Gln Leu. Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser 1 5 10 15 Asn He Val Arg Thr He Val Asn Glu Leu Arg Ser Asp Pro Arg He 20 25 30 Ala Ala Ser - He He Arg Leu His Phe His Asp Cys Phe Val Asn Gly 35 40 45 Cys Asp Ala. Be He Leu Leu Asp Asn Thr Thr Be Phe Arg Thr Glu 50 55 60 Lys Asp Ala. Phe Gly Asn Wing Asn Be Wing Arg Gly Phe Pro Val He 65 70 75 80 Asp Arg Met Lys Wing Wing Val Glu Be Wing Cys Pro Arg Thr Val Ser 85 90 95 Cys Ala Asp - Leu Leu Thr He Ala Wing Gln Gln Ser Val Thr Leu Wing 100 105 110 Gly Gly Proi Ser Trp A g Val Pro Leu Gly Arg Arg Asp Ser Leu Gln 115 120 125 Wing Phe Leu. Asp Leu Wing Asn Wing Asn Leu Pro Wing Pro Phe Phe Thr 130 135 140 Leu Pro Gln. Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg Ser 145 150 155 160 Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn Gln Cys 165 170 175 Arg Phe He Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu Pro 180 185 190 Asp Pro Thr • Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly Leu Cys 1S5 200 205 Pro Leu Asn. Gly Asn Leu Be Ala Leu Val Asp Phe Asp Leu Arg Thr 210 215 220 Pro Thr He. Phe Asp Asn Lys Tyr Tyr Val Asn Read Glu Glu Gln Lys 225 230 235 240 Gly Leu He: Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asn Ala Thr 245 250 255 Asp Thr He. Pro Leu Val Arg Ser Phe Wing Asn Ser Thr Gln Thr Phe 260 265 270 Phe Asn Wing. Phe Val Glu Ala Met Asp Arg Met Gly Asn He Thr Pro 275 280 285 Leu Thr Gly Thr Gln Gly Gln He Arg Leu Asn Cys Arg Val Val Asn 290 295 300 Ser Asn Ser 305 < 210 > 7 < 21 1 > 927 < 21 2 > DNA < 213 > Escherichia coli < 400 > 7 atgcagttaa cccctacatt ctacgacaat agctgtccca acgtgtccaa catcgttcgc 60 gacacaatcg tcaacgagct cagatccgat cccaggatcg ctgcttcaat aatacgtctg 120 cacttccatg actgcttcgt gaatggttgc gacgctagca tattactgga caacaccacc 180 agtttccgca ctgaaaagga tgcattcggg aacgctaaca gcgccagggg ctttccagtg 240 atcgatcgca tgaaggctgc cgttgagtca gcatgcccac gaacagtcag ttgtgcagac 300 tagctgcgca ctgctgacta acagagcgtg actcttgcag gcggaccgtc ctggagagtg 360 ccgctcggtc gacgtgactc cctacaggca ttcctagatc tggccaacgc caacttgcct 420 gctccattct tcaccctgcc ccagctgaag gatagcttta gaaacgtggg tctgaatcgc 480 tcgagtgacc ttgtggctct gtccggagga cacacatttg gaaagaacca gtgtaggttc 540 ggctctacaa atcatggata tttcagcaac actgggttac ctgaccccac gctgaacact 600 acgtatctcc agacactgag aggcttgtgc ccactgaatg gcaacctcag tgcactagtg 660 gactttgatc tgcggacccc aaccatcttc gataacatgt actatgtgaa tctagaggag 720 cagaaaggcc tgatacagag tgatcaagaa ctgtttagca gtccaaacgc cactgacacc 780 atcccactgg tgagaagttt tgctaactct actcaaacct tctttaacgc cttcgtggaa 840 gccatggacc gtatgggt aa cattacccct ctgacgggta cccaaggcca gattcgtctg 900 aactgcagag tggtcaacag caactct 927 < 210 > 8 < 211 > 309 < 212 > PRT < 213 > Escherichia coli < 400 > 8 Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser 1 5 10 15 Asn He Val Arg Asp Thr He Val Asn Glu Leu Arg Ser Asp Pro Arg 20 25 30 He Wing Wing Being He He Arg Leu His Phe His Asp Cys Phe Val Asn 35 40 45 Gly Cys Asp Wing Being He Leu Leu Asp Asn Thr Thr Being Phe Arg Thr 50 '55 60 Glu Lys Asp Wing Phe Gly Asn Wing Asn Being Wing Arg Gly Phe Pro Val 65 70 75 80 He Asp Arg Met Lys Ala Ala Val Glu Be Ala Cys Pro Arg Thr Val 85 90 95 Be Cys Wing Asp Leu Leu Thr lie Wing Gln Gln Ser Val Thr Leu 100 105 110 Wing Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg Arg Asp Ser Leu 115 120 125 Gln Ala Phe Leu Asp Leu Ala Asn Ala Asn Leu Pro Wing Pro Phe Phe 130 135 140 Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg 145 150 155 160 Ser Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn 165 170 175 Gln Cys Arg Phe He Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly 180 185 190 Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly 