CA2331777A1 - Expression of functional eukaryotic proteins - Google Patents

Abstract

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

Description

I~.\1'RI:SSION U1~ l~ UNC'1'IUNA1. I?UKrIR~'OTIC PROTEINS
" _ The Government has cetlain rights to this invention pursuant to Grant Nos.

96-1-U34U and N00014-98-1-0657, awarded by the United States Navy.
'I~his application claims priority from U.S. application No. 60/094,403 filed on July i ?5, I~)~)S and No. ~U/1U6,84U filed November 3, 1998.
I3ACKGRUUND UI~ TI-iC INVENTION
Field of the Invention I 0 'this invention relates to methods for the selection and production of polynucleotides that encode functional poiypeptidcs or proteins, especially eukaryotic proteins, and particularly in facile host cell expression systems. Facile expression systems include robust prokaryotic cells (e.g. bacteria) and eukaryotic systems (e.g. yeast). In particular, the invention concerns the recombinant production of expression-resistant functional eukaryotic 15 proteins by host cells, in high yield, and without deactivation, denaturation, inclusion bodies, or other loss of structure or function. In preferred embodiments, the expressed proteins are secreted by the host cells. Preferred proteins of the invention include peroxidases and heme-containing proteins, such as horseradish peroxidase (HRP) and cytochrome c peroxidase (CCP). Polynucleotides which encode and express these proteins in recombinant host cell 20 expression systems are also encompassed by the invention.
Description of Related Art The publications and reference materials noted herein and listed in the appended Bibliography are each incorporated by reference in their entirety. They are referenced 25 numerically in the text and the Bibliography below.
Many proteins of interest are produced by organisms having "eukaryotic" cells.
These are cells having a nucleus surrounded by its own membrane and containing DNA on structures called chromosomes. All multicellular organisms, such as humans and animals, and many single-cell animals, have eukaryotic cells. Other single-cell organisms, such as SUBSTITUTE SHEET (RULE 26) bacteria have "prokaryotic" cells. These cells have a primitive nucleus with DNA in a defined structure, but without chromosomes and a nuclear membrane that is characteristic, _ of eukaryotes. Prokaryotic organisms are generally mucl7 easier and less costly to grow, maintain and manipulate than eukaryotic cells.
Genetic engineering and recombinant DNA and RNA technologies have made it possible to produce proteins, hormones and enzymes that arc native to one organism, by using the cells of a different organism as "factories" or host cell expression systems. In particular, it is often desirable to express a protein ofeukaryotic origin in a prokaryotic host cell, because the prokaryotes can be grown in large quantities of identical cells, to product large amounts of the desired foreign protein. For example, certain human proteins may be useful as drugs if they can be supplied in sufficient quantity to patients who have a protein deficiency. Such proteins may not easily or ethically be obtained by isolating them from human cells, nor can they easily be made by direct chemical synthesis or by growing them in isolated tissue cultures. Other proteins and enzymes are useful in industry. For example, certain enzymes can break down food products, and are useful in laundry detergent.
However, commercial applications require large amounts of protein and a high degree of quality control.
To solve some of these problems, recombinant genetic engineering techniques have been developed to use genetic machinery of other cells, such as bacteria and yeast, to produce human or other proteins. Selected genetic material, such as a polynucleotide that encodes a desired protein, is "recombined" with genetic material in a host cell, so that the host cell expresses the introduced foreign genetic material and produces the desired polypeptide or protein. Bacteria and yeast can be suitable host cells because they are easy and economical to grow and maintain in large quantities, and can be used to reliably and repeatably produce foreign proteins.
However, many proteins can not easily be expressed in foreign host cells, including bacteria and yeast. Such expression-resistant polypeptides or proteins may not be expressed at all, or are expressed inefficiently, e.g. in low yield. The protein may be expressed, but can lose some or all of its or function. In some cases the expressed protein may lose some or all of its active folded structure, and may even become denatured or completely inactive.
Expressed proteins may also be encapsulated inside inclusion bodies within a host cell.
These are discrete particles or globules inside and separate from the rest of the cell, and _2_ SUBSTITUTE SHEET (RULE 26) which contain expressed protein, perhaps in agglomerated or inactive form.
This makes it difficult to harvest the produced protein from the host cells, as the isolation and purification' techniques can be difficult, inefficient, time-consuming and costly. Efforts to, produce expression-resistant polypeptides in active or functional form and at relatively high yields have spanned many years and have been markedly unsuccessful. In particular, expression-resistant enzymes that arc commercially important, such as peroxidase enzymes like horseradish peroxidase, have not been functionally expressed in reasonably high yield or in convenient, economical or facile host cells. These enzymes arc instead produced in non-funetional or inactive form, for example as inclusion bodies, and are laboriously manipulated and reconstituted to obtain active enzymes at relatively poor yields.
Some proteins that are made by cells can be secreted or delivered outside the cell, which can improve the yield and the efficiency of subsequent isolation and purification steps. However, many proteins are not naturally secreted, and are difficult to secrete artificially, for example because they contain chemical groups that do not easily cross the cell membrane. in particular, it is difficult to engineer a compatible protein and host cell system to secrete a protein that has a tendency to form inclusion bodies.
Therefore, improved techniques for expressing foreign proteins are needed, particularly proteins of eukaryotic origin, and particularly recombinant proteins which can be secreted by host cells in high yield, and without loss of activity or function.
As discussed, a particular challenge when producing foreign proteins in a host cell expression system is the inability of many foreign proteins to fold properly into functional proteins when using common recombinant hosts such as E. coli and yeast ( 1-4).
As a result, the polypeptide chains that are produced in a recombinant host cell system are often degraded upon synthesis or accumulate in inclusion bodies. This is particularly true for eukaryotic proteins that contain disulfide bonds or are glycosylated in the native form. The underlying reasons, which are not clearly understood and are probably multifactorial, may include the "unnatural" recombinant environments in which the proteins accumulate (35) and the lack of proper folding cofactors such as molecular chaperones in the E. coli host (3).
Additionally, glycosylation has been implicated in protein folding in eukaryotic organisms (36), which function is absent in bacteria.
The folding problem presents a challenging roadblock to the large-scale production of proteins for pharmaceutical or industrial applications. The lack of high-efficiency SUBSTITUTE SHEET (RULE 26) functional expression systems has also become one of the bottlenecks in applying directed evolution techniques Cor optimizing proteins and reaction conditions for desired uses. ~ _ Employing random mutagcnesis and Lenc recombination follow~cd by screening or selection, directed evolution has been successfully applied to improve a variety of enzyme properties, _S such as substrate specificity, activity in organic solvents, and stability at high temperatures, which arc often critical for industrial applications {5). Eukaryotic enzymes have a myriad of existing and potential applications, but improvement of these proteins by directed evolution had been limited by tl~c inability to functionally express them in a facile recombinant host.
For example, the difficulty of expressing peroxidase enzymes in a facile expression host has posed at least two technical challenges for realizing the potential of peroxidases as biocatalysts. First, efforts to modi fy these enzymes for industrial applications by protein engineering methods have been impeded. Directed evolution, for example, exploits expression in a host such as E. colt or S. cerevisiae, organisms in which large libraries of mutants or variants can be made. Second, the lack of efficient expression in an appropriate foreign (heterologous) host prevents the mass production of some of these proteins on an economical scale.
