KR20010083086A - Expression of Functional Eukaryotic Proteins - Google Patents

Abstract

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

Description

Expression of Functional Eukaryotic Proteins

The publications and references mentioned herein and listed in the Annex Bibliography are intact and included in the Bibliography, respectively. References are made to numbers in the text and in the following bibliography.

Many proteins of interest are produced by individuals with "eukaryotic" cells. It is a cell with a nucleus surrounded by a membrane and containing DNA in a structure called a chromosome. All multicellular individuals, such as humans and animals, and many unicellular animals have eukaryotic cells. Single cell entities, such as bacteria, are "prokaryotic" cells. The cell has a primitive nucleus with DNA in a bounded structure, but lacks a nuclear membrane that is characteristic of chromosomes and eukaryotic cells. Prokaryotic individuals are generally much easier and less expensive than eukaryotic cells to grow, maintain, and manipulate.

Genetic engineering and recombinant DNA and RNA techniques have made it possible to produce proteins, hormones and enzymes unique to any individual, using cells of another individual as a "factory" or host cell expression system. In particular, it is desirable to express proteins of eukaryotic origin in prokaryotic host cells because prokaryotic cells can grow the same cells in very large amounts to produce large amounts of the desired foreign protein. For example, if a human protein can be applied in a sufficient amount to a patient with protein deficiency it can be useful as a drug. Such proteins cannot be easily or ethically obtained by separation from human cells, and cannot be easily produced by tissue culture or direct chemical synthesis. Other proteins and enzymes are useful in the industry. For example, some enzymes can degrade food products and are useful as laundry detergents. However, commercial applications require large amounts of protein and a high degree of control.

To address this problem, recombinant genetic engineering techniques have been developed to use the genetic machinery of cells such as bacteria and yeast to produce human and other proteins. Genetic materials such as polynucleotides encoding a desired protein are recombined with the genetic material in the host cell to express the foreign genetic material into which the host cell is introduced, and to produce the desired polypeptide and protein. Because bacteria and yeast are easy and economical to grow and maintain in large quantities, they are suitable as host cells and can be used to reliably and repeatedly produce foreign proteins.

However, many proteins cannot be easily expressed in host cells, including bacteria and yeast. Such expression resistant polypeptides or proteins are not expressed at all or are inefficiently expressed in very low yields. Even if the protein is expressed, some or all of its function may be lost. In some cases, the expressed protein may lose some or all of its active conformation and even become denatured and completely inactivated. The expressed protein may also be surrounded by inclusion bodies in the host cell. It is a particle or globule containing agglomerates or inactive expression proteins isolated from the rest of the cell. This makes recovery of production proteins from host cells difficult because separation and purification techniques are difficult, inefficient, time consuming and expensive. Efforts to produce high levels of expression resistant proteins as active and functional forms have been in the past few years and have not been successful. In particular, commercially important expression resistant enzymes, such as horseradish peroxidase, have not been expressed in high yield or simply, economically and easily in host cells. Instead, these enzymes are produced in nonfunctional and inactive forms such as inclusion bodies and are manipulated and restored to obtain activating enzymes in relatively poor yields.

Some proteins made by cells are secreted or delivered extracellularly, which improves the yield and efficiency of subsequent separation and purification steps. However, many proteins are not secreted intrinsically and are difficult to artificially secrete because they do not contain chemical groups that easily cross the cell membrane. In particular, it is difficult to design host cells that secrete proteins that tend to form inclusion bodies and proteins that are compatible with them. Therefore, there is a need for the development of more advanced techniques for expressing exogenous proteins, particularly proteins of eukaryotic origin, recombinant proteins secreted by host cells in high yield without loss of function or activity.

As discussed above, a particular challenge in producing foreign proteins in host cell expression systems is that when using conventional recombinant hosts such as E. coli or yeast, many foreign proteins do not fold correctly into functional protein structures. In ability (cf. 1-4). As a result, polypeptides produced in recombinant host cell systems are often degraded upon synthesis or accumulation in inclusion bodies. This is true for eukaryotic proteins with disulfide bonds or sugar chains. Underlying reasons, which are not clearly understood and possibly due to various factors, are the correct protein folding cofactors, such as the unnatural recombination environment in which proteins accumulate (35) and the molecular chaperone in the E. coli host. lack of cofactors (3). In addition, glycosylation has been implicated in protein folding that is absent in bacteria in eukaryotic cells (36).

Folding problems can impede the production of large scale proteins for pharmaceutical or industrial applications. The lack of high efficiency expression systems has also been a bottleneck in applying directed evolution techniques to optimize proteins and reaction conditions for desired applications. Using random mutations and genetic recombination, followed by selection and screening, regulatory improvement techniques have been successful in improving the properties of enzymes necessary for industrial applications (eg substrate specificity, activity in organic solvents and stability at elevated temperatures). Has been applied (cf. 5). Eukaryotic enzymes can be used in numerous applications, but the improvement of electrical proteins by regulatory improvement techniques is limited by their ability to not functionally express it in easy recombinant hosts.

For example, the difficulties of expression of peroxidase enzymes in easy expression hosts have presented at least two technical challenges in realizing the potential of peroxidase as a biocatalyst. First, the modification of electroenzymes by protein engineering for industrial applications has been hindered. For example, regulatory improvements have developed expressions in hosts such as E. coli and S. cerevisiae , in which a large number of mutations and variants can be made. Second, due to the lack of efficient expression in a suitable foreign host, it is impossible to mass produce such proteins economically.

One way to obtain an active form of recombinant expression protein is to refold the recombinant expression protein from the inclusion body under laboratory conditions, but this process is labor intensive and inefficient (cf. 1-3). Furthermore, this is not a viable condition for directed evolution techniques that require the process of screening tens of thousands of mutants. A more preferred way to solve this problem is to identify mutations in the target gene that facilitate folding in the host cell environment. Numerous studies have gradually revealed that some residues in the amino acid sequence have profound effects on the protein folding itself. Thus, it would be advantageous if scientists could identify mutations in the target gene that facilitate protein folding in the host cell environment. This can avoid inclusion body obstructions, but such techniques require the discovery, identification and use of special mutations.

For example, in a series of studies by King and co-workers, several amino acid substitutions have been shown to interfere with the folding of the phage p22 tail protein at the limit of in vivo conditions, and a second site suppressor mutation It has been found that mutations can compensate for the lack of folding mutations (6). In another study, the replacement of tyrosine 35 with leucine in a bovine pancreatic trypsin inhibitor has been shown to remove kinetic traps in the folding process under laboratory conditions. ). Furthermore, while several mutations of human interleukin 1 due to cassette mutagenesis of several selective amino acid residues are expressed in lysed form in E. coli , the wild type is usually insoluble and forms inclusion bodies. Reported (8). In another study, one site-directed mutation was found to improve the folding yield of recombinant antibodies (9).

It is difficult to predict which amino acid residues are necessary for the function or stability of the protein, not to mention the folding. Thus, it would be desirable to have a way to systematically find mutations that benefit the expression and folding of proteins without loss of biological activity. Intentional improvement techniques may be useful to achieve this goal. This improved approach uses DNA shuffling to simultaneously generate random mutations and recombination to produce variants with improved desired properties for wild-type proteins. Point mutations occur due to the intrinsic inaccuracy of Taq polymerase chain reaction (PCR) associated with reassembly of nucleic acid sequences. For example, Stemmer and co-workers applied the technique to genes encoding green fluorescence protein (GFP) to produce proteins that fold much better than wild-type in E. coli . Reference: 10).

One group of proteins of particular interest is heme proteins, that is, proteins that contain iron, including heme groups. This protein has many biological and biochemical uses and includes an enzyme called peroxidase that facilitates the oxidation or reduction of hydrogen peroxide as a reactant. Peroxide refers to a compound in which oxygen atoms are connected to each other in addition to molecular oxygen. For example, horseradish peroxidase (HRP), a heme enzyme, is widely used as a reporter in diagnostic assays. HRP chemically binds the initial reactant or substrate in the presence of peroxides such as hydrogen peroxide (H ₂ O ₂ ) and catalyzes the reaction to produce water (H ₂ O) as a byproduct. This reaction can be used to suggest whether other reactions of interest have occurred, or whether some substance such as the HRP initial reactant is present in the mixture or sample. It would be beneficial for everyone to provide a method for producing large amounts of HRP and other heme or peroxidase enzymes using efficient and inexpensive systems such as prokaryotic expression systems. However, native HRP contains four disulfide bonds and has sugar chains (~ 21%), although the sugar moiety does not significantly affect activity or stability (11). As a result, previous efforts to express HRP in bacteria have produced inclusion bodies with no functional expression. Prior to the present invention, no effort for successful expression in yeast was achieved (cf. 12-14).

Thus, in order to eliminate the need for laborious refolding methods in laboratory conditions, there is an urgent need for the development of new and improved methods for expressing proteins that are difficult to express under normal conditions. In particular, there is a need for protein expression methods that are well suited for use in connection with intentional improvement techniques.

In particular, the present invention relates to error prone polymerase chain reaction (PCR) and to identify mutations that facilitate functional expression in bacteria ( E. coli, B. subtilis ) and yeast ( S. cerevisiae ) and A method of screening a library of HRP mutations generated by DNA shuffling is described. In one embodiment of the invention, the variant of the invention is a functional and active HRP expressed in E. coli without formation of inclusion bodies in an amount of about 110 μg / L. This is comparable to the amount obtained from refolding techniques in laboratory conditions, which are used in the prior art to recover HRP enzymes from inclusion bodies, and at a much higher cost, time and effort.

Summary of the Invention

The limitations observed so far for the use of native proteins are believed to be the result of improvements. Proteins have been modified in the environment and background of living individuals to perform specific biological functions in living conditions, not laboratory or industrial conditions. In some cases, improvements are not much preferred or needed over enzymes of optimized efficiency. The output, efficiency, working conditions, stability, and other properties of known expression systems are not expected to change, and there are no limitations that appear to be inherent in the nature of cell expression systems. It is possible that the proteins used in these systems can be improved under laboratory conditions, alternatively to alter or improve the properties of the protein, for example, to obtain more efficient expression, folding and secretion while maintaining protein activity. It is possible for proteins to be developed. Improved proteins can also be obtained by screening cultures of native individuals or expressed gene libraries (3).

When expressed using an easy expression system such as E. coli , many proteins form inclusion bodies and become inert by failing to fold correctly. The present invention utilizes intentional refinement techniques to create new polynucleotides encoding functional mutant proteins with increased production capacity in expression systems without inactivation or inclusion body formation. In a preferred embodiment, the protein is secreted extracellularly.

In many cases, secreting proteins from the bacteria into the culture has several advantages because the desired substrate does not readily cross the cell membranes of E. coli . Enzyme secretion facilitates screening in intentional improvement studies because the secreted enzyme promotes the reaction in the culture medium, so that substrates that cannot enter the cell can be used. If the degree of secretion is very sufficient, enzyme secretion greatly simplifies the production of recombinant proteins, since the culture supernatant is usually free of contaminants. Nevertheless, protein secretion from bacteria to cultures remains a very difficult problem for enzymes with large subgroups such as heme.