195 200 205 Leu Cys Pro Leu Asn Gly Asn Leu Ser Wing Leu Val Asp Phe Asp Leu 210 215 220 Arg Thr Pro Thr He Phe Asp Asn Met Tyr Tyr Val Asn Leu Glu Glu 225 230 235 240 Gln Lys Gly Leu He Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asn 245 250 255 Wing Thr Asp Thr He Pro Leu Val Arg Ser Phe Wing Asn Ser Thr Gln 260 265 270 Thr Phe Phe Asn Wing Phe Val Glu Wing Met Asp Arg Met Gly Asn He 275 280 285 Thr Pro Leu Thr Gly Thr Gln Gly Gln He Arg Leu Asn Cys Arg Va l 290 295 300 Val Asn Ser Asn Ser 305 < 210 > 9 < 21 í > 927 < 21 2 > DNA < 213 > Escherichia col < 400 > 9 atgcagttaa cccctacatt ctacgacaat agctgtccca acgtgtccaa catcgttcgc 60 gacacaatcg tcaacgagct cagatccgat cccaggatcg ctgcttcaat aatacgtctg 120 cacttccatg actgcttcgt gaatgg tgc gacgctagca tattactgga caacaccacc 180 agtttccgca ctgaaaagga tgcattcggg aacgctaaca gcgccagggg ctttccagtg 240 atcgatcgca tgaaggctgc cgttgagtca gcatgcccac gaacagtcag ttgtgcagac 300 tagctgcgca ctgctgacta acagagcgtg actcttgcag gcggaccgtc ctggagagtg 360 ccgctcggtc gacgtgactc cctacaggca ttcctagatc tggccaacgc caacttgcct 420 gctccattct tcaccctgcc ccagctgaag gatagcttta gaaacgtggg tctgaatcgc 480 tcgagtgacc ttgtggctct gtccggagga cacacatttg gaaagaacca gtgtaggttc 540 ggctctacaa atcatggata tttcagcaac actgggttac ctgaccccac gctgaacact 600 acgtatctcc agacactgag aggcttgtgc ccactgaatg gcaacctcag tgcactagtg 660 gacttagatc tgcggacccc aaccatcttc gataacaagt actatgtgaa tctagaggag 720 cagaaaggcc tgatacagag tgatcaagaa ctgtttagca gtccaaacgc cactgacacc 780 atcccactgg tgagaagttt tgctaactct actcaaacct tctttaacgc cttcgtggaa 840 gccatggacc gtatgggt aa cattacccct ctgacgggta cccaaggcca gattcgtctg 900 aactgcagag tggtcaacag caactct - 927 < 210 > 10 < 211 > 309 < 212 > PRT < 213 > Eschepclhia coli < 400 > 10 Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser 1 5 10 15 Asn He Val Arg Asp Thr He Val Asn Glu Leu Arg Ser Asp Pro Arg 20 25 30 He Ala Ala Ser He He Arg Leu His Phe His Asp Cys Phe Val Asn 35 40 45 Gly Cys Asp Wing Being He Leu Leu Asp Asn Thr Thr Being Phe Arg Thr 50 55 60 Glu Lys Asp Wing Phe Gly Asn Wing Asn Being Wing Arg Gly Phe Pro Val 65 70 75 80 He Asp Arg Met Lys Wing Wing Val Glu Be Wing Cys Pro Arg Thr Val 85 90 95 Ser Cys Wing Asp Leu Leu Thr Wing Wing Gln Gln Ser Val Thr Leu 100 105 110 Wing Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg Arg Asp Ser Leu 115 120 125 Gln Wing Phe Leu Asp Leu Wing Asn Wing Asn Leu Pro Wing Pro Phe Phe 130 135 140 Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg 145 150 155 160 Ser Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn 165 170 175 Gln Cys Arg Phe He Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly 180 185 190 Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly 195 200 205 L eu Cys Pro Leu Asn Gly Asn Leu Be Wing Leu Val Asp Leu Asp Leu 210 215 220 Arg Thr Pro Thr He Phe Asp Asn Lys Tyr Tyr Val Asn Leu Glu Glu 225 230 235 240 Gln Lys Gly Leu He Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asn 245 250 255 Wing Thr Asp Thr He Pro Leu Val Arg Ser Phe Wing Asn Ser Thr Gln 260 265 270 Thr Phe Phe Asn Wing Phe Val Glu Wing Met Asp Arg Met Gly Asn He 275 280 285 Thr Pro Leu Thr Gly Thr Gln Gly Gln He Arg Leu Asn Cys Arg Val 290 295 300 Val Asn Ser Asn Ser 305 < 210 > 11 < 211 > 927 < 212 > DNA < 213 > Eschepchia coli < 400 > 11 atgcagttaa cccctacatt ctacgacaat agctgtccca acgtgtccaa catcgttcgc 60 gacacaatcg tcaacgagct cagatccgat cccaggatcg ctgcttcaat aatacgtctg 120 cacttccatg actgcttcgt gaatggttgc gacgctagca ta tactgga caacaccacc 180 agtttccgca ctgaaaagga aacgctaaca tgcattcggg gcgccagggg ctttccagtg 240 atcgatcgca tgaaggctgc cgttgagtca gcatgcccac gaatragtcag ttgtgcagac 300 tagctgcgca