One way to obtain the active form of recombinantly expressed proteins is by refolding them in vitro from inclusion bodies, but these processes are often laborious and inefficient (1-3). Additionally, this is not a viable option for directed evolution in which screening of tens of thousands of mutants is required. A more advantageous means to resolve the problem may be to identify mutations in a target gene that can facilitate folding in host environments. Evidence from a number of studies increasingly suggests that certain residues of an amino acid sequence have a profound influence on the folding per se of the protein. Thus, it would be highly advantageous if scientists could identify mutations in a target gene that facilitate folding in the host environment. This may avoid the inclusion body obstacle, but such techniques require the discovery, identification, and use ofparticular beneficial mutants.
For example, a series of studies by King and coworkers have shown that several single amino acid substitutions interfered with the productive folding of the phage p22 tailspike protein at restrictive temperature iu vivo, and that second-site suppresser mutations were able to rescue the defective folding mutants (6). In another study, the replacement of SUBSTITUTE SHEET (RULE 26) tyrosine 35 with leucine in bovine pancreatic trypsin inhibitor (BPTI) eliminated kinetic traps in the folding pathway i~t vitro (7). Furthem~ore, it was reported that several mutantb _ of human intcrleukin 1 (3, created by cassette mutagenesis of a tew selected residues, were expressed in E. coli in soluble form, while the wild type was largely insoluble and formed inclusion bodies (8). In a separate study, a single site-directed mutation was found to improve the folding yield of a recombinant antibody (9).
It is difficult to predict which residues arc critical for protein function or stability, let alone folding. Thus, it would be advantageous if there was a method for systematically searching for beneficial mutations that affect the folding and expression of proteins, without l0 compromising biological activity. Directed evolution techniques may prove useful in the accomplishment of this goal. This evolutionary approach uses DNA shuffling, for simultaneous random mutagenesis and recombination, to generate a variant having an improved desirable property over the existing wild type protein. Point mutations arc generated due to the intrinsic infidelity of Taq-based polymerase chain reactions (PCR) associated with reassembly of nucleic acid sequences. In one example, Stemmer and coworkers applied this technique to the gene encoding for green fluorescence protein (GFP), which resulted in a protein that folded better than the wild type in E. coli (10).
One group of proteins of particular interest are heme proteins, that is, they have iron containing heme groups. These proteins have many biological and biochemical uses, and include certain enzymes called peroxidases, which are enzymes that facilitate oxidation or reduction reactions in which a peroxide (e.g. hydrogen peroxide) is one of the reactants.
Peroxides are compounds, other than molecular Oz, in which oxygen atoms are joined to each other. For example, the heme enzyme horseradish peroxidase (HRP) is widely used as a reporter in diagnostic assays. HRP catalyzes a reaction in which starting materials or substrates are chemically combined in the presence of a peroxide, such as hydrogen peroxide (HzOz), with water (Hz0) as a byproduct. This reaction can be exploited to indicate whether another reaction of interest has occurred, or whether certain materials, such as HRP starting materials, are present in a mixture or sample. It would be beneficial to provide a means of producing large quantities of HR.P, and other heme or peroxidase enzymes, using efficient and cost-effective systems such as prokaryotic expression systems. However, native HRP
contains four disulfides and is highly giycosylated (--21%), although the carbohydrate moiety has no apparent effect on the activity or stability (11). As a consequence, previous SUBSTITUTE SHEET (RULE 26) ~ ~vi vv ~ v vv ~ ~Vdv attempts to express HRP in bacteria have yielded inclusion bodies, with no functional expression (12-14). Successful expression in yeast has also not been achieved prior to this»
i nvenuon.
Accordingly, there is a need to develop new and improved methods for expressing S groteins which ordinarily have difficulty being expressed in order to obviate the need for laborious iu vitro folding protocols. In particular, there is a need for protein expression methods which arc well-suited for use in connection with directed evolution techniques.
In particular, this invention describes methods torscrecning libraries of HRP
mutants produced by error-prone PCR and DNA shuffling to identify mutations that facilitate functional expression in bacteria (E. coli, 13. siibtilis) and yeast (S.
cc~reusine). In one exemplary embodiment, the variant of the invention is a functional and active horseradish peroxidase (HRP) that is expressed in E. coli without inclusion bodies at levels of about 110 p.g/L. This is comparable to amounts previously obtained from much more costly, time-consuming and laborious in vitro refolding techniques used to recover other HRP enzymes from inclusion bodies.
SUMMARY OF THE INVENTION
The observed constraints on the use of native proteins are thought to be a consequence of evolution. Proteins have evolved in the, context and environment of a living organism, to carry out specific biological functions under conditions conducive to life - not in the laboratory or under industrial conditions. In some cases, evolution may favor or even require less than optimally efficient enzymes. The output, efficiency, working conditions, stability and other properties of known expression systems are not thought to be unalterable, nor are they limitations which should be seen as intrinsic to the nature of cellular expression systems. It is possible that the proteins used in these systems can be evolved in vitro, or that analogous proteins can be otherwise developed, to alter or enhance the protein's properties, for example, to obtain much more efficient expression, folding, and secretion, while maintaining activity of the protein. Improved proteins can also be obtained by screening cultures of native organisms or expressed gene libraries (3).
Many proteins, when expressed using facile expression systems (e.g., E. coli) result in inclusion bodies or are inactive due to an inability to properly fold. The invention takes advantage of directed evolution techniques to create novel polynucleotides encoding for SUBSTITUTE SHEET (RULE 26) mutated functional proteins which have an increased ability to be produced in an expression system, without inactivation or inclusion bodies. In preferred embodiments the protein is -secreted outside of the cell.
There are several advantages to secreting proteins from bacteria into the culture media. since in many cases desired substrates cannot readily pass through the membranes of E. coli. Secretion can facilitate screening in directed evolution studies, because, by allowing the secreted enzyme to catalyze a reaction in the culture medium, substrates that cannot enter the cells can be used. It can also significantly simplify the production of recombinant proteins, as the culture supernatant is largely free of contaminating substances, lU if the secretion level is high enough. Nonetheless, secretion of proteins from bacteria into culture media remains a di fficult task, particularly for enzymes that contain bulky prosthetic groups such as heme.
This problem can be solved by using a suitable signal peptide, such as the signal from the pectate lyase B (PeIB) of Em~inia carorovora (27), to efficiently direct the secretion of a peroxidase such as I-iRP or CCP into the culture medium. This signal peptide is also generally applicable to outer proteins containing heme prosthetic groups, such as cytochrome P450 enzymes and other peroxidases.
According to one embodiment of the invention, directed evolution or random mutagenesis is used to produce in vitro proteins which readily fold after expression, even in yeast and in prokaryotic expression systems such as E. call, and are easily secreted outside the host cell in quantities expected for proteins produced by such expression systems. Furthermore, activity of these proteins is not compromised by the mutagenic step after appropriate selection is made.
Thus, the invention provides a method for improving the expression of a ~ polynucleotide encoding peroxidase enzymes by using directed evolution, and polynucleotides encoding for variant horseradish peroxidase which have improved expression in conventional expression systems.
The above features and many other attendant advantages of the invention will become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.
_7_ SUBSTITUTE SHEET (RULE 26) FIG. 1 A is a schematic map of an E. coli HRP expression vector pETHRP, the' -plasmid pETpeIBI~RP. The 1-iRP gene (with an extra methioninc residue at the N-terminus) was inserted into pET-22b(+), immediately downstream of the signal sequence from the pectate lyase B (PeIB) of Crwiuia caro~oaora for periplasmic localization.