In order to effectively regulate secretion of peroxidases such as HRP or CCP into cultures, this can be achieved by using appropriate signal peptides such as signals of pectate lyase B (PelB) from Erwinia carotovora . You can solve the problem. This signal peptide generally applies to other proteins, including heme complements such as cytochrome P450 peroxidase and other peroxidases.

According to one embodiment of the present invention, a probiotic cell expression system such as E. coli and proteins that are easily folded after expression in yeast and that are readily secreted out of the host cell by the expected amount of protein produced by such expression system are in a laboratory condition. Intentional refinement or random mutagenesis is used for production in Furthermore, the activity of these proteins is not achieved by the mutagenesis step after proper selection.

Accordingly, the present invention improves the expression of polynucleotides encoding peroxidase enzymes and polynucleotides encoding horseradish peroxidase variants that exhibit improved expression in conventional expression systems by using intentional refinement techniques. Provide a method.

These and many additional advantages of the present invention can be further understood by reference to the following detailed description in conjunction with the accompanying drawings.

The Government has the rights under the support numbers N0014-96-1-0340 and N00014-98-1-0657 awarded by the United States Navy to the present invention.

This application claims priority under United States Patent Application No. 60 / 106,840, filed November 3, 1998 and United States Patent Application No. 60 / 094,403, filed July 28, 1998.

The present invention relates to the selection and preparation of functional polypeptides or proteins, in particular polynucleotides encoding eukaryotic proteins, in particularly facile host cell expression systems. Easy expression systems include strong prokaryotic cells (eg, bacteria) and eukaryotic cells (eg, yeast). In particular, the present invention relates to the recombinant production of expression resistant functional eukaryotic proteins by host cells in high yields without reduced activity, denaturation, inclusion bodies or other structures or functions. In a preferred embodiment, the expressed protein is secreted by the host cell. Examples of preferred proteins of the present invention include proteins having horseradish peroxidase, peroxidase such as cytochrome c peroxidase (CCP) and heme do. Polynucleotides that encode and express electric proteins in recombinant host cell expression systems are also included in the present invention.

Figure 1A is a schematic diagram showing the plasmid pETpelBHRP, which is an E. coli HRP expression vector pETHRP. The HRP gene (with an additional methionine residue at the N-terminus) is placed on pET-22b (+) so that it is directly below the signal sequence of pectate lyase B (PelB) of Erwinia carotovora . Inserted. Expression is under the control of the T7 promoter.

1B is Pichia. It is a schematic diagram showing P. pastoris expression vector pPICZαB-HRP. The HRP gene was inserted so that it was directly below the α-factor signal of the plasmid. Expression is under the control of the P _aox1 promoter induced by methanol.

Figure 2 shows the nucleic acid and amino acid sequence of the pelB signal peptide (SEQ ID NO: 1 and SEQ ID NO: 2).

Figure 3 is a diagram showing the nucleic acid and amino acid sequence encoding the HRP enzyme variant named HRP1A6 (SEQ ID NO: 3 and SEQ ID NO: 4).

4 is a map of the expression vector pETpelBHRP1A6.

5A is a graph showing the relative activity of improved HRP mutations (1A6) in wild type and E. coli .

5B is a graph showing the prospects of first generation HRP mutations sorted by decreasing activity. Activity was normalized to wild type.

6 is a graph showing the activity level of mutant HRP1A6 at various IPTG concentrations.

7 is a structural diagram of HRP showing the substitution position of Asn255 with Asp in the surface loop of the HRP1A6 mutant. This schematic was generated from an HRP equivalent (34) published using Insight II (Molecular Biosystems) program.

8 is a map of the pYEXS1-HRP expression vector containing the coding sequence of HRP cloned into the secretion plasmid pYEX-S1.

9 is a graph showing the activity levels of HRP1A6 and three other mutations (HRP1-77E2, HRP1-117G4, HRP2-28D6) obtained in S. cerevisiae by intentional improvement. In this graph, HRP1A6 is the parent of HRP1-77E2 and HRP1-117G4, while HRP1-117G4 is the parent of HRP2-28D6.

10 is a graph showing the residual activity of several HRP mutations as a function of temperature in a thermal inactivation curve showing the relative thermal stability of the mutations.

FIG. 11 is a graph showing the residual activity of several HRP mutations as a function of hydrogen peroxide concentration in a titration curve showing relative degradation resistance in the presence of hydrogen peroxide of the mutation.

Figure 12 shows the nucleotide and amino acid sequences of the HRP enzyme variant named HRP1-77E2 (SEQ ID NO: 5 and SEQ ID NO: 6).

Figure 13 is a diagram showing the nucleotide and amino acid sequence of the HRP enzyme variant named HRP1-4B6 (SEQ ID NO: 7 and SEQ ID NO: 8).

Figure 14 shows the nucleotide and amino acid sequence of the HRP enzyme variant named HRP1-28B11 (SEQ ID NO: 9 and SEQ ID NO: 10).

Figure 15 shows the nucleotide and amino acid sequence of the HRP enzyme variant named HRP1-24D11 (SEQ ID NO: 11 and SEQ ID NO: 12).

Figure 16 shows the nucleotide and amino acid sequence of the HRP enzyme variant named HRP1-117G4 (SEQ ID NO: 12 and SEQ ID NO: 13).

Figure 17 shows the nucleotide and amino acid sequence of the HRP enzyme variant named HRP1-80C12 (SEQ ID NO: 17 and SEQ ID NO: 18).

Figure 18 shows the nucleotide and amino acid sequences of the HRP enzyme variant named HRP2-28D6 (SEQ ID NO: 19 and SEQ ID NO: 20).

Figure 19 shows the nucleotide and amino acid sequence of the HRP enzyme variant named HRP2-13A10 (SEQ ID NO: 21 and SEQ ID NO: 22).

20 is a diagram showing the nucleotide and amino acid sequence of the HRP enzyme variant named HRP3-17E12 (SEQ ID NO: 23 and SEQ ID NO: 24).

21 is a graph showing the activity of improved HRP mutations in wild type, parent (HRP1A6) and S. cerevisiae strain BJ5465. The measured value was measured by ABTS assay. Cells were grown in shaking flasks at 30 ^° C. for 64 hours.

22 is a graph showing the correlation between reactivity and stability (A _resid / A _i ) of HRP mutations.

FIG. 23 is a graph showing the reactivity of HRP mutations in an organic solvent / water system.

24 is a family tree showing the lineage of the mutations. Nucleotide substitutions are shown in parentheses immediately following equivalent amino acid substitutions. For each generation, new mutations are marked with "*".

25 is a graph showing secreted HRP activity accumulation for variant HRP3-17E2 from Pichia .

Fig. 26 is a schematic diagram of the yeast cytochrome c peroxidase expression vector pETCCP.

The present invention relates to methods of improving protein expression that degrade as soon as they are synthesized, using conventional expression systems, to form inclusion bodies or fail to fold correctly in the expression system environment.

Justice

As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, more preferably within 5% of a given value or range. .

The term "substrate" means any substance or compound that is interpreted as being converted or changed into another compound by an enzyme catalyst. The term includes aliphatic and aromatic compounds and includes combinations of compounds, such as solutions, mixtures and other materials containing not only one compound but at least one substrate.

As used herein, the term "oxidation reaction" or "oxygenation reaction" refers to chemicals that involve adding oxygen or supplying oxygen to a substrate to form an oxidized substrate or product. Or a biochemical reaction. Oxidation reactions typically occur like reduction reactions (hence the term "redox" for both oxidation and reduction). Compounds are said to be "oxidized" when they gain oxygen or lose electrons. When a compound loses oxygen or gains electrons it is said to be "reduced".

The term “enzyme” refers to a substance that is more or less entirely composed of proteins or polypeptides that more or less specifically promote one or more chemical or biochemical reactions.

The term "polypeptide" is a chain of chemical building blocks called amino acids linked together by chemical bonds called peptide bonds. Proteins or polypeptides, including enzymes, may be "native" or "wild-type", meaning the forms present in nature of the protein or polypeptide; Or the protein or polypeptide may be “mutant”, “variant” or “modified”, which may be made, modified, derived, slightly different or changed from a natural protein or other mutant protein. Means that. A “parent” polypeptide or enzyme is any polypeptide or enzyme from which other polypeptides or enzymes are derived or made from any method, tool or technique and the parent itself may be a natural or mutant polypeptide or enzyme. A parent polynucleotide is one that encodes a parent polypeptide. A "test enzyme" is a substance that contains a protein that tests whether it has the properties of an enzyme. The term "enzyme" may also refer to catalytic polynucleotides (DNA or RNA).

An “activity” of an enzyme is a measure of the ability to catalyze a reaction and can be expressed as the rate at which the reaction product is produced. For example, enzymatic activity can be expressed as the amount of product produced per time unit, enzyme unit (concentration and weight). "Stability" of an enzyme refers to its ability to act over time under particular circumstances and conditions. One way to assess stability is to measure the resistance to activity loss over time in a given environment. Enzyme stability can also be assessed by other methods, such as determining relative activity in the folded or unfolded state of the enzyme. Thus, when one enzyme has much less activity loss under the same conditions than another enzyme, has resistance to denaturation or is more durable by other suitable methods, the enzyme is more stable or has improved stability than other enzymes. For example, more "thermostable" enzymes have greater resistance to unfolding or loss of function when exposed to heat or temperature rise. One way to evaluate this is to determine the "melting temperature" or T _m of the protein. The melting temperature, called the midpoint, is the temperature at which half of the protein is fully folded and denatured. The midpoint can typically be determined by calculating the midpoint of the protein denaturation titration curve as a function of temperature. Thus, proteins with higher T _m require more heat and are more thermostable to cause denaturation. Stated another way, a protein with a higher T _m has a much smaller number of protein molecules denatured at the same temperature than a protein with a lower T _m , meaning that a protein with resistance to denaturation is more stable. (Much less denaturation occurs at the same temperature). Another measure of stability is T _1/2, which is the middle transition point of the protein inactivation curve as a function of temperature. T _1/2 is the temperature at which a protein loses half its activity. Thus, a protein with a higher T _1/2 requires more heat and is more thermostable to inactivate it. Stated another way, a protein with a higher T _1/2 has a much smaller number of protein molecules that are inactivated at the same temperature than a protein with a lower T _1/2 and a protein that is resistant to inactivation. More stable (more active at the same temperature). Such assays are called "thermal shift" assays because the inactivation or denaturation curves plotted against temperature "shift" to higher or lower temperatures when stability increases or decreases. Because. Thermal stability can also be measured in other ways. For example, the longer half-life (t _1/2 ) for enzymatic activity at high temperatures is an indicator of thermal stability.