ctgctgacta acagagcgtg actcttgcag gcggaccgtc ctggagagtg 360 ccgctcggtc gacgtgactc cctacaggca ttcccagatc tggccaacgc caacttgcct 420 gctccattct tcaccctgcc ccagctgaag gatagcttta gaaacgtggg tctgaatcgc 480 tcgagtgacc ttgtggctct gtccggagga cacacatttg gaaagaacca gtgtaggttc 540 at-catggata ggctctacaa tttcagcaac actgggttac ctgaccccac gctgaacact 600 acgtatctcc agacactgag aggcttgtgc ccactgaatg gcaacctcag tgcactagtg 660 gactttgatc tgcggacccc aaccatcttc gataacaagt actatgtgaa tctagaggag 720 cagaaaggcc tgatacagag tgatcaagaa ctgtttagca gtccaaacgc cactgacacc 780 atcccactgg tgagaagttt tgctaactct actcaaacct tctttaacgc cttcgtggaa 840 gccatggacc gtatg ggtaa cattacccct ctgacgggta cccaaggcca gattcgtctg 900 aactgcagag tggtcaacag caactct 927 < 210 > 1 2 < 21 1 > 312 < 212 > PRT <; 213 > Escherichia coli < 400 > 12 Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser 1 5 10 15 Asn He Val Arg Asp Thr He Val Asn Glu Leu Arg Ser Asp Pro Arg 20 25 30 lie Ala Ala Ser He He Arg Leu His Phe His Asp Cys Phe Val Asn 35 40 45 Gly Cys Asp Wing Being He Leu Leu Asp Asn Thr Thr Being Phe Arg Thr 50 55 60 Glu Lys Asp Wing Phe Gly Asn Wing Asn Being Wing Arg Gly Phe Pro Val 65 70 75 80 He Asp Arg Met Lys Wing Wing Val Glu Be Wing Cys Pro Arg Thr Val 85 90 95 Ser Cys Wing Asp Leu Leu Thr Wing Wing Gln Gln Ser Val Thr Leu 100 105 110 Wing Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg Arg Asp Ser Leu 115 120 125 Gln Wing Phe Pro Asp Leu Wing Asn Wing Asn Leu Pro Wing Pro Phe Phe 130 135 140 Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg 145 150 155 160 Ser Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn 155 170 175 Gln Cys Arg Phe Wing Cys Thr He Met Asp Arg Leu Tyr Asn Phe Ser 180 185 190 Asn Thr Gly Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr 195 200 205 Leu Arg Gly Leu Cys Pro Leu Asn Gly Asn Leu Be Wing Leu Val Asp 210 215 220 Phe Asp Leu Arg Thr Pro Thr He Phe Asp Asn Lys Tyr Tyr Val Asn 225 230 235 240 Leu Glu Glu Gln Lys Gly Leu He Gln Ser Asp Gln Glu Leu Phe Ser 245 250 255 Ser Pro Asn Wing Thr Asp Thr He Pro Leu Val Arg Ser Phe Wing Asn 260 265 270 Be Thr Gln Thr Phe Phe Asn Wing Phe Val Glu Wing Met Asp Arg Met 275 280 285 Gly Asn He Thr Pro Leu Thr Gly Thr Gln Gly Gln He Arg Leu Asn 290 295 300- Cys Arg Val Val Asn Ser Asn Ser 305 310 < 210 > 13 < 211 > 927 < 212 > DNA < 21 3 > Escherichia coh < 400 > 13 atgcagttaa cccctacatt ctacgacaat agctgtccca acgtgtccaa catcgttcgc 60 gacacaatcg tcaacgagct cagatccgat cccaggatcg ctgcttcaat aatacgtctg 120 cacttccatg actgcttcgt gaatggttgc gacgctagca tattactgga caacaccacc 180 agtttccgca ctgaaaagga tgcattcggg aacgctaaca gcgccagggg ctttccagtg 240 atcgatcgca tgaaggctgc cgttgagtca gcatgcccac gaacagtcag ttgtgcagac 300 tagctgcgca ctgctgacta acagagcgtg actcttgcag gcggaccgtc ctggagagtg 360 ccgctcggtc gacgtgactc cctacaggca ttcctagatc tggccaacgc caacttgcct 420 gctccattct tcaccctgcc ccagctgaag gatagcttta gaaacgtggg tctgaatcgc 480 tcgagtgacc ttgtggctct gtccggagga cacacatttg gaaagaacca gtgtaggttc 540 ggctctacaa atcatggata tttcagcaac actgggttac ctgaccccac gctgaacact 600 acgtatctcc agacactgag aggcttgtgc ccactgaatg gcaacctcag tgcactagtg 660 gactttgatc tgcggacccc aaccatcttc gataacaagt actatgtgaa tctagaggag 720 cagaaaggcc tgatacagag tgatcaagaa ctgtttagca gtccaaacgc cacagacacc 780 atcccactgg tgagaagttt tgctaactct actcaaacct tctttaacgc cttcgtggaa 840 gccatggacc gtatggg taa cattacccct ctgacgggta cccaaggcca gattcgtctg 900 aactgcagag tggtcaacag caactct 927 < 210 > 14 < 211 > 307 < 212 > PRT < 213 > Escherichia col < 400 > 14 Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser 1 5 10 15 Asn He Val Arg Thr He Val Asn Glu Leu Arg Ser Asp Pro Arg He 20 25 30 Ala Ala Ser He He Arg Leu