Expression is under the control of the T7 promoter.
f IG. 1 Q is a schematic map of a I'. pasioris expression vector pPICZaB-HRP.
The 1-1 RP gene was inscricd immediately downstream of the plasmid's a-factor signal.
Expression is under the control of the methanol-inducible P~"~, promoter.
FIG. 2 shows the nucleic acid and x1111110 aCld sequences of the pelB signal peptide 1SEQ. ID. NO. 1 and SEQ. 1D. NO. 2).
F1G. 3 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant designated HRP1A6 (~SEQ. ID NO. 3 and SEQ. ID. NO. 4]).
FIC. 4 is a map of the expression vector pETpeIBHRP 1 AG.
FIG. SA shows the relative activities ofwild-type and an HRP mutant (I AG) evolved in E. coli.
FIG. 5B shows a representative landscape of first generation HRP mutants sorted by activity in descending order. Activities are normalized to that of wild-type.
FIG. 6 shows activity levels of the mutant HRP 1 AG at various ITPG
concentrations.
FIG. 7 is a representation of the structure of HRP, showing the location of the Asn255 to Asp mutation in a surface loop of HRP mutant I AG. This figure was generated from published HRP coordinates (34), using Insight II software (Molecular Biosystems).
FIG. 8 is a map of the expression vector pYEXS 1-HRP containing a coding sequence for HRP cloned into the secretion plasmid pYEX-S I .
FIG. 9 shows the activity levels of HRPIA6 and three other mutants obtained by directed evolution in S. cerevisiae: HRP 1-77E2, HRP I -11764, and HRP2-28DG.
In this example HRP 1 A6 was the parent of HRP I -77E2 and HRP 1- I I 764. while HRP I

was the parent of HRP2-28D6.
FIG. 10 shows the residual activity of several HRP mutants as a function of temperature, in a thermal inactivation curve that indicates the relative thermostability of the mutants.
_g_ SUBSTITUTE SHEET (RULE 26) FIG. I1 shows the residual activity of several HRP mutants as a function of hvdroeen peroxide concentration, in a titration curve that indicates the relative ability of the, _ mutants to resist degradation in the presence of hydrogen peroxide.
FIG. 12 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant dcsi~:natcd HRP1-77E2 ([SEQ. 1D NO. 5 and SEQ. 1D. NO. 6]).
hIG. 13 shows a nucleotide and amino acid sequence encoding an I-1RP enzyme variant designated HRP1-4B6 ((SEQ. ID NO. 7 and SEQ. ID. NO. 8J).
f IG. 14 shows a nucleotide and amino acid sequence encodine an HRP C117y111C
variant c9csi~.:natcd I-iRPI-2881 1 ([SEQ. ID NO. 9 and SEQ. ID. NO. 10]).
hIG. 15 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant dcsi~~nated HRPi-24D11 ((SEQ. 1D NO. 11 and SEQ. ID. NO. 12J).
FIG. 16 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant designated HRP1-1 1764 ((SEQ. ID NO. 12 and SEQ. ID. NO. 13]).
FIG. 17 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant designated HRP1-80C12 ([SEQ. ID NO. 17 and SEQ. ID. NO. 18]).
FIG. 18 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant designated HRP2-28D6 ([SEQ. ID NO. 19 and SEQ. ID. NO. 20]).
FIG. 19 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant designated HRP2-13A10 ([SEQ. ID NO. 21 and SEQ. ID. NO. 22]).
FIG. 20 shows a nucleotide and amino acid sequence encoding an HRP enzyme variant designated HRP3-17E12 ([SEQ. ID NO. 23 and SEQ. ID. NO. 24J).
FIG. 21 shows the activities of wild-type, parent (HRP1A6) and evolved HRP
mutants in S. cerevisiae strain BJ5465. The values were obtained with the ABTS
assay .
Cells were grown in shaking flasks at 30°C for 64h.
FIG. 22 shows A) The correlation between reactivity and stability (A,~s;~/A;) ofHRP
mutants.
FIG. 23 shows reactivity of HRP mutants in organic solvent / water systems.
F1G. 24 shows the lineage of the mutants. Nucleotide substitutions are shown in parentheses following the corresponding amino acid substitutions, and synonymous mutations in Italics. For each generation new mutations are donated with "*".
FIG. 25 shows the accumulation of secreted HRP activity from Pichia for the variant HRP3-17E2.

SUBSTITUTE SHEET (RULE 26) FIG. 26 is a schematic map of the yeast cytochrome c peroxidase expression vector pETCCP
DETAILED DESCRIPTION OF THE INVENTION
_5 This in~~ention concerns methods for improving the expression of proteins using conventional expression systems, which proteins would ordinarily result in inclusion bodies or arc degraded upon synthesis due to an inability to fold properly in the environment of the expression system.
Definilion.s.
As used herein, "about" or "approximately" shall mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range.
The term "substrate" means any substance or compound that is convened or meant to be converted into another compound by the action of an enzyme catalyst. The tern includes aromatic and aliphatic compounds, and includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate.
An "oxidation reaction" or "oxygenation reaction", as used herein, is a chemical or biochemical reaction involving the addition of oxygen to a substrate, to form an oxygenated or oxidized substrate or product. An oxidation reaction is typically accompanied by a reduction reaction (hence the term "redox" reaction, for oxidation and reduction). A
compound is "oxidized" when it receives oxygen or loses electrons. A compound is "reduced" (it loses oxygen or gains electrons).
The term "enzyme" means any substance composed wholly or largely of protein or polypeptides that catalyzes or promotes, more or less speci fically, one or more chemical or biochemical reactions.
A "polypeptide" (one or more peptides) is a chain of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds. A
protein or polypeptide, including an enzyme, may be "native" or "wild-type", meaning that it occurs in nature; or it may be a "mutant", "variant" or "modified", meaning that it has been made, altered, derived, or is in some way different or changed from a native protein, or from another mutant. A "parent" polypeptide or enzyme is any polypeptide or enzyme from which any other polypeptide or enzyme is derived or made, using any methods, tools or SUBSTITUTE SHEET (RULE 26) techniques, and whether or not the parent is itself a native or mutant polypeptide or enzyme.
A parent polynucleotide is one that encodes a parent polypeptide. A "test enzyme" is a~ _ protein-containing substance that is tested to dctcm~ine whether it has properties of an enzyme. The term "enzyme" can also refer to a catalytic polynucleotide (e.g.
RNA or DNA).
The "activity" of an enzyme is a measure of its ability to catalyze a reaction, and may be expressed as the rate at which the product of the reaction is produced. For example, enzyme activity can be represented as the amount of product produced per unit of time, per unit (e.g. concentration or weight) ofcnzyme. The "stability" ofan enzyme means its ability to function, over time, in a particular environment or under particular conditions. One way l0 to evaluate stability is to assess its ability to resist a loss of activity over time, under given conditions. Enzyme stability can also be evaluated in other ways, for example, by determining the relative degree to which the enzyme is in a folded or unfolded state. Thus, one enzyme is more stable than another, or has improved stability, when it is more resistant than the other enzyme to a loss of activity under the same conditions, is more resistant to unfolding, or is more durable by any suitable measure. For example, a more "thermally stable" or "thermostable" enzyme is one that is more resistant to loss of structure (unfolding) or function (enzyme activity) when exposed to heat or an elevated temperature.