An "oxidation enzyme" is an enzyme that typically promotes one or more oxidation reactions by adding, inserting, investing or transferring oxygen from a donor to a substrate. Such enzymes are also called oxidoreductase or redox enzymes, and are oxygenase, hydrogenase or reductase, oxidase and peroxidase. Peroxidase.

The terms "oxygen donor" and "oxidizing agent (oxidant)" refer to a substance, molecule or compound that supplies oxygen to a substrate in an oxidation reaction. Typically, the oxidant is reduced (accepts electrons). Examples of oxygen providers include, but are not limited to, oxygen molecules or alkyl peroxides including t-butyl peroxide and most preferably hydrogen peroxide (H ₂ O ₂ ). Peroxide refers to a compound having two oxygen molecules connected to each other.

A "luminescent" material refers to electromagnetic radiation that can be detected by mechanisms including color changes, ultraviolet absorption, fluorescence and phosphorescence, or changes in electromagnetic waves, most notably visible light. Means the material produced. Preferably, the luminescent material according to the invention exhibits detectable color, fluorescence and ultraviolet absorption.

By "chemiluminescent agent" is meant a substance that improves the detectability of a luminescent signal (such as fluorescence) by increasing the intensity or duration of the signal. One example and preferred chemiluminescent agent is 5-amino-2,3-dihydro-1,4-phthalazinedione (5-amino-2,3-dihydro-1,4-phthalazinedione, luminol) and their analogs. have. Other chemiluminescent agents are tetramethyl-1,2-dioxetane (TMD), 1,2-dioxetanones and 1,2-dioxetanedione (1). 1,2-dioxetane, such as, 2-dioxetanedione).

The term "polymer" means a compound in which two or more building blocks (mers) are repeatedly connected to each other. For example, a "dimer" is a compound in which two structural units are linked to each other.

"Cofactor" means a nonprotein material that is essential for the activity of an enzyme. "Coenzyme" refers to a cofactor that interacts directly with an enzyme or promotes an enzyme mediated reaction. Many coenzymes serve as carriers. For example, NAD ⁺ and NADP ⁺ carry hydrogen atoms from one enzyme to another. "Ancillary protein" means a protein essential for the activity of an enzyme.

The term “host cell” is selected, modified and transformed in any way for the production of a substance by a cell (eg, the expression of a gene, DNA or RNA sequence, protein or enzyme by a cell). Cell of an individual being grown, grown, used and manipulated.

"DNA (deoxyribonucleic acid)" refers to a sequence or chain in which chemical structural units, called nucleotide bases, adenine (A), guanine (G), cytosine (C) and thymine (T) are connected to each other at the backbone of the deoxyribose sugar. . DNA has one strand of nucleotide base or two complementary strands forming a double helix. "RNA (ribonucleic acid)" refers to a sequence or chain in which chemical structural units, called nucleotide bases, adenine (A), guanine (G), cytosine (C), and uracil (U) are connected to each other on the backbone of a ribose sugar. RNA typically has one nucleotide base.

A "polynucleotide" or "nucleotide sequence" is a series of consecutive nucleotide bases (also called "nucleotides") on DNA and RNA and refers to two or more nucleotide chains. Nucleotide sequences typically carry genetic information, including information used to make proteins and enzymes by cellular machinery. The term includes two or one strand of genome or cDNA, RNA, genetically engineered synthetic nucleotides, sense and anti-sense polynucleotides (although here only the sense strands are shown). This includes DNA-DNA, DNA-RNA, RNA-RNA hybrids as well as one- and two-stranded molecules, ie, "protein nucleic acids" (PNAs) in which bases are conjugated to amino acid backbones. It also includes nucleic acids with modified bases such as thio-uracil, thio-guanine and fluoro-uracil.

Polynucleotides herein may be exposed to natural regulatory sequences, and may also include promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5 'and 3 Heterologous sequences, such as non-coding regions. Nucleic acids can also be modified by many methods known in the art. Examples of such modifications include methylation, "cap", substitution of one or more naturally occurring nucleotides with analogues, non-electrical linkages (methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates) and electrically linkages. nucleotide-to-nucleotide modifications such as (phosphorothioates, phosphorodithioates), including but not limited to. Polynucleotides include proteins (nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (acridine, psoralen, etc.), chelators (metals, radioactive metals, iron, oxidative metals, etc.) And additional moieties joined by one or more covalent bonds, such as alkylators. Polynucleotides may also be induced by the formation of methyl or ethyl phosphotriester bonds or alkyl phosphoramidate linkages. Furthermore, the polynucleotide herein can be modified to have a tag that can provide a detection signal either directly or indirectly. Examples of tags include radioisotopes, fluorescent materials, biotin, and the like.

Proteins and enzymes are made in the host cell by DNA and RNA information according to the genetic code. In general, DNA sequences with information of particular proteins and enzymes are "transcribed" into the RNA of that sequence. Again the RNA sequence is "translated" to an amino acid sequence that forms a protein or enzyme. An "amino acid sequence" is a chain of two or more amino acids. Each amino acid is represented by one or more nucleotide triplets in DNA or RNA. Each triplet produces one codon corresponding to one amino acid. For example, the amino acid lysine is encoded as a nucleotide triplet or codon AAA or codon AAG (the genetic code has some repeatability called degeneracy, which means that most amino acids have one or more corresponding codons). Means there is). Since the nucleotides of the DNA and RNA sequences are translated into three groups for protein production, it is important to start sequencing at the correct amino acids so that the correct triplet is translated. The manner in which nucleotide sequences are grouped into codons is called a "reading frame."

The term "gene" or "structural gene" refers to a DNA sequence that corresponds to or encodes a specific sequence of amino acids that includes all or part of one or more proteins, and the conditions under which the gene is expressed. It may or may not include regulatory DNA sequences, such as promoters, which determine. Some genes that are not structural genes can be transcribed from DNA into RNA but are not translated into amino acid sequences. Other genes can act as regulators of structural genes or regulators of DNA transcription.

A “coding sequence” or sequence that “encodes” a polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, results in the generation of the electrical polypeptide, protein, or enzyme. That is, the nucleotide sequence encoding the amino acid sequence for the polypeptide, protein, enzyme. When an RNA polymerase transcribes a coding sequence to make an mRNA, and then the mRNA is spliced into trans-RNA and translated into a protein by the coding sequence, the coding sequence is transcribed and translated in the cell. Under the control of the transcriptional and translational control sequence. Preferably, the coding sequence is double-stranded DNA that is transcribed and translated into protein in vitro or in vivo cells under the control of appropriate regulatory sequences. The boundary of the coding sequence is determined by the 5 'end codon and the 3' end translation codon. Coding sequences include, but are not limited to, prokaryotic sequences, cDNAs from eukaryotic mRNAs, genomic DNA sequences of eukaryotic (eg, mammalian) DNA, and synthetic DNA sequences. In order for the coding sequence to be expressed in eukaryotic cells, the polyadenylation signal and transcription termination sequence are usually located at the 3 'end of the coding sequence.

Transcription and translation control sequences are DNA regulatory sequences provided for expression of coding sequences in host cells such as promoters, enhancers, terminators. In eukaryotic cells, the polyadenylation sequence is a control sequence.

A "promoter sequence" is a DNA regulatory region capable of incorporating RNA polymerase in a cell to initiate transcription in the 3 'direction of the coding sequence. For purposes of defining the present invention, the promoter sequence is bounded by a transcription initiation site at the 3 'end and includes a minimum number of bases and elements necessary to initiate transcription above the background level. 'Expand in the direction. Within the promoter sequence, transcriptional initiation sites (as easily defined by the nuclease S1 mapping) are found, as well as protein consensus sequences to which RNA polymerase binds. As above, promoter DNA is a DNA sequence that initiates, regulates, mediates and controls the expression of DNA. Promoters can be "inducible", meaning that they are affected by the presence and amount of other compounds called "inducers." For example, an inducible promoter includes a promoter that initiates or increases the expression of the bottom coding sequence in the presence of a specific inducing compound. A "leaky" inducible promoter is a promoter that provides very high expression levels in the presence of an inducible compound and provides relatively very low or minimal expression levels in the absence of inducers.

A "signal sequence" can be included at the beginning of a protein coding sequence that is expressed in extracellular or periplasmic space. This sequence is present at the N-terminus of the mature polypeptide and encodes a signal peptide so that the host cell regulates polypeptide migration. The term "translocation signal squence" is also used to refer to a signal sequence. Transfer signal sequences are found in association with a variety of proteins unique to prokaryotic and eukaryotic cells, and also function in both kinds of individuals. By adding sequences that direct the secretion of proteins out of the host cell, the proteins of the invention can be further modified and improved. The addition of the signal sequence does not interfere with the folding of the secreted protein, and its evidence can be readily verified according to the protein using techniques well known in the art (eg, testing the activity of a given protein after modification).

Preferred signal sequences of the present invention include pelB signal sequences, which lead to proteins in the peripheral cytoplasmic space between the inner and outer cell membranes of bacteria. Other signal sequences include, for example, ompA and ompT (see 52). The signal sequence is linked to the top (5 'side) of the nucleotide sequence encoding the protein so that it is present at the protein N-terminus after expression. Conventional cloning techniques can be used as described. In order to optimize the addition of signal sequences to any given protein, some repetitive experimentation is needed within the scope of those skilled in the art.

The terms “express” and “expression” are used to produce information by expressing information in a gene or DNA sequence, for example, by activating cellular functions involved in the transcription and translation of an electrical gene or DNA sequence. It means to do. DNA sequences are expressed intracellularly or by cells to form "expression products" such as proteins. The expression product itself may be said to be "expressed" by the cell. For example, when a polynucleotide or polypeptide is expressed or produced in an exogenous host cell under the control of an external or intrinsic promoter, or when it is expressed or produced in the native host cell under the control of an external promoter, it is said to be expressed recombinantly.

The term "over-expressed" is used when polynucleotides and polypeptides are expressed or produced in amounts or yields significantly higher than the basic yields such as those in nature. For example, when a polypeptide is overexpressed, the yield is significantly greater than the standard, average or base yield of the polypeptide expressed in the native host cell under conditions suitable for the life history of the native host cell. Overexpression of polypeptides can be obtained, for example, by altering one or more of the following conditions: (a) growth and living environment of host cells; (b) a polynucleotide encoding a polypeptide to be overexpressed; (c) a promoter that regulates expression of the polynucleotide; And (d) the host cell itself. This is relative and thus "overexpression" can be used to compare and distinguish between expression levels of either polypeptide relative to another polypeptide, whether the polypeptide is native or encoded by the native polynucleotide. Typically, overexpression refers to yields that are at least about twice or more than standard, average, or basic yields. Thus, the polypeptide is said to be overexpressed when produced in an amount or yield that is significantly higher than the amount or yield of the parent polypeptide at the same conditions. Similarly, it is said that a polypeptide is under-expressed when produced in an amount or yield, for example half the base yield, which is significantly lower than the amount or yield of the parent polypeptide at the same conditions. In this context, expression level or yield refers to the amount or concentration of the expressed polypeptide, or generated polypeptide (ie, expression product) (whether active or not). As an example, polynucleotides and polypeptides are said to be underexpressed when expressed in an amount detectable only under the control of an inducible promoter without induction in the absence of an inducer.