His Phe His Asp Cys Phe Val Asn Gly 35 40 45 Cys Asp Wing Be He Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr Glu 50 55 60 Lys Asp Wing Phe Gly Asn Wing Asn Being Wing Arg Gly Phe Pro Val He 65 70 75 80 Asp Arg Met Lys Wing Wing Val Glu Be Wing Cys Pro Arg Thr Val Ser 85 90 95 Cys Wing Asp Leu Leu Thr He Wing Wing Gln Gln Ser Val Thr Leu Wing 100 105 110 Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg Arg Asp Ser Leu Gln 115 120 125 Wing Phe Leu Asp Leu Wing Asn Wing Asn Leu Pro Wing Pro Phe Phe Thr 130 135 140 Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg Ser 145 150 155 160 Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn Gln Cys 165 170 175 Arg Phe He Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu Pro 180 185 190 Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly Leu Cys 195 200 205 Pro Leu Asn Gly Asn Leu Be Wing Leu Val Asp Phe Asp Leu Arg Thr 210 215 220 Pro Thr He Phe Asp Asn Lys Tyr Tyr Val Asn Leu Glu Glu Gln Lys 225 230 235 240 Gly Leu He Gln Ser Asp Gln Glu Leu Phe Ser Pro Asn Ala Thr 245 250 255 Asp Thr He Pro Leu Val Arg Ser Phe Wing Asn Ser Thr Gln Thr Phe 260 265 270 Phe Asn Wing Phe Val Glu Wing Met Asp Arg Met Gly Asn He Thr Pro 275 280 285 Leu Thr Gly Thr Gln Gly Gln He Arg Leu Asn Cys Arg Val Val Asn 290 295 300 Ser Asn Ser 305 < 210 > 15 < 211 > 24 < 212 > DNA < 213 > "Artificial sequence" < 220 > < 223 > Oligonucleotide initiator < 400 > 15 ttattgctca gcggtggcag cagc 24 < 210 > 16 < 21 1 > 24 < 212 > DNA < 21 3 > "Artificial sequence" < 220 > < 223 > Oligonucleotide primer < 400 > 16 aagcgctcat gagcccgaag tggc 24 < 210 > 17 < 211 > 927 < 212 > DNA < 213 > Escherichia coli < 400 > 17 atgcagttaa cccctacatt ctacgacaat agctgtccca acgtgtccaa catcgttcgc 60 gacacaatcg tcaacgagct cagatccgat cccaggatcg ctgcttcaat attacgtctg 120 cacttccatg actgcttcgt gaatggttgc gacgctagca tattactgga caacaccacc 180 agtttccgca ctgaaaagga tgcattcggg aacgctaaca gcgccagggg ctttccagtg 240 atcgatcgca tgaaggctgc cgttgagtca gcatgcccac gaacagtcag ttgtgcagac 300 tagctgcgca ctgctgacta acagagcgtg actcttgcag gcggaccgtc ctggagagtg 360 ccgctcggtc gacgtgactc cctacaggca ttcccagatc tggccaacgc caacttgcct 420 gctccattct tcaccctgcc ccagctgaag gatagcttta gaaacgtggg tctgaatcgc 480 tcgagtgacc ttgtggctct gtccggagga cacacatttg gaaagaacca gtgtaggttc 540 ggctctacaa atcatggata tttcagcaac actgggttac ctgaccccac gctgaacact 600 acgtatctcc agacactgag aggcttgtgc ccactgaatg gcáacctcag tgcactagtg 660 gactttgatc tgcggacccc aaccatcttc gataacaagt actatgtgaa tctagaggag 720 cagaaaggcc tgatacagag tgatcaagaa ctgtttagca gtccaaacgc cactgacacc 780 atcccactgg tgagaagttt tgctaactct actcaaacct tctttaacgc cttcgtggaa 840 gccatggacc gtatgg gtaa cattacccct ctgacgggta cccaa.ggcca gattcgtctg 900 aactgcagag tggtcaacag caactct 927 < 210 > • 18 < 211 > • 309 < 212 > • PRT < 213 > - Escherichia coli < 400 > 18 Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser 1 5 10 15 Asn He Val Arg Asp Thr lie Val Asn Glu Leu Arg Ser Asp Pro Arg 20 25 30 He Ala Ala Be He Leu Arg Leu His Phe His Asp Cys Phe Val Asn 35 40 45 Gly Cys Asp Wing Being He Leu Leu Asp Asn Thr Thr Being Phe Arg Thr 50 55 60 Glu Lys Asp Wing Phe Gly Asn Wing Asn Being Wing Arg Gly Phe Pro Val 65 70 75 80 He Asp Arg Met Lys Wing Wing Val Glu Be Wing Cys Pro Arg Thr Val 85 90 95 Ser Cys Wing Asp Leu Leu Thr Wing Wing Gln Gln Ser Val Thr Leu 100 105 110 Wing Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg Arg Asp Ser Leu 115 120 125 Gln Wing Phe Pro Asp Leu Wing Asn Wing Asn Leu Pro Wing Pro Phe Phe 130 135 140 Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg 145 150 155 160 Ser Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn 165 170 175 