One way to evaluate this is fo determine the "melting temperature" or Tm for the protein. The melting temperature, also called a midpoint, is the temperature at which half of the protein is unfolded from its fully folded state. This midpoint is typically determined by calculating the midpoint of a titration curve that plots protein unfolding as a function of temperature.
Thus, a protein with a higher Tm requires more heat to cause unfolding and is more stable or more thermostable. Stated another way, a protein with a higher Tm indicates that fewer molecules of that protein are unfolded at the same temperature as a protein with a lower Tm, again meaning that the protein which is more resistant to unfolding is more stable (it has less unfolding at the same temperature). Another measure of stability is T"~, which is the transition midpoint of the inactivation curve of the protein as a function of temperature. T"~
is the temperature at which the protein loses half of its activity. Thus, a protein with a higher T"~ requires more heat to deactivate it, and is more stable or more thermostable.
Stated another way, a protein with a higher T"~ indicates that fewer molecules of that protein are inactive at the same temperature as a protein with a lower T,n, again meaning that the protein which is more resistant to deactivation is more stable (it has more activity at the SUBSTITUTE SHEET (RULE 26) same temperature). These assays are also called "thermal shift" assays, because the inactivation or unfolding curve, plotted against temperature, is "shifted" to higher or lower w _ temperatures w~hcn stability increases or decreases. Thcnnostability can also be measured in other ways: For example, a longer half-life (t,;,} for the enzyme's activity at elevated temperature is an indication of thcmlostability.
An "oxidation enzyme" is an enzyme that catalyzes one or more oxidation reactions, typically by adding, inserting, contributing or transferring oxygen from a source or donor to a substrate. Such enzymes arc also called oxidorcductases or rcdox enzymes, and encompasses oxygcnascs, hydrogcnascs or reductases, oxidascs and pcroxidascs.
The terms "oxygen donor", "oxidizing agent" and "oxidant" mean a substance, molecule or compound which donates oxygen to a substrate in an oxidation reaction.
Typically, the oxygen donor is reduced (accepts electrons). Exemplary oxygen donors, which are not limiting, include molecular oxygen ordioxygen (02) and peroxides, including alkyl peroxides such as t-butyl peroxide, and most preferably hydrogen peroxide (Hz02).
A peroxide is any compound having two oxygen atoms bound to each other.
A "luminescent" substance means any substance which produces detectable electromagnetic radiation, or a change in electromagnetic radiation, most notably visible light, by any mechanism, including color change, UV absorbance, fluorescence and phosphorescence. Preferably, a luminescent substance according to the invention produces a detectable color, fluorescence or W absorbance.
The term "chemiluminescent agent" means any substance which enhances the delectability of a luminescent (e.g., fluorescent) signal, for example by increasing the strength or lifetime of the signal. One exemplary and preferred chemiluminescent agent is 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) and analogs.
Otherchemiluminescent agents include 1,2-dioxetanes such as tetramethyl-1,2-dioxetane (TMD),1,2-dioxetanones, and 1,2-dioxetanediones.
The term "polymer" means any substance or compound that is composed of two or more building blocks ('mgrs') that are repetitively linked to each other. For example, a "dimer" is a compound in which two building blocks have been joined together.
The term "cofactor" means any non-protein substance that is necessary or beneficial to the activity ofan enzyme. A "coenzyme" means a cofactor that interacts directly with and serves to promote a reaction catalyzed by an enzyme. Many coenzymes serve as carriers.

SUBSTITUTE SHEET (RULE 26) For example, NAD' and NADP~ cant' hydrogen atoms from one enzyme to another.
An "ancillary protein" means any protein substance that is necessary or.beneficial to the activity, _ of an enzyme.
The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gent, a DNA or RNA
sequence, a protein or an enzyme.
"DNA" (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that arc linked together on a deoxyribose sugar backbone. DNA can have one strand ofnucleotide bases, or two complimentary strands which may form a double helix structure.
"RNA" (ribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and uracil (U), called nucleotide bases, that arc linked together on a ribose sugar backbone. RNA typically has one strand of nucleotide bases.
A "polynucleotide" or "nucleotide sequence" is a series of nucleotide bases (also called "nucleotides") in DNA and RNA, and means any chain of two or more nucleotides.
A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide (although only sense stands are being represented herein). This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil.
The polynucleotides herein may be flanked by natural regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, poiyadenylation sequences, introns, 5'- and 3'-non-coding regions, and the like. The nucleic acids may also be modified by many means known in the ari. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl SUBSTITUTE SHEET (RULE 26) phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, ctc.}. Polynucieotides may contain» -one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., $ acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, ctc.}, and alkylators. The polynuclcotides may be dcrivatized by formation of a methyl or ethyl phosphotricster or an alkyl phosphoramidatc linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, eithec directly or indirectly. Lxemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
Proteins and enzymes are made in the host cell using instructions in DNA and RNA, according to the genetic code. Generally, a DNA sequence having instructions for a particular protein or enzyme is "transcribed" into a corresponding sequence of RNA. The RNA sequence in tum is "translated" into the sequence of amino acids which forn~ the protein or enzyme. An "amino acid sequence" is any chain of two or more amino acids.
Each amino acid is represented in DNA or RNA by one or more triplets of nucleotides.
Each triplet forms a codon, corresponding to an amino acid. For example, the amino acid lysine (Lys) can be coded by the nucleotide triplet or codon AAA or by the codon AAG.
(The genetic code has some redundancy, also called degeneracy, meaning that most amino acids have more than one corresponding codon.) Because the nucleotides in DNA
and RNA
sequences are read in groups of three for protein production, it is important to begin reading the sequence at the correct amino acid, so that the correct triplets are read.
The way that a nucleotide sequence is grouped into codons is called the "reading frame."
The term "gene", also called a "structural gene" means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or pan of one or more proteins or enzymes, and may or may not include regulatory DNA
sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA
to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription.
A "coding sequence" or a sequence "encoding" a polypeptide, protein or enzyme is a nucleotide sequence that, when expressed, results in the production of that polypeptide, SUBSTITUTE SHEET (RULE 26) protein or enzyme, i.e.. the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme- A coding sequence is "under the control" of transcriptional~ -and translational control sequences in a cell when RNA polymerise transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein _S encoded by the coding sequence. Preferably,.the coding sequence is a double-stranded DNA
sequence which is transcribed and translated into a polypeptide in a cell iu vitro or irr vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence arc determined by a start codon at the 5' (amino) terminus and a translation S(Ol) COdOn 1l the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukarvotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
Transcriptional and translational control sequences are DNA regulatory sequences, I5 such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.
A "promoter sequence" is a DNA regulatory region capable of binding RNA
polymerise in a cell arid initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining this invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA
polymerise. As described above, promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA.
A promoter may be "inducible", meaning that it is influenced by the presence or amount of another compound (an "inducer"). For example, an inducible promoter includes those which initiate or increase the expression of a downstream coding sequence in the presence of a particular inducer compound. A "leaky" inducible promoter is a promoter that provides a high expression level in the presence of an inducer compound and a comparatively very low SUBSTITUTE SHEET (RULE 26) expression level, and at minimum a detectable expression level, in the absence of the _ " _ inducer.