Expression products may be characterized as intracellular, extracellular or secreted. The term "intracellular" means that something is in the cell. The term "extracellular" means that something is outside the cell. When a substance is transferred from somewhere within the cell to the extracellular or periplasm, it is said to be "secreted" by the cell.

As used herein, the terms "expression-resistant polypeptide" and "resistant to functional expression" are synonymous and refer to polypeptides that are difficult to be functionally expressed in the host cell of choice. For example, expression resistant polypeptides are not produced, produced in very low yields, or produced in non-functional form when the polynucleotides encoding them are introduced or transformed into an easy host cell expression system.

Examples of such polypeptides are those that have disulfide bonds or require glycosylation, which consists of many subunits. Expression resistant polypeptides include polypeptides that are sensitive to folding and non-folding conditions, particularly intracellular conditions such as temperature, pH and protein concentration, cofactors, coenzymes, coproteins, or the absence or the like. Expression resistant polypeptides also include polypeptides encoded by polynucleotides that are sensitive to a particular promoter or signal sequence in a particular expression system. In addition, expression resistant polypeptides include polypeptides that tend to agglomerate with one another to form inclusion bodies or are produced in an inactive or unfolded form.

Polypeptides suitable for use as the expression resistant parent polypeptide of the present invention are inactive (eg, aggregated) when expressed in very high yields (e.g., overexpressed) but expressed in very low yields (e.g., aggregated). For example, when expressed low). Examples thereof include the following polypeptides: (a) when expressed in the presence of an inducer by a polynucleotide under the control of an inducible promoter, they tend to aggregate to form inclusion bodies or to be inactive or unfolded; And (b) polypeptides that do not aggregate or tend to be active when expressed without inducing agents by polynucleotides under the control of an inducible promoter. Such promoters are known and are called "leaky" promoters.

Polypeptides comprising, merging or associated with heme groups are also examples of expression resistant polypeptides. Examples of specific expression resistant polypeptides of the invention are peroxidase enzymes, such as horseradish peroxidase enzymes. An "expression resistant polynucleotide" is a polynucleotide encoding an expression resistant polypeptide.

Whether genomic DNA or cDNA, the gene encoding a protein of the invention for use in an expression system can be isolated from any source, in particular human cDNA or genomic libraries. Methods for obtaining genes are well known in the art in techniques such as Sambrook et al. (19).

Thus, any animal cell can be a source of nucleic acid for cloning a gene of interest. DNA is well known in the art, such as cloned DNA (eg, DNA libraries), cDNA libraries prepared from tissues expressing proteins, chemical synthesis, cDNA cloning, genomic DNA, or cloning of fragments thereof isolated from desired cells. Can be obtained by known standard procedures (19, 51). Clones derived from genomic DNA may include regulatory and intron DNA regions in addition to coding regions; Clones derived from cDNA do not include intron sequences.

In cloning genes from genomic DNA, DNA fragments occur, some of which encode the desired genes. DNA can be cut at specific sites using a variety of restriction enzymes. Alternatively, DNAse can be used in the presence of manganese to fragment the DNA. Alternatively, DNA may be physically cut by sonication. Linear DNA fragments can be separated by size by standard techniques, including but not limited to agarose and polyacrylamide gel electrophoresis and column chromatography.

The term "transformation" refers to the introduction of foreign genes, DNA, RNA sequences into the host cell, expressing the host cell to produce a desired substance, such as a protein or enzyme encoded by the introduced gene or sequence. . The introduced gene or sequence is called a "cloned" or "foreign" gene or sequence, and controls such as initiation, termination, promoter, signal, secretion or other sequences used by the genetic machinery of the cell or Control sequences may be included. The gene or sequence may comprise a nonfunctional sequence and a sequence with unknown functions. Host cells that receive and express the introduced DNA or RNA are referred to as "transformed" and are referred to as "transformant" or "clone". 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 other genus or species.

Terms such as "vector", "cloning vector" and "expression vector" are used to transform a host cell to promote expression (e.g., transcription and translation) of the introduced sequence. By a carrier that carries a DNA or RNA sequence (eg, an external gene) that can be introduced into it.

Vectors typically include delivery DNA into which external DNA can be inserted. The usual way to insert one DNA fragment into another DNA fragment involves the use of an enzyme called a restriction enzyme that cuts DNA at a specific site (a specific group of nucleotides) called a restriction site. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA and delivered by the vector into a host cell, such as a transfer vector DNA. DNA having a DNA fragment or sequence inserted or added together with an expression vector is called a "DNA construct".

The general form of the vector is a "plasmid," which is generally a circular double-stranded DNA molecule that can readily accept foreign DNA and be easily introduced into suitable host cells. Plasmid vectors contain coding DNA and promoter DNA and have one or more restriction sites suitable for inserting external DNA. Promoter DNA and coding DNA may come from the same gene or from different genes, and may come from the same or different individuals. Numerous vectors, including plasmids and fungal vectors, have been described for replication and / or expression in a variety of prokaryotic and eukaryotic hosts. Examples of plasmids include, but are not limited to, pKK plasmid (Clonetech), pUC plasmid, pET plasmid (Novagen, Inc., Madison, WI), pRSET or pREP plasmid (Invitrogen, San Diego, CA), or pMAL plasmid (New England Biolabs, Beverly, Mass.), And many suitable host cells are disclosed or cited herein and are otherwise well known to those skilled in the art. Recombinant cloning vectors usually include one or more replication systems for cloning or expression, markers such as antibiotic resistance for selection in host cells, and one or more expression cassettes. Preferred vectors are described in the Examples and include pcWori, pET-26b (+), pXTD14, pYEX-S1, pMAL and pET22-b (+). Other vectors can be used as desired by one of ordinary skill in the art. Although different from that described in the examples, iterative testing of biotechnology can be used to determine the vector that is most suitable for the present invention. In general, the choice of vector depends on the size of the polynucleotide and the host cell to be used in the method of the invention.

"Cassette" refers to a segment of DNA that can be inserted at a specific restriction site of a vector. DNA fragments encode polypeptides of interest, and cassettes and restriction sites are designed to ensure cassette insertion in the correct translation frame for transcription and translation.

An "expression system" means a vector compatible with a host cell under appropriate expression conditions of a protein encoded by an external DNA on the vector introduced into the host cell. Common expression systems include bacteria ( E. coli and B. subtilis ) or yeast ( S. cerevisiae ) host cells and plasmid cells, insect host cells and baculoviruses. As used herein, “facile expression system” is foreign and heterologous to the selected polynucleotide or polypeptide, and native or heterologous to the electrical polynucleotide and peptide. By an expression system that can use a host cell that can be grown or maintained more advantageously than a normal cell, or can produce a polypeptide more efficiently or in a higher yield. For example, the use of robust prokaryotic cells expressing eukaryotic protein may be an easy expression system. Preferred easy expression systems include E. coli, B. subtilis and S. cerevisiae host cells and suitable vectors.

The terms "mutant" and "mutation" refer to the detected change in the genetic material, such as DNA, or the process, mechanism or result of such a change. This includes gene mutations in which the genetic structure (eg DNA sequence) is altered, genes or DNA resulting from the mutation process and expression products (eg proteins or enzymes) expressed by the modified gene or DNA sequence. The term "variant" can also be used to imply any type of mutant, such as a modified or altered gene, DNA sequence, enzyme, cell, or the like.

A "sequence-conservative variant" of a polynucleotide sequence is a variant in which one or more nucleotide changes within a given codon position have no change in the amino acid encoded at that position.

"Function-conservative variant" means that a given amino acid residue of a protein or enzyme is replaced with another amino acid (eg, acidic, basic, hydrophobic) with similar properties without altering the overall structure and function of the polypeptide. Changes, including but not limited to, have occurred. Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic base amino acids and may be interchanged. Similarly, the hydrophobic amino acid isoleucine may be replaced with leucine, methionine or valine. Amino acids other than conservatively implied amino acids differ between proteins or enzymes, and therefore the percentage of amino acid sequence similarity between two proteins with similar functions changes, and the similarity is determined by an alignment method such as the Cluster Method based on the MEGALIGN algorithm. As such, the percentage is between 70% and 99%. The functional preservation variant has an amino acid identity of at least 60%, preferably at least 75%, more preferably at least 85%, most preferably at least 90%, as determined by the BLAST or FASTA algorithm, and can be compared with the electrical variant. Polypeptides or enzymes with the same or substantially similar properties and functions as natural or maternal proteins or enzymes are included.

The term "DNA reassembly" is used when recombination occurs between the same sequences. The term “DNA shuffling” suggests recombination between fairly similar sequences except for sequences that are not identical.

"Isolation" or "purification" of a polypeptide or enzyme refers to the removal of a polypeptide from its initial environment and induction of it (e.g., from a natural environment, if it is naturally occurring, by recombinant DNA methods). From host cells if produced). Polypeptide purification methods include preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed phase HPLC, gel filtration, ion exchange and distribution chromatography, countercurrent distribution Methods are well known in the art, including but not limited to. For some purposes, it is desirable to generate, but is not limited to, polypeptides comprising additional sequence tags that facilitate purification, such as polyhistidine sequences. The polypeptide can be purified from crude lysate of the host cell by chromatography on a suitable solid phase medium. Alternatively, antibodies generated against proteins or peptides derived therefrom can be used as purification materials. Other purification methods are also possible. Purified polynucleotides and polypeptides comprise less than about 50%, preferably about 75%, most preferably less than about 90% cellular constructs. "Substantially pure" enzymes suggest the highest degree of purity that can be achieved using conventional purification techniques well known in the art.

Polynucleotides are said to “hybridize” with each other when a strand of polynucleotides can anneal to another polynucleotide under stringent conditions. Stringent conditions of hybridization are determined by the following examples: (a) the temperature at which hybridization and / or washing is performed; (b) Ionization strength and polarity of hybridization and wash solutions, not to mention other measures (eg formamide). Hybridization occurs only when two polynucleotides contain complementary sequences; However, depending on the stringent conditions of hybridization, inappropriate hybridization may also be tolerated. Typically, in order to hybridize two sequences under very stringent conditions (eg, 0.5 × SSC aqueous solution at 65 ^° C.), the two sequences must have a fairly high degree of complementarity to the entire sequence. Under moderate stringency conditions (eg 0.2X SSC aqueous solution at 65 ^o C) and low stringency conditions (eg 0.2X SSC aqueous solution at 55 ^o C), lower complementarity between hybridization sequences need. (1X SSC is 0.15M NaCl, 0.015M Na citrate) Here, the polynucleotide "hybridizing" the polynucleotide may be any length. In one embodiment such polynucleotides are at least 10, preferably at least 15, most preferably at least 20 in length. In another embodiment, the polynucleotides that hybridize are about the same length. In another embodiment, the hybridizing polynucleotide encodes a polypeptide or enzyme having the ability to promote oxidation, such as an oxygenase or a coupling reaction of the present invention, and a poly that can be annealed under moderately stringent conditions. Nucleotides.