Gln Cys Arg Phe He Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly 180 185 190 Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly 195 200 205 Leu Cys Pro Leu Asn Gly Asn Leu Be Ala Leu Val Asp Phe Asp Leu 210 215 220 Arg Thr Pro Thr He Phe Asp Asn Lys Tyr Tyr Val Asn Leu Glu Glu 225 230 235 240 Gln Lys Gly Leu He Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asn 245 250 255 Wing Thr Asp Thr He Pro Leu Val Arg Ser Phe Wing Asn Ser Thr Gln 260 265 270 Thr Phe Phe Asn Wing Phe Val Glu Wing Met Asp Arg Met Gly Asn He 275 280 285 Thr Pro Leu Thr Gly Thr Gln Gly Gln He Arg Leu Asn Cys Arg Val 290 295 300 Val Asn Ser Asn Ser 305 < 210 > 19 < 211 > 928 < 212 > DNA < 213 > Escherichia col < 400 > 19 atgcagttaa cccctacatt ctacgacaat agctgtccca acgtgtccaa catcgttcgc 60 gacacaatcg tcaacgagct cagatccgat cccaggatcg ctgcttcaat attacgtctg 120 cacttccatg actgcttcgt gaatggttgc gacgctagca tattactgga caacaccacc 180 hagtttccgc actgaaaagg atgcattcgg gaacgctaac agcgccaggg gctttccagt 240 gatcgatcgc atgaaggctg ccgttgagtc cgaacagtca agcatgccca gttgtgcaga 300 cctgctggct atagctgcgc aacagagcgt gactcttgca ggcggaccgt cctggagagt 360 gccgctcggt cgacgtgact ccctacaggc attcccagat ctggccaatg ccaacttgcc 420 tgctccattc ttcaccctgc cccagctgaa ggatagcttt agaaacgtgg gtctgaatcg 480 ctcgagtgac cttgtggctc tgtccggagg acacacattt ggaaagaacc agtgtaggtt 540 aggctctaca catcatggat atttcagcaa cactgggtta c? tgacccca cgctgaacac 600 tacgtatstc cagacactga gaggcttgtg cccactgaat ggcaacctca gtgcactagt 660 ggactttgat cagcggaccc aaaccatctt cgataacaag atctagagga tactatgtga 720 ctgatacaga gcagaaaggc gtgatcaaga actgtttagc agtccaaacg ccacagacac 780 catcccactg gtgagaagtt ttgctaactc tactcaaacc ttctttaacg ccttcgtgga 840 agccatggac cgtatgg gta acattacccc tctgacgggt acccaaggcc agattcgtct 900 gaactgcaga gtggtcaaca gcaactct 928 < 210 > 20 < 211 > 309 1 < 212 > PRT < 213 > Escheri chiei col i < 400 > : 20 Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser 1 5 10 15 Asn He Val Arg Asp Thr He Val Asn Glu Leu Arg Ser Asp Pro Arg 20 25 30 He Ala Ala Be He Leu Arg Leu His Phe His Asp Cys Phe Val Asn '35 40 45 Gly Cys Asp Wing Being He Leu Leu Asp Asn Thr Thr "Ser Phe Arg Thr 50 55 60 Glu Lys Asp Wing Phe Gly Asn Wing Asn Being Wing Arg Gly Phe Pro Val 65 70 75 80 He Asp Arg Met Lys Wing Wing Val Glu Be Wing Cys Pro Arg Thr Val 85 90 95 Ser Cys Wing Asp Leu Leu Wing Wing Wing Gln Gln Ser Val Thr Leu 100 105 110 Wing Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg Arg Asp Ser Leu 115 12D 125 Gln Wing Phe Pro Asp Leu Wing Asn Wing Asn Leu Pro Wing Pro Phe Phe 130 135 140 Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg 145 150 155 160 Ser Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn 165 170 175 Gln Cys Arg Phe He Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly 180 185 190 Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly 195 200 2 05 Leu Cys Pro Leu Asn Gly Asn Leu Be Ala Leu Val Asp Phe Asp Gln 210 215 220 Arg Thr Gln Thr He Phe Asp Asn Lys Tyr Tyr Val Asn Leu Glu Glu 225 230 235 240 Gln Lys Gly Leu He Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asn 245 250 255 Wing Thr Asp Thr He Pro Leu Val Arg Ser Phe Wing Asn Ser Thr Gln 260 265 270 Thr Phe Phe Asn Wing Phe Val Glu Wing Met Asp Arg Met Gly Asn He 275 280 285 Thr Pro Leu Thr Gly Thr Gln Gly Gln He Arg Leu Asn Cys Arg Val 290 295 300 Val Asn Ser Asn Ser 305 < 210 > 21 < 211 > 928 < 212 > DNA < 213 > Escherichia coli < 400 > 21 atgcagttaa cccctacatt ctacgacaat agctgtccca acgtgtccaa catcgttcgc 60 mgacacaatc gtcaacgagc tcagatccga tcccaggatc gctgcttcaa tattacgtct 120 gcacttccat gactgcttcg tgaatggttg cgacgctagc atattactgg acaacaccac 180 cagtttccgc actgaaaagg atgcattcgg