A "signal sequence" is included at the beeinnint of the Iodine sequence of a protein to be expressed in the periplasmic space, or outside the cell. This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptidc. The tcmt "translocation signal sequence" is also used to refer to a signal sequence. Translocation signal sequences can be found associated with a variety ofproteins native to eukaryotes and prokaryotes, and arc often functional in both types of organisms.
Proteins of llte invention may be further modified and improved by adding a sequence which 1 p directs the secretion of the protein outside the host cell. The addition of the signal sequence does not interfere with the folding of the secreted protein, and evidence thereof is easily tested for using techniques known in the aru and depending on the protein (e.g., tests for activity of a given protein after modification).
Preferred signal sequences ofthe invention include the pelB signal sequence, which normally directs a protein to the periplasmic space between the inner and outer membranes ofbacteria. Other signal sequences include, for example ompA and ompT (52).
The signal sequence is iigated upstream of the nucleotide sequence encoding the protein, such that the sequence is present at the N-tem~inus of the protein after expression.
Conventional cloning techniques can be used as described. Some routine experimentation within the scope of one skilled in the art may be necessary to optimize addition of the signal sequence to any given protein.
The terms "express" and "expression" mean allowing or causing the information in a gene or DNA sequence to became manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product" such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be "expressed" by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.
A polynucleotide or polypeptide is "over-expressed" when it is expressed or produced in an amount or yield that is substantially higher than a given base-line yield, e.g.

SUBSTITUTE SHEET (RULE 26) a yield that occurs in nature. For example, a polypeptide is over-expressed when the yield is substantially greater than the normal, average or base-line yield of the native -polypol~~peptide in native host cells under given conditions, for example conditions suitable to the life cycle of the native host cells. Over-expression ofa polypeptide can be obtained, for example, by altering any one or more of: (a) the growth or living conditions of the host cells; (b) the polynucleotide encoding the polypeptide to be over-expressed;
(c) the promoter used to control expression of the polynucleotide; and (d) the host cells themselves. This is a relative, and thus "over-expression" can also be used to compare or distinguish the expression level of one polypcptide to another, without regard for whether either polypcptidc is a native polypcptide or is encoded by a native polynucleotide.
Typically, over-expression means a yield that is at least about two times a normal, average or given base-line yield. Thus, a polypeptide is over-expressed when it is produced in an amount or yield that is substantially higher than the amount or yield of a parent polypeptide or under parent conditions. Likewise, a poiypeptide is "under-expressed" when it is produced in an amount or yield that is substantially lower than the amount or yield of a parent polypeptide or under parent conditions, e.g. at least half the base-line yield. In this context, the expression level or yield refers to the amount or concentration of polynucleotide that is expressed, or polypeptide that is produced (i.e. expression product), whether or not in an active or functional form. As one example, a polynucleotide or polypeptide may be said to he under-expressed when it is expressed in detectable amounts under the control of an inducible promoter, but without induction, i.e. in the absence of an inducer compound.
An expression product can be characterized as intracellular, extracellular or secreted.
The term "intracellular" means something that is inside a cell. The term "extracellular"
means something that is outside a cell. A substance is "secreted" by a cell if it delivered to the periplasm or outside the cell, from somewhere on or inside the cell.
As used herein, the terms "expression-resistant polypeptide" and "resistant to functional expression" are synonymous and refer to a polypeptide that is difficult to functionally express in selected host cells. For example, an expression-resistant polypeptide is not produced, or is produced in very low yield or in non-functional form, when a polynucleotide encoding that polypeptide is transformed or introduced into host cells, e.g.
into a facile host cell expression system.

SUBSTITUTE SHEET (RULE 2C) These polypeptides include, for example, those which have disulfide bridges, which arc composed of mutiplc subunits. or which require glycosylation. Expression-resistant, polypeptides also include those which arc sensitive to folding and unfolding conditions, particularly intracellular conditions (inside the cell), such as temperature, pH, protein _5 concentration, and the presence or absence of certain cofactors, coenzymes, ancillary proteins, ctc. Expression-resistant polypcptidcs also include polypeptides that are encoded by polynucleotides which arc sensitive to particular promoters or signal sequences in particular expression systems. In addition, expression-resistant polypeptides include those which tend to agglomerate, form inclusion bodies, or which arc produced in a non-active or unfolded fom~.
Particularly suitable for use as expression-resistant parent polypeptides in the invention are polypeptides that are inactive (e.g. they agglomerate, etc.) when produced at a high yield (e.g. when they are over-expressed), but which are active (e.g.
they do nor agglomerate, etc.) when produced at a very low yield (e.g. when they are under-expressed).
These include, for example, polypeptides that: (a) tend to agglomerate, form inclusion bodies, or are inactive or unfolded, when expressed in the presence of an inducer, by a polynucleotide that is under the control of an inducible promoter; and (b) tend not to agglomerate, etc., and are active, when expressed without inducer, by a polynucleotide that is under the control of the inducible promoter. Such promoters are known and can be called "leaky" promoters.
Polypeptides that include, incorporate or are associated with heme groups are also examples of expression-resistant polypeptides. Particular expression-resistant polypeptides of the invention are prexidase enzymes, such as horseradish peroxidase enzymes. An "expression-resistant polynucleotide" is a polynucleotide that encodes an expression resistant polypeptide.
A gene encoding a protein of the invention for use in an expression system, whether genomic DNA or cDNA, can be isolated from any source, particularly from a human eDNA
or genomic library. Methods for obtaining genes are well known in the art, e.g., Sambrook et al. ( 19).
Accordingly, any animal cell potentially can serve as the nucleic acid source for the molecular cloning of the gene of interest. The DNA may be obtained by standard procedures known in the art, such as from cloned DNA (e.g., a DNA "library"), from cDNA
_18_ SUBSTITUTE SHEET (RULE 26) library prepared from tissues with high level expression of the protein, by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof,, _ puri<<ed from the desired cell (19,51). Clones derived from genomic DNA may contain regulatory and intros DNA regions in addition to coding regions; clones derived from $ cDNA will not contain intros sequences.
In the molecular cloning of the gene from genomic DNA, DNA fragments arc Lcncrated, sonic of which will encode the desired gene. The DNA may be cleaved at specific sifts using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for 1 p example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.
The tem "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express I S the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a "cloned" or "foreign" gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or 20 sequences with no known function. A host cell that receives and expresses introduced DNA
or RNA has been "transformed" and is a "transformant" or a "clone." The DNA or RNA
introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.
The terms "vector", "cloning vector" and "expression vector" mean the vehicle by 25 which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA is inserted. A common way to insert one segment of DNA into another segment of 30 DNA involves the use of enzymes called restriction enzymes that cleave DNA
at specif c sites (specific groups of nucleotides) called restriction sites. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector SUBSTITUTE SHEET (RULE 26) into a host cell along with the transmissible vector DNA. A segment or sequence of DNA
having inserted or added DNA, such as an expression vector, can also be called a "DNA ~ -construct."