General genetic engineering tools and techniques discussed herein are well known in the art, including transformation and expression, use of host cells, vectors, expression systems, and the like.

Mutation Induction and Intentional Improvement of Proteins

In order to improve protein expression using conventional expression systems, the present invention is intentionally refined to generate polynucleotide mutant libraries that express functional proteins with similar or much higher activity than natural proteins in conventional or easy expression systems. It has been found that the technique can be used. Inclusion bodies, which are often formed when expressing proteins with disulfide bonds and require an expensive refolding process, can be removed by intentional refinement techniques.

According to the present invention, proteins that can be more easily expressed in an easy gene expression system can be obtained by intentional refinement to generate mutant polynucleotides in the form of libraries for selection. General methods for generating libraries and separating and identifying improved proteins (also referred to as "variants") in accordance with the present invention using intentional refinements are briefly described below, as described in US Pat. Nos. 5,741,691 and 5,811,238. It is disclosed in detail. It should be understood that any method may be used for mutagenesis of the polynucleotide sequence to provide an improved polynucleotide for use in the expression system. Proteins produced by intentional improvement techniques are screened for improved expression, folding, secretion and function according to conventional methods.

Nucleic acid in purified form from any source can be used as the starting nucleic acid. Thus, one or two strands of DNA or RNA including mRNA can be used. Additionally, DNA-RNA hybrids, each present in one strand, can be used. Nucleic acid sequences vary in length depending on the size of the nucleic acid being mutated. Preferably the specific nucleic acid sequence has 50 to 50,000 base pairs. Consideration should be given to whether a vector comprising a nucleic acid encoding a protein of interest can be used in the methods of the invention.

Any particular nucleic acid sequence can be used to generate a mutation group by the present invention. An initial group of specific nucleic acid sequences with mutations can be made by several different known methods, including those described below.

Error-prone polymerase chain reaction (PCR) (see 20, 45, 46) and cassette mutation induction (38-44) are used to replace optimized specific regions with synthetic mutant oligonucleotides. Used in the present invention. Error-pron PCR can be used to mutate fragment mixtures of unknown sequences. This technique can be used in low accuracy polymerization conditions to introduce low levels of random point mutations on long sequences or to mutate fragment mixtures of unknown sequences.

Oligonucleotide-directed mutagenesis, which replaces short sequences with synthesized mutant oligonucleotides, can be used to generate improved polynucleotides with improved expression.

Optionally, the nucleic acid fragments or shuffling methods of nucleic acids or DNA using in vivo or in vitro homologous recombination methods of polynucleotide pools are polynucleotides having the variant sequences of the present invention. Can be used to generate sequences.

Parallel PCR can be used to improve polynucleotides for improved expression in a conventional expression system, which involves numerous different PCRs occurring in parallel in the same vessel so that the product of one reaction initiates another reaction. Use the reaction. Reassembly of fragments by random cleavage and mutual priming can result in random mutation of sequences at various levels. Site-directed mutations can be introduced in long sequences by randomly cutting the template in the presence of mutation-inducing oligonucleotides and recombining the fragments.

A particularly useful application of parallel PCR that can be used in the present invention is called sexual PCR. In voiced PCR, also known as DNA randomization, parallel PCR is used to perform in vitro recombination of a pool of DNA sequences. Sexual PCR can also be used to build gene chimera libraries from different species.

Polynucleotides used in the present invention can also be modified by chemical mutagenesis. For example, chemical mutagens include sodium bisulfite, nitrous acid, hydroxylamine, hydrazine and formic acid. Other compounds that are analogs of nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally, such compounds are added to the PCR reaction instead of nucleotide precursors to mutate the sequence. Intercalating agents such as proflavin, acriflavin, quinacrine and the like can also be used. Random mutations in polynucleotide sequences can be achieved by replicating polynucleotides in a host (eg, E. coli ) lacking X-ray or ultraviolet radiation or DNA damage repair machinery. Generally, such mutated plasmid DNA or DNA fragments are introduced into E. coli and replicated as a pool or library of mutant plasmids.

Alternatively, a mixed group of specific nucleic acids may be found in nature consisting of different alleles of the same gene or cognate genes from different species that are associated with each other. Alternatively, the mixed group of specific nucleic acids may be interrelated DNA sequences found within one species, such as a group of peroxidase genes. Once a mixed group of specific nucleic acid sequences occurs, the polynucleotides can be inserted into suitable cloning vectors using techniques well known in the art.

Once the improved polynucleotide molecule occurs, it can be cloned into a suitable vector selected by a specialist according to methods well known in the art.

When a mixed group of specific nucleic acids is cloned into a vector, the mixed group of specific nucleic acids can be amplified by inserting each vector into a host cell and allowing the host cell to amplify the vector. The selection method depends on the DNA fragment desired. For example, in the present invention, DNA fragments encoding proteins having improved folding properties can be determined by test results for the functional activity of the protein and the absence of inclusion body formation. Such assays are well known in the art.

Using intentional refinements, the present invention provides new methods for producing correctly folded, functional and soluble proteins in conventional or easy expression systems such as E. coli or yeast. Conventional assays can be used to determine whether the protein of interest produced from the expression system has improved expression, folding and / or functional properties. For example, in order to determine whether a polynucleotide expressed in an external host cell produces a protein with improved folds by applying intentional refinement techniques, experts in the art perform experiments designed to test the functional activity of the protein. can do. In short, the improved protein can be quickly screened and easily isolated and purified from the secreted expression system or medium. It can be immediately applied to assays designed to test the functional activity of particular proteins in their natural form. Such assays for various proteins are well known in the art and are discussed in the Examples below.

In one specific embodiment, the present invention contemplates the use of a polynucleotide encoding a variant of heme protein. Thus, the present invention is intentional to generate new peroxidase enzymes such as HRP that fold correctly, maintain functional activity, and eliminate inclusion body formation problems in host cells (eg, E. coli ) used in expression systems. Use improved technology.

The invention can also be applied to select or optimize expression systems, including the selection of host cells, promoters and signal sequences. Expression conditions can also be optimized in accordance with the present invention.

The following examples describe the methods of the present invention, and in particular, teach the use of intentional refinements to generate HRP variants that, when expressed using conventional expression systems, do not form inclusion bodies and maintain functional activity. Usually, the same natural protein forms a post-expression inclusion body in a conventional expression system and maintains little functional activity.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Functional Expression of Horseradish Peroxidase (HRP) in E. coli and Yeast

Much attention has been focused on developing eukaryotic peroxidases that are used as industrial biocatalysts. However, protein engineering and intentional improvement techniques to improve certain properties have been difficult due to the lack of easy recombinant expression systems. In an effort to develop a bacterial expression system suitable for mass screening of mutant HRP, an embodiment of the present invention describes the development of a bacterial expression system for its expression by inserting a heme protein gene such as HRP into the signal peptide pelB. In addition, the application of these genes to intentional retrofitting allows the heme protein to fold more effectively in E. coli to resist heat and hydrogen peroxide (H ₂ O ₂ ). This example provides an approach that makes it very easy to fine tune many enzymes that are promising as industrial biocatalysts but lack adequate bacterial or yeast expression systems due to inclusion body formation and inefficient secretion by cells.

Cloning of HRP

The HRP gene (with methionine added to the N-terminus) was cloned from plasmid pBBG10, which introduced the AatII site to the initiation codon and the HindIII site just below the termination codon by PCR. This plasmid contains the synthetic HRP gene described in Smith et al. (13), whose DNA sequence is based on the amino acid sequence for the HRP protein (49). pBBG10 was made by inserting an HRP sequence between the polylinker HindIII and EcoRI sites of the well known plasmid pUC19. PCR products obtained from this plasmid were cut with AatII, blunt-end with t4DNA polymerase, and cut with HindIII. The cleaved product was linked to McsI and HindIII treated pET-22b (+) (Novagen) to create the vector pETpelBHRP. A map of this expression vector is shown in FIG. In this construct, the HRP gene is under the control of the T7 promoter and theoretically fused to fit the reading frame with the periplasmic space, the pelB signal sequence (27) that guides the transport of proteins out of the cytoplasm. (See SEQ ID NO: 1, SEQ ID NO: 2 and FIG. 2). The ligation product was transformed into E. coli strain BL21 (DE3) for expression of the protein in the presence or absence of induction by 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside).

In IPTG-induced cells, even if HRP polypeptides make up more than 20% of the total cellular protein, background or more peroxidase against BL21 (DE3) cells and BL21 (DE3) cells with pET-22b (+) No activity was detected. This is in good agreement with the observations of the prior art (cf. 12-14).

In cells not induced with IPTG, clones were found that showed weak but measurable activity against azino-di- (ethylbenzthiazoline sulfonate).

The T7 promoter of the pET-22b (+) vector is known to be leaky (31), and it is possible that part of the HRP polypeptide produced theoretically at baseline can be folded into its natural form. In contrast, the addition of IPTG induces high levels of HRP synthesis but prevents correct folding due to aggregation of the chains. As a result, random mutations and screening were used to identify mutations that lead to higher HRP activity expression.

Thus, one aspect of the invention involves the use of a promoter that produces a small amount of polypeptide under some conditions and a large amount of polypeptide under other conditions. For example, a "leaky" inducible promoter can be used. This type of promoter produces a large amount of a specific protein in the presence of an inducer, but in the absence of an inducer it produces much less protein. In one specific embodiment, the polypeptide may be overexpressed under certain conditions (eg in the presence of an inducing agent) and underexpressed under other conditions (eg without the inducing agent). Polypeptides that are inert when under or overexpressed or active when underexpressed are particularly suitable for use as the parent polypeptide of the invention. Such expression resistant polypeptides can be improved to provide functional and active expression that exhibits high yields and activities using the methods of the invention.

Generation and Screening of Random Libraries

One of the HRP clones showing detectable peroxidase activity was used as the first generation of error-pron PCR mutagenesis. Random libraries were generated by the modifications of the error-pron PCR assay described above (20, 21, 22) (0.15 mM MnCl ₂ was used in place of 0.5 mMnCl ₂ ). This assay incorporates manganese ions and unbalanced nucleotides and gives rise to both transition and transversion to provide a broader range of amino acid changes (50).