gaacgctaac agcgccaggg gctttccagt 240 gatcgatcgc atgaaggctg ccgttgagtc ctaacagtca agcatgccca gttgtgcaga 300 cctgctggct atagctgcgc aacagagcgt gactcttgca ggcggaccgt cctggagagt 360 gccgctcggt cgacgtgact ccctacaggc attcccagat ctggccaatg ccaacttgcc 420 tgctccattc ttcaccctgc cccagctgaa ggatagcttt agaaacgtgg gtctgaatcg 480 ctcgagtgac cttgtggctc tgtscggagg acacacattt ggaaagaacc agtgtaggtt 540 aggctctaca catcatggat atttcagcaa cactgggtta cctgacccca cgctgaacac 600 tacgtatctc cagacactga gaggcttgtg cccactgaat ggcaacctca gtgcactagt 660 ggactttgat cagcggaccc caaccatctt cgataacaag atctagagga tactatgtga 720 ctgatacaga gcagacaggc gtgatcaaga actgtttagc agtccaaacg ccacagacac 780 catcccactg gtgagaagtt ttgctaactc tactcaaacc ttctttaacg ccttcgtgga 840 agccatggac cgtatgg gta acattacccc tctgacgggt acccaaggcc agattcgtct 900 gaactgcaga gaggtcaaca gcaactct 928 < 210 > 22 < 211 > £ 30) < 212 > PRT < 213 > Escherichisi coli < < 100 > 22 Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser 1 5 10 15 Asn He Val Arg Asp Thr He Val Asn Glu Leu Arg Ser Asp Pro Arg 20 25 30 He Ala Ala Be He Leu Arg Leu His Phe His Asp Cys Phe Val Asn 35 40 45 Gly Cys Asp Wing Being He Leu Leu Asp Asn Thr Thr Being Phe Arg Thr 50 55 60 Glu Lys Asp Wing Phe Gly Asn Wing Asn Being Wing Arg Gly Phe Pro Val 65 70 75 80 He Asp Arg Met Lys Wing Wing Val Glu Be Wing Cys Pro Leu Thr Val 85 90 95 Ser Cys Wing Asp Leu Leu Wing Wing Wing Gln Gln Ser Val Thr Leu 100 105 110 Wing Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg Arg Asp Ser Leu 115 120 125 Gln Wing Phe Pro Asp Leu Wing Asn Wing Asn Leu Pro Wing Pro Phe Phe 130 135 140 Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg 145 150 155 160 Ser Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn 165 170 175 Gln Cys Arg Phe He Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly 180 185 190 Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly 195 200 205 L eu Cys Pro Leu Asn Gly Asn Leu Be Wing Leu Val Asp Phe Asp Gln 210 215 220 Arg Thr Pro Thr He Phe Asp Asn Lys Tyr Tyr Val Asn Leu Glu Glu 225 230 235 240 Gln Lys Gly Leu He Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asn 245 250 255 Wing Thr Asp Thr He Pro Leu Val Arg Ser Phe Wing Asn Ser Thr Gln 260 265 270 Thr Phe Phe Asn Wing Phe Val Glu Wing Met Asp Arg Met Gly Asn He 275 280 '285 Thr Pro Leu Thr Gly Thr Gln Gly Gln He Arg Leu Asn Cys Arg Glu 290 295 300 Val Asn Ser Asn Ser 305 < 210 > 23 < 21 1 > 927 < 21 2 > DNA < 213 > Escherichia coli < 400 > 23 atgcagttaa cccctacatt ctacgacaat agctgtccca acgtgtccaa catcgttcgc 60 gacacaatcg tcaacgagct cagatccgat cccaggatcg ctgcttcaat attacgtctg 120 cacttccatg actgcttcgt gagtggttgc gacgctagca tattactgga caacaccacc 180 agtttccgca ctgaaaagga tgcattcggg aacgctaaca gcgccagggg ctttccagtg 240 atcgatcgca tgaaggctgc cgttgagtca gcatgcccac gaacagtcag ttgtgcagac 300 tagctgcgca ctgctggcta acagagcgtg actcttgcag gcggaccgtc ctggagagtg 360 ccgctcggcc gacgtgactc cctacaggca ttcccagatc tggccaatgc caacttgcct 420 gctccatñct tcaccctgcc ccagctgaag gatagcttta gaaacgtggg tctgaatcgc 480 tcgagtgacc ttgtggctct gtccggagga cacacatttg gaaagaacca gtgtaggttc 540 ggctctacaa atcatggata tttcagcaac actgggttac ctgaccccac gctgaacact 600 acgtatctcc agacactgag aggcttgtgc ccactgaatg gcaacctcag tgcactagtg 660 agcggaccca gactttgatc aaccatcttc gataacaagt actatgtgaa tctagaggag 720 cagacaggcc tgatacagag tgatcaagaa ctgtttagca gtccaaacgc cacagacacc 780 atcccactgg tgagaagttt tgctaactct actcaaacct tctttaacgc cttcgtggaa 840 gccatggacc gtatgg gtaa cattacccct ctgacgggta cccaaggcca gattcgtctg 900 aactgcagag tggtcaacag caactct - 927 < 210 > 24 < 211 > 309 < 212 > PRT < 213 > Escherichia coli < 400 > 24 Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser 1 5 10"15 Asn He Val Arg Asp Thr He Val Asn Glu Leu Arg Ser Asp Pro Arg 20 25 30 He Ala Ala Ser He Leu Arg Leu His Phe His Asp Cys Phe Val Asn 35 40 ~ 45 Gly Cys Asp Ala Ser He Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr 50 55 60 Glu Lys Asp Ala Phe Gly Asn Ala Asn Ser Ala Arg Gly Phe Pro Val 65 70 75 80 He Asp Arg Met Lys Ala Ala Val Glu Be Ala Cys Pro Arg Thr Val 85 90 95 Being Cys Wing Asp Leu Leu Wing He Wing Wing Gln Gln Being Val Thr Leu 100 105 110 Wing Gly Gly Pro Being Trp Arg Val Pro Leu Gly Arg Arg Asp Being Leu 115 120 125 Gln Wing Phe Pro Asp Leu Wing Asn Wing Asn Leu Pro Wing Pro Phe Phe 130 135 140 Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg 145 150 155 160 Be Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn 165 170 175 Gln Cys Arg Phe He Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly 180 185 190 Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly 195 200 205 Leu Cys Pro Leu Asn Gly Asn Leu Ser Ala Leu Val Asp Phe Asp Gln 210 215 220 Arg Thr Gln Thr He Phe Asp Asn Lys Tyr Tyr Val Asn Leu Glu Glu 225 230 235 240 Gln Lys Gly Leu He Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asn 245 250 255 Wing Thr Asp Thr He Pro Leu Val Arg Ser Phe Wing Asn Ser Thr Gln 260 265 270 Thr Phe Phe Asn Wing Phe Val Glu Wing Met Asp Arg Met Gly Asn He 275 280 285 Thr Pro Leu Thr Gly Thr Gln Gly Gln He Arg Leu Asn Cys Arg Val 290 295 300 Val Asn Ser Asn Ser 305 < 210 > • 25 < 211 > • 18 < 212 > • DNA < 213 > • "Artificial sequence" < 220 > < 223 > Oligoonucleotide Initiative < 400 > 25 cagttaaccc ctacattc < 21 0 > 26 < 21 1 > 21 < 2 1 2 > D NA < 21 3 > 'Artificial Secu nce < 220 > < 223 > I nucleotide oligonucleotide < 400 > 26 tcattaagag ttgctgttga c 21 < 210 > 27 < 211 > 21 < 212 > DNA < 213 > "Artificial sequence" < 220 > < 223 > Oligonucleotide primer < 400 > 27 cgtagttttt caagttctta g 21 < 210 > 28 < 211 > 20 < 212 > DNA < 213 > "Artificial Sequence <220> <223> Oligonucleotide Initiator <400> 28 tccttacctt ccaataattc 20 < 210 > 29 < 211 > 20 < 212 > DNA < 213 > "Artificial sequence" < 220 > < 223 > Oligonucleotide primer < 400 > 29 tgatgctgtc gccgaagaag 20 < 210 > 30 < 211 > 19 < 212 > DNA < 213 > "Artificial Sequence <220> <223> Oligonucleotide Initiator <400> 30 tcagttaacc cctacattc 19 < 210 > 31 < 211 > 21 < 212 > DNA < 213 > "Artificial sequence" < 220 > < 223 > Oligonucleotide primer < 400 > 31 ccaccaccag tagagacatg g 21 < 210 > 32 < 211 > 22 < 212 > DNA < 213 > "Artificial Sequence <220> <223> Oligonucleotide Initiator <400> 32 gagaaaagag aggctgaagc te 22 < 210 > 33 < 211 > 20 < 212 > DNA < 213 > "Artificial Sequence <220> <223> Oligonucleotide Initiator <400> 33 tccttacctt ccaataattc 20

Claims (1)

1. CLAIMS 1 . A method for obtaining and improving the production of an expression-resistant polypeptide comprising the steps of: providing at least one parent polynucleotide encoding a parent polypeptide that is resistant to functional expression in selected host cells, altering the nucleotide sequence of the parent polynucleotide to produce a population of mutant polypeptides; transforming the host cells to express the mutant polypeptides; and classifying functional mutants produced by the host cells and having at least one doubling or improved expression without inclusion bodies. 2. A method of claim 1, wherein the parent polypeptide forms inclusion bodies when they are overexpressed in the host cells. 3. A method of claim 1, wherein the parent polypeptide has at least one of a disulphide bridge structure and a glycosylated structure. 4. A method of claim 1, wherein the parent polypeptide has or is associated with at least one heme group. 