A common type of vector is a "plasmid", which generally is a self contained _S molecule of double-stranded DNA, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. A large number of i 0 vectors, including plasmid alld fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, lnc., Madison, WI), pRSET or pREP plasmids (lnvitrogen, San Diego, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), and many appropriate host cells, using methods disclosed or cited 15 herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. Preferred vectors are described in the Examples, and include without limitations pcWori, pET-26b(+), pXTD 14, pYEX-S 1, pMAL, and pET22-b(+). Other vectors may be 20 employed as desired by one skilled in the art. Routine experimentation in biotechnology can be used to determine which vectors are best suited for used with the invention, if different than as described in the Examples. In general, the choice of vector depends on the size of the polynucleotide sequence and the host cell to be employed in the methods of this invention.
25 A "cassette" refers to a segment of DNA that can be inserted into a vector at specific restriction sites. The segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
The term "expression system" means a host cell and compatible vector under suitable 30 conditions, e.g. for the expression of a protein coded for by foreign DNA
carried by the vector and introduced to the host cell. Common expression systems include bacteria (e.g.
E. coli and B. subtilis) or yeast (e.g. S. cerevisiae) host cells and plasmid vectors, and insect SUBSTITUTE SHEET (RULE 26~

host cells and Baculovims vectors. As used herein, a "facile expression system" means any expression system that is foreign or heterologous to a selected polynucleotide or, -polypeptide, and which employs host cells that can be grown or maintained more advantageously than cells that are native or heterologous to the selected polynucleotide or polypeptide, or which can produce the polypeptide more efficiently or in higher yield. For example, the use of robust prokaryotic cells to express a protein of eukaryotic origin would be a facile expression system. Preferred facile expression systems include !:.
colt, I3. snhlilis alld S. cereoisiae host cells and any suitable vector.
The terns "mutant" and "mutation" mean any detectable chanLC in gcnetic.material, l0 e.g. DNA, or any process, mechanism, or result of such a change. l~his includes gear mutations, in which the structure (e.g. DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g.
protein or enzyme) expressed by a modified gene or DNA sequence. The term "variant" may also be used to indicate a modified or altered gene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant.
"Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position.
"Function-conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, acidic, basic, hydrophobic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable.
Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99%
as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A "function-conservative variant" also includes a polypeptide or enzyme which has at least 60 % amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75 %, most preferably at least 85%, and SUBSTITUTE SHEET (RULE 26) even more preferably at least 90%, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared. , _ The teen "DNA reassembly" is used when recombination occurs between identical sequences. The term "DNA shuffling" indicates recombination between substantially homologous but non-identical sequences.
"isolation" or "purification" of a polypcptidc or enzyme refers to the derivation of the polypcptide by removing it from its original environment (for example, from its natural environment if it is naturally occurring, or form the host cell if it is produced by fCC0111b111a17t DNA I11C1110(15). Methods for polypcptidc purification arc wcll-known 111 the art, including, ~~~ithout limitation, preparative disc-gel electrophoresis, isoelectric focusing, 1-IPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution. For some puposes, it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against the protein or against peptides derived therefrom can be used as purification reagents.
Other purification methods are possible. A purified polynucleotide or polypeptide may contain less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated. A
"substantially pure" enzyme indicates the highest degree of purity which can be achieved using conventional purification techniques known in the art.
Polynucleotides are "hybridizable" to each other when at least one strand of one polynucleotide can anneal to another polynucleotide under defined stringency conditions.
Stringency of hybridization is detetzrtined, e.g., by a) the temperature at which hybridization and/or washing is performed, and b) the ionic strength and polarity (e.g., fotlrtamide) of the hybridization and washing solutions, as well as other parameters.
Hybridization requires that the two polynucleotides contain substantially complementary sequences;
depending on the stringency of hybridization, however, mismatches may be tolerated.
Typically, hybridization of two sequences at high stringency (such as, for example, in an aqueous solution of O.SX SSC at 65 °C) requires that the sequences exhibit some high degree of complementarily over their entire sequence. Conditions of intermediate stringency (such SUBSTITUTE SHEET (RULE 26) as, for example, an aqueous solution of 2X SSC at 65 °C) and low stringency (such as, for example, an aqueous solution of 2X SSC at 55°C), require correspondingly less overall ~ -complemcntarity between the hybridizing sequences. ( 1 X SSC is 0. I 5 M NaCI, 0.015 M
Na citrate.) Poiynucleotidcs that "hybridize" to the polynucleotides herein may be of any length. In one embodiment, such polynucleotides are at least i 0, preferably at least 15 and most preferably at least 20 nucleotides long. In another embodiment, polynucleotides that hybridizes are of about the same length. In another embodiment, polynuclcotides that hybridize include those which anneal under suitable stringency conditions and which encode l7plyl)cptldCS Uf Crl'Ly111CS havine the same function, such as the ability to catalyze an I 0 oxidation, oxygenase, or coupling reaction of the invention.
The general genetic engineering tools and techniques discussed here, including transforn~ation and expression, the use of host cells, vectors, expression systems, etc., arc well known in the art.
Mutagenesis and Direc~ed Evolution oJProtei~ts.
To improve the expression of proteins using conventional expression systems, the invention makes the unexpected discovery that directed evolution can be used to generate mutant libraries of polynucleotides which, when expressed using conventional or facile expression systems, result in functional proteins having normal or even higher activity than the native protein. Inclusion bodies, which commonly form when expressing proteins having disulfide bonds, and laborious in vitro refolding procedures can also be avoided by directed evolution.
According to the invention, proteins that are more easily expressed in facile gene expression systems can be obtained by using directed evolution to generate mutant polynucleotides in a library format for selection. General methods for generating libraries and isolating and identifying improved proteins (also described as "variants") according to the invention using directed evolution are described briefly below and more extensively, for example, in U.S. Patent Nos. 5,741,691 and 5,811,238. It should be understood that any method for generating mutations in polynucleotide sequences to provide an evolved poiynucleotide for use in expression systems can be employed. Proteins produced by directed evolution methods can then be screened for improved expression, folding, secretion, and function according to conventional methods.

SUBSTITUTE SHEET (RULE 26) Anv source of nucleic acid, in purified form can be utilized as the starting nucleic acid. Thusrthe process may employ DNA or RNA including messenger RNA, which DNA _ or RNA may be single or double stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. The nucleic acid sequence may be of various lengths depending on the size of the nucleic acid sequence to be mutated. Preferably the specific nucleic acid sequence is from 50 to 50,000 base pairs. It is contemplated that entire vectors containins~ the nucleic acid encoding the protein of interest may be used in the methods of this invention.
Any specific nucleic acid sequenee can be used to produce the population of mutants by the present process. An initial population of the specific nucleic acid sequences having mutations may be created by a number of di fferent known methods, some of which are set forth below.
Error-prone polymerase chain reaction (20,45,4G) and cassette mutagenesis (38-44), in which the specific region optimized is replaced with a synthetically mutagenized oligonucleotide can be employed in the invention. Error-prone PCR can be used to mutagenize a mixture of fragments of unknown sequences. These techniques can also be employed under low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence, or to mutagenize a mixture of fragments of unknown sequence.
Oligonucleotide-directed mutagenesis, which replaces a short sequence with a synthetically mutagenized oligonucleotide may also be employed to generate evolved polynucleotides having improved expression.
Alternatively, nucleic acid or DNA shuffling, which uses a method of in vitro or in vivo homologous recombination of pools of nucleic acid fragments or polynucleotides, can be employed to generate polynucleotide molecules having variant sequences of the invention.