In brief, the PCR reaction solution contains 20fmole template in 100μl volume, two primers of 30pmole each, 7mM MgCl ₂ , 50mM KCl, 10mM Tris-HCl (pH 8.3), 0.01% gelatin, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 0.15 mM MnCl ₂ and 5 units of Taq polymerase were included. PCR reactions were performed 30 times in an MJ PTC-200 circulator (MJ Research, MA) using the following measures: 1 minute at 94 ^o C, 1 minute at 50 ^o C and 1 minute at 72 ^o C. The primers used are as follows:

5'-TTATTGCTCAGCGGTGGCAGCAGC (SEQ ID NO: 15), and

5'-AAGCGCTCATGAGCCCGAAGTGGC (SEQ ID NO: 16)

PCR products were purified using the Promega Wizard PCR kit and cut with NdeI and HindIII. The truncated product was purified using a QIAEX II gel extraction kit and the HRP fragments were similarly cut and linked to the purified pET-22b (+) vector. The linkage mixture was transformed by electroporation on BL21 (DE3) using Gene Pulser II, Bio-Rad. Cell growth and expression were performed in 96-well or 384-well plates at 30 ^° C. in LB medium. Peroxidase activity was performed with H ₂ O ₂ and ABTS (26).

For each generation, typically 12,000-15,000 colonies were taken and screened in 96 well plates. This number is all accessible values for which one mutation can be sampled at least once with 95% probability (25). Pick colonies by hand or use Caltech, Q-bot (Genetix, UK) automated colony forceps. As shown in FIGS. 5A and 5B, out of 12,000 colonies screened (no IPTG added), a mutation named HRP1A6 showed 10-14 times higher peroxidase activity than the parent clone. As shown in FIG. 6, this mutant clone showed significantly lowered activity when 5 μM of IPTG was added. Sigma reported that 1 mg of purified HRP from horseradish had 1000 units of activity by the ABTS assay. Other researchers reported similar results (13). Based on this data, the concentration of active HRP is estimated to be 100 μg / L. HRP1A6 showed much greater activity than 100 units / L. This is comparable to the yield obtained by refolding aggregated HRP chains under laboratory conditions (13). This level of expression for HRP mutations is comparable to that of the bovine pancreatic trypsin inhibitor, a non-sugar chain protein with three disulfide bonds in E. coli (32). It was found that more than 95% of the HRP activity determined by ABTS activity was in LB culture medium.

Mutant HRP was stable at 4 ^o C for 1 week. IPTG was excluded from all HRP expression experiments unless otherwise specified. The peroxidase activity test for HRP was performed with the classic peroxidase assay, ABTS and hydrogen peroxide (26). In a microplate, 15 μl of cell suspension was mixed with 140 μl of ABTS / H ₂ O ₂ (2.9 mM ABTS, 0.5 mM H ₂ O _2, pH 4.5) and the activity was measured at 25 ^° C. by SpectraMax plate reader, Molecular Devices, The decision was made using a Sunnyvale, CA) reader. One HRP unit is defined as the amount of enzyme that oxidizes 1 μmole of ABTS per minute in assay conditions.

Sequencing of the mutant gene revealed a mutation at position 255 where the codon AAC for amino acid asparagine (Asp or N) is replaced with a codon GAC for amino acid aspartic acid (Asp or D). This residue is a potential glycosylation site and is located on the surface of the protein. The sequence of this mutant HRP1A6 is shown in Figure 3 (SEQ ID NO: 3). A map of plasmid pETpelBHRP1A6 containing this mutation is shown in FIG. 4. The structural diagram of the HRP mutation showing Asn255Asp mutation is shown in FIG. 7.

HRP Functional Expression in Yeast

Natural HRP proteins contain four disulfide bonds, and E. coli has a limited ability to form disulfide bonds. In theory, well-conserved disulfide bonds in HRP (and other plant peroxidases) are considered important for structural integration of proteins and should not be replaced by mutations. Yeast has much more ability to form disulfide bonds. Therefore, if you want to reduce the apparent limit to the HRP folding imposed by the limitations of the E.coli disulfide bond formation in particular yeast it can be used in place of a suitable expression host E.coli. For example, S. cerevisiae can be used as a host for the expression of mutant HRP genes and proteins.

The HRP mutation (HRP1A6) was cloned into the secretion vector pYEX-S1 obtained from Clonetech (Palo Alto, Calif.), Resulting in pYEXS1-HRP (see Figure 8). This vector utilizes a phosphoglycerate kinase promoter and secretory signal peptide of Kluveromyces lactis. The plasmid was first amplified in E. coli and then transformed into S. cerevisiae strain BJ5465 obtained from Yeast Genetic Stock Center (YGSC, University of California, Berkeley) using the LiAc method (36). BJ5465 is known to be suitable for secretion due to a lack of protease.

First generation of HRP error-pron PCR in yeast was performed. Of the 7400 mutations initially screened, four variants showed 400% higher activity than HRP1A6 in yeast. More details and results are shown in Example 2.

Example 2 Functional Expression of HRP in Yeast Through Intentional Improvement Techniques

This example describes the use of intentional refinements to further improve the functional expression of HRP. As described in Example 1, the HRP1A6 variant was isolated. Since HRP has four disulfide bonds and E. coli has limited disulfide bond formation capacity, further improvement of HRP expression in E. coli is limited by the correct pairing of disulfide bonds containing cysteine. . Yeast cells, such as S. cerevisiae, have a much larger disulfide bond forming capacity and are advantageous for accepting disulfide bonds of peroxidase enzymes. In theory, well-preserved disulfide bonds of HRP and other plant peroxidases are considered important for structural integration of proteins and should not be replaced by mutations. Therefore, if you want to reduce the apparent limit to the HRP folding imposed by the limitations of the E.coli disulfide bond formation in particular yeast it can be used in place of a suitable expression host E.coli.

Thus, S. cerevisiae was selected as the selective host for the expression of HRP. S. cerevisiae are microorganisms and eukaryotic cells, with post-translational and secretory mechanisms of eukaryotic proteins such as endoplasmic reticulum (ER) and Golgi complex that can promote disulfide bond formation and glycosylation of polypeptides. have. Genetic engineering techniques (particularly gene transformations) are also readily available. The drawback is that yeast secretes little protein. Moreover, the glycosylation of yeast differs significantly from that of higher eukaryotes, which may present a problem in the secretion of glycoproteins (cf. 4). Nevertheless, several proteins were efficiently secreted from yeast (4). Strategically, the experiments of this example utilized the yeast's ability to promote the formation of disulfide bonds, while fine-tuning glycosylation factors through intentional refinement techniques.

Construction of HRP Yeast Expression System

The HRP mutant HRP1A6 from Example 1 was cloned into the secretion vector pYEX-S1 obtained from Clonetech (Palo Alto, Calif.), Resulting in pYEXS1-HRP (see Figure 8). This vector utilizes a phosphoglycerate kinase promoter and secretory signal peptide of Kluveromyces lactis. pYEX-S1 was cut with SacI to make blunt-ends with T4 DNA polymerase. The mature HRP1A6 gene was cloned from pETpelBHRP1A6 by PCR using a proofreading polymerase pfu (Stratagene, CA) to produce blunt-end products. The front and reverse primers used were 5'-CAGTTAACCCCTACATTC-3 '(SEQ ID NO: 25) and 5'-TCATTAAGAGTTGCTGTTGAC-3' (SEQ ID NO: 26), respectively. PCR fragments were linked to the truncated site of pYEX-S1 and transformed with E. coli DH5α. Numerous colonies were screened for the presence of the HRP gene by colony PCR with the following two primers: 5'-CGTAGTTTTTCAAGTTCTTAG-3 '(SEQ ID NO: 27) and 5'-TCCTTACCTTCCAATAATTC-3' (SEQ ID NO: 28). The correct orientation of the HRP gene was confirmed by sequencing. This yeast expression vector is referred to here as pYEXS1-HRP (see FIG. 8). In this construct, the HRP gene is located directly below the secretory signal peptide of Kluveromyces lactis and the expression is under the control of the phosphoglycerate kinase promoter. The vector also contains the Amp resistance genes of E. coli , as well as leu2-d and URA3, markers of selection in yeast (47).

In expression experiments, plasmids were first amplified in E. coli DH5α, and then in S. cerevisiae strain BJ5465 obtained from Yeast Genetic Stock Center (YGSC, University of California, Berkeley), Gietz et al. (48). Transformation was performed using the LiAc method using single stranded DNA as described. BJ5465 is known to be suitable for secretion due to a lack of protease. After transformation, cells were plated in YNB selective medium to which 20 μg / ml leucine, 20 μg / ml histidine, 20 μg / ml adenine and 20 μg / ml tryptophan were added. Colonies were taken and grown in 96-well microplates in YEPD medium at 30 ^° C. in an air circulation incubator for 16 hours for 2 days. HRP activity evaluation was performed using ABTS and hydrogen peroxide, a classic peroxidase assay (26). The activity of HRP1A6 obtained in yeast was only about 1/10 of that obtained from E. coli , slightly less than that obtained for wild type in this construct.

Generation and Screening of HRP Mutations

The HRP mutation library was constructed by error-pron PCR (20) as described (53), except that the following two primers in contact with the HRP gene were used for the mutant PCR reaction: 5'-CAGTTAACCCCTACATTC- 3 '(SEQ ID NO: 25) and 5'-TGATGCTGTCGCCGAAGAAG-3' (SEQ ID NO: 29). The thermocycling conditions are as follows: 95 ^o C 2 min, (94 ^o C 1 min, 50 ^o C 1 min, 72 ^o C 1 min, 30 cycles).

PCR products were purified using the Promega Wizard PCR kit (Madison, WI) and cut with SacI and BamHI (the first 27 amino acids of HRP were left unmodified). The truncated product was purified using QIAEX II gel extraction kit (QIAGEN, Valencia, Calif.) And the HRP fragments were similarly cut and linked to the purified pYEXS1-HRP1A6 vector. The linkage mixture was transformed by electroporation to E. coli HB101 using Gene Pulser II, Bio-Rad, and selected in LB medium with 100 mg / ml ampicillin. Colonies were harvested directly on LB plates. This plasmid DNA was used to transform yeast BJ5465 as described above.

One colony was removed from the YNB plate by incubating in a 96-well microplate containing YEPD medium (1% yeast extract, 1% peptone, 2% glucose) at 30 ^° C. for 64 hours. The microplates were centrifuged at 1500 g for 10 minutes to transfer 10 ml of supernatant from each well to a new microplate with a Beckman 96-channel pipetting station and assayed for total HRP activity. The overall standard deviation of the test method (including about 2% pipetting error) did not exceed 10%. Improved mutations (which show the highest overall HRP activity) were recovered directly from microplates, washed three times with sterile hydrogen peroxide and grown again in YNB selection medium. Plasmids containing HRP mutations were extracted from yeast cells with the Zymo enzyme plasmid miniprep kit (Zymo Research, Orange, Calif.) And transformed back to E. coli X10-Gold for amplification and preparative separation.

Pre-screening of HRP-expressing yeast clones was performed as follows. Colonies of YNB plates were copied to MS1-supported nitrocellulose membranes (Micron Separations Inc., Westboro, Mass.) And grown at 30 ^° C. for 34 hours on fresh YEPD agar. The membrane was soaked in 100 ml of TMB substrate (0.8 mM TMB, 2.9 mM H ₂ O ₂ , 0.12% (w / v) dextran sulfate as enhancer) for 5 minutes to allow color development. Yeast clones showing bright green were traced in the original YNB plates to pick colonies and grown in YEPD for screening as described above.