5. A method of claim 1, wherein the parent polypeptide is produced in a non-functional manner when it is overexpressed in the host cells, and is produced in a functional form when it is sub-expressed in the cells. the guests. 6. A method of claim 5, wherein the parent polypeptide is overexpressed under the control of an inducible promoter in the presence of an inducer, and is sub-expressed under the control of an inductible promoter in the absence of an inducer. The method of claim 1, comprising repeating the method for a number of cycles, wherein the parent polynucleotide in each cycle is a mutant polynucleotide of a previous cycle. The method of claim 1, wherein the step of altering the nucleotide sequence is performed by at least one of random mutagenesis, site-specific mutagenesis and intermixing of DNA. The method of claim 7, wherein the step of altering the nucleotide sequence is performed by at least one of random mutagenesis, site-specific mutagenesis or intermixing of DNA. 10. A polynucleotide developed according to the method of claim 1. eleven . A polynucleotide developed according to the method of claim 7. 1. A polynucleotide developed according to the method of claim 9. 13. A method of claim 1, wherein the host cells are transformed by a vector having a signal sequence that directs the secretion of polypeptides encoded by the mutant polmucleotide. The method of claim 1, wherein the signal sequence is the Pel B signal sequence. 5. A method of claim 1, wherein the host cells are easy host cells. 16. A method of claim 1, wherein the host cells are selected from yeasts and bacteria. 17. A method of claim 7, wherein the host cells are selected from yeasts and bacteria. 18. A method of claim 9, wherein the host cells are selected from yeasts and bacteria. 9. The method of claim 1, wherein the host cells are E. coli cells. The method of claim 1, wherein the host cells are S. cerevisiae cells. twenty-one . The method of claim 1, wherein the host cells are P. pastoris cells. 22. The method of claim 9, wherein the host cells are E. coli cells. 23. The method of claim 9, wherein the host cells are S. cerevisiae cells. 24. The method of claim 1, wherein the host cells are P. pastoris cells. 25. The method of claim 7, wherein the polypeptide is a protein containing heme 26. The method of claim 9, wherein the polypeptide is a protein containing h emo. 27. The method of claim 1 8, wherein the polypeptide is a protein containing heme. 28. The method of claim 7, wherein the polypeptide is a peroxidase enzyme. 29. The method of claim 9, wherein the polypeptide is a peroxidase enzyme. 30. The method of claim 18, wherein the polypeptide is a peroxidase enzyme. 31 The method of claim 26, wherein the polypeptide is a horseradish peroxidase enzyme. 32. The method of claim 27, wherein the polypeptide is a horseradish peroxidase enzyme. 33. The method of claim 28, wherein the polypeptide is a horseradish peroxidase enzyme. SUMMARY This invention relates to the improved expression of developed polynucleotide and polypeptide sequences encoding eukaryotic enzymes, particularly peroxidase enzymes, in conventional or easy expression systems. Several methods for directed evolution of polynucleotide sequences can be used to obtain the improved sequences. The improved characteristics of the polypeptides or proteins generated in this manner include improved doubling, without formation of inclusion bodies and retained functional activity. In a particular embodiment, the invention relates to improved expression of the non-spicy horseradish peroxidase gene and horseradish peroxidase enzymes. I heard / 22 < F