Parallel PCR is another method that can be used to evolve polynucleotides for improved expression in conventional expression systems, which uses a large number of different PCR reactions that occur in parallel in the same vessel, such that the product of one reaction primes the product of another reaction. Sequences can be randomly mutagenized at various levels by random fragmentation and reassembly of the fragments by mutual priming. Site-specific mutations can be introduced into long sequences by random SUBSTITUTE SHEET (RULE 2fi) fragmentation of the template followed by reassembly of the fragmems in the presence of mutagenic oligonucleotides.
A particularly useful application of parallel PCR, which can be used in the invention, is called sexual PCR. In sexual PCR, also known as DNA shuttling, parallel PCR
is used to perform irr vi~ro recombination on a pool of DNA sequences. Sexual PCR can also be used to construct libraries of chimacras of genes from different species.
The polynucleotide sequences for use in the invention can also be altered by chemical mutagenesis. Chemical mutagens include, for example, sodium bisulfate, nitrous acid, hydroxylarninc, hydrazine or formic acid. Other agents which arc analogues of l0 nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, oracridine.
Generally, these agents are added to the PCR reaction in place of the nucleotide precursor thereby mutating the sequence. Intercalating agents such as profiavine, acriflavine, quinacrine and the like can also be used. Random mutagenesis of the polynucleotide sequence can also be achieved by irradiation with X-rays or ultraviolet light, or by subjecting the polynucieotide to propagation in a host (such as E. colt) that is deficient in thenormal DNA damage repair function. Generally, plasmid DNA or DNA fragments so mutagenized are introduced into E. colt and propagated as a pool or library of mutant piasmids.
Alternatively a mixed population of specific nucleic acids may be found in nature in that they may consist of different alleles of the same gene or the same gene from different related species (i.e., cognate genes). Alternatively, they may be related DNA
sequences found within one species, for example, the peroxidase class of genes. Once the mixed population of the specific nucleic acid sequences is generated, the poiynucleotides can be used directly or inserted into an appropriate cloning vector, using techniques well-known in the art.
Once the evolved polynucleotide molecules are generated they can be cloned into a suitable vector selected by the skilled artisan according to methods well known in the art.
If a mixed population of the specific nucleic acid sequence is cloned into a vector it can be clonally amplified by inserting each vector into a host cell and allowing the host cell to amplify the vector. The mixed population may be tested to identify the desired recombinant nucleic acid fragment. The method of selection will depend on the DNA fragment desired.
For example, in this invention a DNA fragment which encodes for a protein with improved SUBSTITUTE SHEET (RULE 26) WO 00/06718 PCT/US99/1712?
folding properties can be detern~ined by tests for functional activity of the protein and absence of inclusion body fom~ation. Such tests are well known in the art.
Using the methods of directed evolution, the invention provides a novel means for producing properly folded, functional, and soluble proteins in conventional or facile expression systems such as E. toll or yeast. Conventional tests can be used to detern~ine whether a protein of interest produced from an expression system has improved expression, folding and/or functional properties. For example, to dctennine whether a polynuclcotide subjected to directed evolution and expressed in a foreign host cell products a protein with improved folding, one skilled in lhC art Call perform experiments designed to test the functional activity of the protein. f3ric(ly, the evolved protein can be rapidly screened, and is readily isolated and purified from the expression system or media if secreted. It can then be subjected to assays designed to test functional activity of the particular protein in native forn~. Such experiments for various proteins are well known in the art, and are discussed in the Examples below.
In one embodiment, the invention contemplates the use polynucleotides encoding for variants of heme-containing proteins. Thus, the invention employs directed evolution to generate novel peroxidase enzymes, such as HR.P, which fold properly in the host cells (e.g. ~, toll) used in the expression system, retain functional activity, and avoid the problems associated with inclusion body formation.
The invention can also be applied to select or optimize an expression system, including selection of host cells, promoters, and signal sequences. Expression conditions can also be optimized according to the invention.
The Examples below further describe the methods of the invention and, in particular, teach the use ofdirected evolution to generate variants ofHRP which when expressed using conventional expression systems do not form inclusion bodies and retain functional activity.
Ordinarily, the corresponding native proteins form inclusion bodies and show little retained functional activity after expression in conventional expression systems.
Examples of practicing the invention are provided, and are understood to be exemplary only, and do not limit the scope of the invention or the appended claims. A
person of ordinary skill in the art will appreciate that the invention can be practiced in many forms according to the claims and disclosures here.

SUBSTITUTE SHEET (RULE 26) Functional Expression of Horseradish Peroxidase in E..coli and Yeast There is growing interest in exploiting eukaryotic peroxidases for use as industrial biocatalysts. Protein engineering and directed evolution to improve specific properties, however, arc complicated by the lack of facile recombinant expression systems.
In an effort to develop a functional bacterial expression system suitable for large-volume screening of mutants of horseradish peroxidasc (I~RP), the present Example describes the development of a bacterial expression system for heme-associated proteins, such as horseradish l 0 peroxidase (1-lRP), by inserting a corresponding gene as a fusion to the signal peptide Pell3.
In addition, by subjecting these genes to directed evolution heme-associated proteins fold more ef ~ ciently in E. colt and arc rendered more resistant to heat (thennostable) and more resistant to inactivation by H20:. This Example provides an approach for greatly facilitating efforts to "fine-tune" many enzymes that are promising industrial biocatalysts, but for which suitable bacterial or yeast expression systems are currently lacking because the proteins form inclusion bodies or are inefficiently secreted by the cell.
('IoninQ of HRP
The HRP gene (with an extra methionine residue at the N-terminus) was cloned from the plasmid pBBGlO (British Biotechnologies, Ltd., Oxford, UK) by PCR
techniques to introduce an Aat II site at the start codon and a Hind III site immediately downstream from the stop codon. This plasmid contains the synthetic horseradish peroxidase (HRP) gene described in Smith et al. (13), whose DNA sequence is based on a published amino acid sequence for the HRP protein (49). pBBGlO was made by inserting the HRP
sequence between the HindIII and EcoRl sites of the polylinker in the well-known plasmid pUCl9.
The PCR product obtained from this plasmid was digested with Aat II first, blunt-ended with t4 DNA polymerase, and then further restricted with Hind III. The digested product was and ligated into pET-22b(+) (purchased frori~ Novagen) treated with McsI
and Hind III, to yield the vector pETpeIBHRP. A map of this expression vector shown in FIG
1. In this construct, the HRP gene was placed under the control of the T7 promoter and is fused in-frame to the pelB signal sequence (See ~SEQ. ID NO. 1 and SEQ. ID NO. 2J and FIG. 2), which theoretically directs transport of proteins into the periplasmic space, that is, for delivery outside the cell cytoplasm (27). The ligation product was transformed into E. colt SUBSTITUTE SHEET (RULE 26) strain BL21 (DE3) for expression of the protein in cells both with and without induction by 1 mM isopropyl-b-D-thiogalactopyranoside (1PTG).
In the cells that were induced with IPTG, no peroxidase activity about background was detected, for BL21 (DE3) eelis or pET-22b(+)-harboring BL21 (DE3) cells, even though S the level of 1-1RP polypeptides accounted for over 20% of total cellular proteins. This was consistent with previous observations ( 12-14).
In the cells thU were trot induced with IPTG, clones were discovered that showed weak but mcasurahlc activity atainst azino-di-(cthylbcnzthiazoline sulfonate (ABTS).
l'he T7 pre~moter in tl~c pCT-22b(+) vector is known to be Icaky (31 ), and in theory it is therefore possible that sonic of the HRP polypeptide chains produced at this basal level were able to fold into the native form. Conversely, addition of 1PTG leads to high-level HRP synthesis, «~hich instead favors aggregation of chains and prevents their proper folding.