Induction of first generation HRP mutations in yeast for improved expression

The first generation of error-pron PCR of HRP1A6 in yeast is aimed at increasing expression levels. As previously described, error-pron PCR was used that incorporates an imbalance between nucleotide concentration and manganese ions (20, 21). This method generates random mutations that allow a wider range of amino acid changes. The concentration of manganese ions used was 100 μM, which on average represented an error rate of 1 to 2 mutations per gene, approximately 22. PCR products were purified using the Promega Wizard kit and cut with SacI and BamHI (the first 27 amino acids of HRP were left unmodified). The truncated product was purified using a QIAEX II gel extraction kit and the HRP fragments were similarly cut and linked to the purified pEXS1-HRP1A6 vector. The linkage mixture was transformed by electroporation to E. coli HB101 using Gene Pulser II, Bio-Rad. Colonies were scraped from E. coli plates and suspended in LB medium, from which plasmids were prepared. This plasmid was transformed into yeast to obtain yeast colonies and grown as above.

Approximately 14,000 colonies were selected and screened. This number is all accessible values for which one mutation can be sampled at least once with 95% probability (25). Among these colonies, numerous mutations showed significantly higher activity in the yeast than the parent (HRP1A6). Two exemplary improved mutations were named 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 showed 16-fold higher activity than the parent, with a total activity of about 220 units / L (see Figure 9). HRP1-77E2 showed a total activity of about 147 units / L. Both are above the highest levels obtained in E. coli (see Figure 12 (HRP1-77E2) and Figure 16 (HRP1-117G4)).

Induction of Second Generation HRP Mutations in Yeast for Improved Expression

The second generation of error-pron PCR used HRP1-117G4 as a parent. For this generation, higher concentrations of manganese ions were used to increase the mutation rate. This change was made in consideration of the following. Since screening can now handle about 10 ⁴ to 10 ⁵ mutant libraries, mutation rates have been significantly limited in creating one dominant mutation in the past (15). In this example, the fraction of clones more active than the parent for a given generation was relatively constant with error rates of up to six per gene. The advantage of using a higher error rate is that neutral mutations can be present with beneficial mutations isolated during screening. These neutral mutations can be useful for the next generation by bridging new mutations or synergistically interacting with newly created mutations. The concentration of manganese ions used in this generation was 350 μM, which averaged an error rate of 4-5 mutations per gene on average (22).

In addition, sunscreening of colonies with nitrocellulose membranes was performed. This is possible because higher error rates reduce the number of colonies that show similar or better activity than the parent. Colonies were copied to nitrocellulose membranes and grown at 30 ^° C. for 30 hours on YEPD plates. The membrane was recovered from the plate and immersed in a mixture of tetramethylbenzidine (TMB) and hydrogen peroxide. The brightest color colonies were identified and grown in master plates following the same parental colonies. In this generation, about 120,000 colonies were screened (about 5,000 actually picked up) and mutant HRP2-28D6 was obtained. It exhibits 85% higher activity than the parent HRP1-117G4 and has a total activity of 410 units / L (see FIG. 9).

Induction of Third Generation HRP Mutations in Yeast for Improved Expression

Three rounds of random mutations were performed under similar conditions using HRP2-28D6 as parent. In this generation, a total of 90,000 colonies were screened and grown 3,000. The highest mutant HRP3-17E12 showed an expression level of 1080 units / L, 160% higher than the parental HRP2-28D6, and 85 times higher than the starting mutation HRP1A6.

Induction of Third Generation HRP Mutations in Yeast for Improved Stability

A first generation of HRP random mutants to improve thermostability and resistance to hydrogen peroxide were carried out with HRP1-77E2 as parent. Random mutations (100 μM manganese) and cell growth were performed essentially as above (without sunscreening). Thermostability testing was performed using a MJ-PTC-200 cycler (MJ Research, Mass.) For a 10 min incubation time at 73 ^° C. Hydrogen peroxide resistance tests were performed separately by ABTS screening at 25 mM H ₂ O ₂ and preincubation time 30 minutes, 25 mM H ₂ O ₂ at room temperature. More thermostable or chemically stable (H ₂ O ₂ resistant) mutations than the parent were analyzed at various temperatures (for heat stability) or hydrogen peroxide concentration (for H ₂ O ₂ stability).

From among the 3,000 colonies screened, the heat stable mutations HRP1-4B6 the matrix T _1/2 (T _1/2 is an intermediate transition point of the inert HRP curve as a function of temperature) than the 6 ^o C higher T _1/2 value (See FIG. 10). Another mutation, HRP1-24D11, was not as thermally stable as the parent HRP1-77E2, but was more resistant to hydrogen peroxide degradation. (The feedback mechanism common to HRP enzymes is that it is degraded by hydrogen peroxide, the reactant of HRP's enzymatic reaction. Will be). The HRP1-24D11 mutant maintained about 60% activity after incubation for 30 minutes in 25mM H ₂ O ₂ , while the mother showed 42% residual activity under the same conditions (see FIG. 11).

Pichia pastoris HRP Expression Vector

Improved HRP mutations were cloned into Pichia expression vector pPIZaB (Invitrogen Corp., Carlsbad, Calif.) To facilitate the production of mutations for biochemical analysis (54). This vector has a P _aox1 promoter induced by methanol and an a-factor signal peptide comprising four spacer residue sequences Glu-Ala-Glu-Ala in the C-terminal secretion signal. pPIZaB was first cut with PstI, blunt-end by T4 polymerase and then cut with EcoRI. Samples were purified with Promega DNA Purification Kit. The coding sequence of the HRP variant was obtained from pYEXS1-HRP by PCR using calibration polymerase pfu. Two primers were used for the PCR reaction: 5'-TCAGTTAACCCCTACATTC-3 '(front, SEQ ID NO: 30) and 5'-CCACCACCAGTAGAGACATGG-3' (back, SEQ ID NO: 31). The PCR product was cut with EcoRI and linked to purified pPIZaB to make pPIZaB-HRP (FIG. 1B) where the HRP gene is located just below the a-factor signal. The ligation product was first transformed into E. coli XL10-Gold, low salt LB medium (1% tryptophan, 0.5% yeast extract, 0.5% NaCl, pH 7.5) with 25 mg / ml zeocin (Cayla, toulouse cedex, France). ) Was selected. The presence of the HRP gene of the colonies was screened by colony PCR with the following two primers (see 55): 5'-GAGAAAAGAGAGGCTGAA GCTC- 3 '(front, SEQ ID NO: 32) and 5'-TCCTTACCTTCCAATAATTC-3' (Back, SEQ ID NO: 33). The front primer contains the last three nucleotides of the signal sequence and the first nucleotide of the HRP sequence (underlined), which ensures that the positive colonies have the full length of the HRP gene in the right direction. Plasmids were isolated from the culture of positive transformants using the QIAgen miniprep kit and used to transform with Pichia for HRP expression.

Pichia transformation was performed by electroporation according to the manufacturer's method (Invitrogen). Prior to transformation, the plasmids were cut linearly with PmeI, purified using a Promega DNA purification kit, and then treated on a Princeton Centri-Sep column equilibrated with secondary distilled water to remove residual impurities.

Linearization DNA was incorporated into the Pichia genome by homologous recombination between the transforming DNA and the Pichia genome. Transformed cells were plated in YPDS (1% yeast extract, 2% peptone, 2% glucose, 1M sorbitol) to which 100 mg / ml zeocin was added. For each construct containing the characteristic HRP mutation, 4 to 6 transformants were plated on new YPDS plates (with 100 mg / ml zeocin added) to separate the Sangle colonies, showing the highest expression levels. Screened to identify colonies. Pichia X-33 was used in all expression tests. In early experiments, Pichia X-33 (Mut +) strains showed much better HRP expression than KM17 (Muts).

Pichia pastoris HRP expression in

Pichia cell growth was performed in a 30 ^° C shaker. Cells with pPIZaB-HRP were OD in BMGY (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4X10-5% biotin, 1% glycerol) medium supplemented with 1% casamino acid. Growing until ₆₀₀ 1.2-1.6. The pellets of cells were collected and suspended in BMMY with 1% casamino acid (the same as BMGY but with 0.5% methanol instead of 1% glycerol) to OD ₆₀₀ 1.0. Cells were grown for 54-72 hours. Sterilized methanol was added every 24 hours to maintain induction conditions. The HRP activity of the supernatant peaked at 54 to 60 hours post-induction (the time at which the OD ₆₀₀ reached 8.0 to 10.0). If possible, 1.0 mM vitamin B1, 1.0 mM d-ALA and 0.5 ml with induction / L Rare Element Mixture (0.5g / L MgCl ₂ , 30g / L FeCl ₂ 6H ₂ O, 1g / L ZnCl ₂ 4H ₂ O, 0.2g / LCoCl ₂ 6H ₂ O, 1g / L Na ₂ MoO ₄ 2H ₂ O , 0.5 g / L CaCl ₂ 2H ₂ O, 1 g / L CuCl ₂ and 0.2 g / LH ₂ BO ₃ ) were added to the growth medium.

Peroxidase Activity Assay

Peroxidase activity evaluation on HRP was performed using the classic peroxidase assay ABTS and hydrogen peroxide (26). 10 μl (or 15 μl) of cell suspension was mixed with 140 μl (or 150 μl) of ABTS / H ₂ O ₂ (0.5 mM, 2.9 mM, pH 4.5) in a microplate to increase absorbance at 405 nm (e value of oxidized ABTS was the 34.700cm ^-1 M ^-1) to spectra max plate reader (SpectraMax plate reader, Molecular Devices, Sunnyvale, CA) was determined at 25 ^O C. One unit of HRP is defined as the amount of enzyme that oxidizes 1 μmole of ABTS per minute under assay conditions.

Guajacol Assay

This assay is performed with 1 mM H ₂ O ₂ , 5 mM Guajacol in 50 mM phosphate buffer (pH 7.0), and the addition of the yeast supernatant determines the increase in absorbance at 470 nm (ε value of oxidized product at 470 nm is 26.000 cm ^-1 M ^-1 .)

Stability of the mutations is assessed using assays for initial activity (A _i ) and residual activity (A _resid ), performed as described above with ABTS as substrate. A _resid is measured after incubating HRP mutations at 50 ^o C for 10 minutes in NaOAc buffer (pH 4.5) without H ₂ O ₂ or with 1 mM H ₂ O ₂ .

Stability assay in an organic solvent / buffer (NaOAc buffer 50 mM pH 4.5) mixture was carried out using HRP mutant supernatant (10 μl) expressed in yeast in dioxane / buffer (20/80) with 1 mM H ₂ O ₂ and 2 mM ABTS do.

Pichia Production of HRP Mutants in the

To obtain a sufficient amount of purified enzyme, Pichia was used in an effort to increase the production of HRP mutations. 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 Pichia secretion vector pPICZaB. In this construct (pPICZaB-HRP, FIG. 1B), HRP was fused to a-factor signal peptide and expression was directed to methanol. Typical expression curves are shown in FIG. 25. For HRP3-17E12, after 55 hours of incubation, 6,500 units / L of HRP activity was detected in the supernatant (FIG. 25, white square), 6.5 times that obtained from yeast. After a as well as the laboratory was added in studies by other investigators, a rare metal element (trace metal element), heme synthesis intermediate amino LES disadvantage Nick acid (aminolevulinic acid), and (as thiamin (thiamine)) vitamin additives to the culture medium E There was a tremendous increase in the yield of heme-containing proteins in .coli (59-62). The addition of these additives to the Pichia culture medium in this experiment resulted in a 32% increase in HRP3-17E12 activity in the supernatant (FIG. 25, black square).

Sequence Determination

As a result of sequencing, HRP1-77E2, the parent for the thermal and hydrogen peroxide stability studies, had D255 of N255 (GAC → AAC) and the second mutation L371 (TTA → ATA). This residue is part of helix 2 and is near the heme sac (see 34; Fig. 12, SEQ ID NO: 5 and SEQ ID NO: 6).

Mutant HRP1-4B6 has K232M (AAG → ATG) as well as L371. This residue is part of Helix 14 and is exposed to a surface solvent (see Figure 13, SEQ ID NO: 7 and SEQ ID NO: 8).

The thermostable mutation HRP1-28B11 between HRP1-77E2 and HRP1-4B6 has a mutation of F221L (TTT → TTA) in addition to L371. This residue is in a structural loop and is part of the substrate access channel (FIG. 14, SEQ ID NO: 9 and SEQ ID NO: 10).

Mutant HRP1-24D11 has a mutation of L131P (CTA-> CCA) in addition to L371. This residue is at the end of helix 7 and is exposed on the surface (see Figure 15, SEQ ID NO: 11 and SEQ ID NO: 12).

HRP1-117G4, the most preferred mutation in the first generation in terms of total activity, contains five mutations for the parent: (1) D255 → N255 (GAC → AAC) (wild-type sequence); (2) L131P (CTA-> CCA); (3) L223Q (CTG → CAG); silent mutations (4) N135 (AAC → AAT) and (5) T257 (ACT → ACA). For the L223Q mutation, this amino acid residue is in a loop and exposed to the solvent (see Figure 16, SEQ ID NO: 13 and SEQ ID NO: 14).

Surprisingly, the improved HRP mutations, HRP1-80C12 (see Figure 17, SEQ ID NO: 17 and SEQ ID NO: 18) and HRP1-77E2 (see Figure 20, SEQ ID NO: 23 and SEQ ID NO: 24) are also D255 → N255 (GAC → AAC) In addition, HRP1-80C12 contains L131P (CTA-> CCA) found in HRP1-77G4. HRP-77E2, on the other hand, has a L371 (TTA → ATA) mutation, which is part of helix B, the heme pouch and is probably solvent accessible.

HRP2-28D6 (see FIG. 18, SEQ ID NO: 19 and SEQ ID NO: 20) contains two additional mutations for HRP1-117G4: T102A (ACT → GCT) and P226Q (CCA → CAA). T102A is part of Helix D and is the only mutation buried inside the structure. P226Q is in the same loop as the L223Q. In contrast, HRP2-13A10 further comprises four mutations for HRP1-117G4: R93L (CGA → CTA); T102A (ACT → GCT); K241 (AAA → ACA); And V303E (GTG → GAG). Solvent accessible R93L is in the structural loop connecting Helix C and D. K241T is in a structural loop that connects Helix G and H. This residue is again exposed to the solvent. Finally, V303E is part of a long strand that extends from Helix G to the C terminus of the protein. These three mutations seem to contribute to the mutant's increased stability over others.

HRP3-17E12 has three more mutations compared to HRP2-28D6: N47S (AAT → AGT); K241T (AAA → ACA); And silent mutation G121 (GGT → GGC). It is noteworthy that K241T is also found in HRP2-13A10. N47S is located in the structural loop connecting Helix B and a3-helix and is solvent accessible.

Analysis of the mutation

Three improved mutations in the first generation had D255N compared to the parental HRP1A6. This suggests that glycosylation sites on HRP benefit folding and expression. The function of sugar chains in proteins is an interesting problem, but its role in protein folding, modification and secretion is increasingly recognized (56-58).

Three identical mutations cannot be easily explained by changes in codon usage (63). N135 (AAC → AAT) and T257 (ACT → ACA) show little change in frequency of use, but in G121 (GGT → GGC), GGT (61%), which is a more commonly used codon, has a lower frequency of use. %). However, it is unclear how this substitution affects the translation of HRP mRNA.

Analysis of mutations related to reactivity and stability

In addition to the ABTS assay, the guajacol system, a second independent activity assay for HRP mutations, was used (see Figure 21). Both assays show a very good correlation with the activity of the mutations.

Figure 22a and Figure 22b is a time when the H ₂ O ₂ is not (a) and 1mM H ₂ O ₂ is (b) after incubation at 50 ^o C shows a correlation between the reactivity and stability. In both cases, the HRP2-13A10 mutation showed the highest stability in combination with good reactivity. As revealed by sequencing, three amino acid changes appear to exhibit this stability.

A similar pattern of stability was observed in organic solvent systems (FIG. 23), where the HRP2-13A10 mutation showed the best activity ratio in buffer to dioxane / buffer.

Example 3 Expression and Secretion of CCP in E. coli

CCP expression vector construction

S.cerevisiae cytochrome c peroxidase from pT7CCP provided by Dr. Dave Goodin (The Scripps Research Institute, La Jolla, Calif.) Was shown by HindIII directly below the MscI site and termination codon at the initiation codon by PCR. Recloning to introduce site. PCR products were cut with McsI and HindIII and linked to similarly cut pET-22b (+) to make pETCCP (see FIG. 26). pT7CCP has a CCP gene whose N-terminal sequence is modified to encode amino acid Met-Lys-Thr, as described by Goodin et al. (17) and Fitzgerald et al. (16). Thus, in this construct, the CCP gene is under the control of the T7 promoter and has been fused to the translation frame with the pelB signal sequence located in the periplasm.

Expression of CCP

CCP expression experiments in E. coli BL21 (DE3) were performed in LB medium containing 100 μg / ml ampicillin. The cells were grown at 37 ^° C. until A ₆₀₀ became 0.7-0.8, at which time IPTG was added to a final concentration of 1 mM to induce the synthesis of CCP from the T7 promoter. The cells were further grown at 30 ^° C. for 20 hours, and cells and supernatants were harvested by centrifugation.

It is known that CCP is folded directly in E. coli . Surprisingly, more than 95% of the CCP protein was found in LB medium culture at very high levels (approximately 100 mg / L by SDS-PAGE). The protein was active against ABTS, indicating that the secreted CCP is correctly folded and contains heme.

While the embodiments of the present invention have been described so far, those disclosed herein are merely exemplary, and it will be apparent to those skilled in the art that the present invention may be variously modified, selected, and applied within the scope of the present invention. It will be true. For example, in order to practice the present invention, the steps of any method of the present invention may be in any order, including at the same time, unless a particular order is necessary, inherently equipped, or absolute. The implementer of should understand. Accordingly, the present invention is not limited to any particular embodiment or illustration herein. The invention is defined in accordance with the claims and is limited only by the claims.

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Claims (33)

A method for producing and improving expression-resistant polypeptides comprising the following steps: (i) providing at least one parent polynucleotide encoding a functional expression resistant parent polypeptide in a selected host cell; (ii) altering the nucleotide sequence of the parent polynucleotide to produce a population mutant polypeptide; (iii) transforming the host cell to express the mutant polypeptide; And (iv) screening for functional mutations caused by host cells having at least one property of improved expression or folding without inclusion body formation. - The method of claim 1, The parent polypeptide forms an inclusion body when overexpressed in the host cell. Way. The method of claim 1, The parent polypeptide has at least one of a disulfide bond structure and a sugar chain structure Way. The method of claim 1, The parent polypeptide has or is associated with at least one heme group Way. The method of claim 1, The parent polypeptide is produced in a nonfunctional form when overexpressed in the host cell, and is produced in a functional form when it is underexpressed in the host cell. Way. The method of claim 5, The parent polypeptide is overexpressed under the control of an inducible promoter in the presence of an inducer and underexpressed under the control of an inducible promoter in the absence of an inducer. Way. The method of claim 1, The parental polynucleotide of each cycle is characterized by repeating the method of claim 1, which is a mutant polynucleotide from a previous cycle, in a number of cycles. Way. The method of claim 1, Altering the nucleotide sequence is carried out in at least one method of random mutation, site-specific mutation and DNA randomization. Way. The method of claim 7, wherein Altering the nucleotide sequence is carried out in at least one method of random mutation, site-specific mutation and DNA randomization. Way. A polynucleotide improved according to the method of claim 1. A polynucleotide improved according to the method of claim 7. A polynucleotide modified according to the method of claim 9. The method of claim 1, The host cell is transformed with a vector having a signal sequence that directs the secretion of the polypeptide encoded by the mutant polynucleotides. Way. The method of claim 13, The signal sequence is characterized in that the PelB Way. The method of claim 1, The host cell is characterized in that the facile host cell Way. The method of claim 1, The host cell is characterized in that selected from yeast and bacteria Way. The method of claim 7, wherein The host cell is characterized in that selected from yeast and bacteria Way. The method of claim 9, The host cell is characterized in that selected from yeast and bacteria Way. The method of claim 1, The host cell is characterized in that the E. coli cells Way. The method of claim 1, The host cell is characterized in that the S. cerevisiae cells Way. The method of claim 1, The host cell is characterized in that the P. pastoris cells Way. The method of claim 9, Characterized in that the host cell is an E. coli cell. Way. The method of claim 9, The host cell is characterized in that the S. cerevisiae cells Way. The method of claim 9, The host cell is characterized in that the P. pastoris cells Way. The method of claim 7, wherein The polypeptide is characterized in that the protein containing heme (heme) Way. The method of claim 9, The polypeptide is characterized in that the protein containing heme (heme) Way. The method of claim 18, The polypeptide is characterized in that the protein containing heme (heme) Way. The method of claim 7, wherein The polypeptide is characterized in that the peroxidase enzyme Way. The method of claim 9, The polypeptide is characterized in that the peroxidase enzyme Way. The method of claim 18, The polypeptide is characterized in that the peroxidase enzyme Way. The method of claim 26, The polypeptide is characterized in that the horseradish peroxidase Way. The method of claim 27, The polypeptide is characterized in that the horseradish peroxidase Way. The method of claim 28, The polypeptide is characterized in that the horseradish peroxidase Way.