Subsequently, random mutagenesis and screening were used to identify mutations that lead to higher expression of HRP activity. , Thus, one aspect of the invention includes the use of a promoter that can regulate production of small amounts of polypeptide under some conditions, and larger amounts under other conditions. For example, a "leaky" inducible promoters can be used. This type of promoter produces high levels of a particular protein or proteins in the presence of an inducer compound, and much lower levels in the absence of inducer. In some embodiments, a polypeptide can be over-expressed under certain conditions (e.g. in the presence of inducer) and under-expressed in other conditions (e.g. without inducer).
Polypeptides that are inactive when expressed at normal levels or when over-expressed, but are active when under-expressed, are particularly suitable for use as parent polypeptides of the invention.
Such expression-resistant polypeptides can be improved, using the methods ofthe invention, to provide functional, active expression at suitably high yields and activity levels.
Random library Qerreration and screening One of the HRP clones that showed detectable peroxidase activity was used in the first generation of error-prone PCR mutagenesis. The random libraries were generated by a modification of the error-prone PCR protocol described above (20,21,22), in which 0.15 mM of MnCl2 was used instead of 0.5 mM MnCIZ. This protocol incorporates both SUBSTITUTE SHEET (RULE 26) manganese ions and unbalanced nucleotides, and has been shown to generate both transitions and transversions and therefore a broader spectrum of amino acid changes (50).
" _ Briefly, the PCR reaction solution contained 20 fmolcs template, 30 pmoles of each of two primers, 7 mM MgCli, 50 mM KCI, 10 mM Tris-HC1 (pH 8.3), 0.01 %
gelatin, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, I mM dTTP, 0.15 mM MnCli, and 5 unit of Taq polymerase in a 100 pi volume. PCR reactions were perfom~ed in a MJ PTC-200 cycler (MJ Research, MA) for 30 cycles with the following parameters: 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min. The primers used were:
S'-TTATTGCTCAGCGGTGGCAGCAGC [SEQ. ID NO. 15(, and 5'-AAGCGCTCATGAGCCCGAAGTGGC (SEQ. 1D. NO. 16[.
The PCR products were purified with a Promega Wizard PCR kit, and digested with Nde 1 and Hind III. The digestion products were subjected to gel-purifica:ion with a QIAEX
I1 gel extraction kit, and the HRP fragments were ligated back into the similarly digested and gel-puri feed pET-22b(+) vector. Ligation mixtures were transformed in the BL21 (DE3) I S cells by electroporation with a Gene Pulser II (Bio-Rad). Cell growth and expression was carried out in either 96-well or 384-well microplates in LB medium at 30° C. Peroxidase activity tests were performed with HiOi and ABTS (26).
For each generation, typically 12,000-15,000 colonies were picked and screened in 96-well plates. This number represents an exhaustive search of all accessible single mutants, with a probability of 95% for any mutant to be sampled at least once (25).
Colonies were either picked manually, or using an automated colony picker at Caltech, Q-bot (Genetix, LJK). Of the 12,000 colonies that were screened (no IPTG added), a mutant designated HRP1A6 showed 10-14 fold higher peroxidase activity than the parent clone.
FIG. SA and SB. This mutant clone also showed markedly decreased activity when as little as 5 pM of IPTG was added. FIG. 6. Sigma reports that 1 mg of highly purified HRP from horseradish has a total activity of 1,000 units, as determined by the ABTS
assay. Other workers reported similar results (13). Based on this data, the concentration of active HRP
was estimated to be 100 ug/L. HRP 1 A6 shows a total activity of greater than 100 unitslL.
This compares favorably with the yield obtained from refolding of aggregated HRP chains in vitro ( 13). This level of expression for the HRP mutant is also similar to that for bovine pancreatic trypsin inhibitor (BPTI) in E. coh (32), an unglyeosylated protein with three SUBSTITUTE SHEET (RULE 26) disulfide bonds. Greater than 95% of the HRP activity was found in the LB
culture medium as judged by the ABTS activity.
The mutant HRP remained stable for up to a week at 4°C. IPTG was omitted in all HRP expression experiments, unless otherwise specified. Peroxidase activity tests for HRP
were performed with a classical peroxidase assay, ABTS and hydrogen peroxide (2G).
Fifteen pl of cell suspension was mixed with 140 pl of ABTS/HiOz (2.9 mM ABTS, 0.5 mM 1-IiO, , pH 4.5) in microplates, and the activity was detcnnined with a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA) at 25°C. A unit of HRP is defined as the amount of enzyme that oxidizes 1 ymole of ABTS per min at the assay conditions.
Sequencing of the mutant gene found a mutation at position 255, in which the codon AAC for the amino acid asparagine (Asn or N) was changed to the codon GAC for the amino acid aspartic acid (Asp or D). This residue is a putative glycosylation site, and is located at the surface of the protein. The sequence of this mutant (HRP 1 AG) is shown in FIG. 3 (SEQ. ID NO. 3]. A map of a plasmid pETpeIBHRP 1 AG containing this mutant is shown in F1G. 4.
A representation of the structure of this HRP mutant, showing the Asn255Asp mutation ~s shown in FIG. 7.
Functional Expression of HRP in Yeast The native HRP protein contains four disulfide bonds, and E. coli has only a limited capability to support disulfide formation. In theory, these well-conserved disulfides in HRP
(and other plant peroxidases) are likely to be important for the structural integrity of the protein, and may not be replaceable by mutations elsewhere. Yeast has a much greater ability to support the formation of disulfide bonds. Thus, yeast can be used as suitable expression host, in place of E. coli, particularly if it is desired to relieve the apparent limitation on the folding of HRP imposed by any constraints on disulfide formation in E.
coli. For example, S. cerevisiae can be used as a host for the expression of mutant HRP
genes and proteins.
The HRP mutant (HRP 1 AG) was cloned into the secretion vector pYEX-S 1 obtained from Clontech (Palo Alto, CA} (35), yielding pYEXS 1-HRP (FIG. 8). This vector utilizes the constitutive phosphoglycerate kinase promoter and a secretion signal peptide from Kluveronryces lactic . The plasmid was first propagated in E. coli, and then transformed into SUBSTITUTE SHEET (RULE 26) S. cerevisiae srrai~r BJ54G5, obtained from the Feast Genetic Stock Center (YGSC), University of California, Derkeley using the LiAc method as described (3G).
BJ5465 is~ -protcasc deficient, and has been found to be generally suitable for secretion.
A first generation of error-prone PCR of HRP in yeast was perfom~ed. Anyone the $ first 7,400 mutants screened, four variants showed 400% higher activity than HRP l AG in yeast. Additional details and results arc given in Example 2.
EaA>\1P1.E 2 IU
Functional Expression of 1-1RP in 1'cast through Directed Evolution This example describes the use of directed evolution to further improve the functional expression of HRP. As explained m txampte i, a vanam m ~wm~a~m~
peroxidase (HRP lAG) was isolated. Since HRP contains four well-conserved disulfides, and E. coli has only limited ability to support disulfide bond forniation, the further 15 improvement in bacterial expression of HRP in E. coli maybe constrained by correct pairing of disulfide-containing cysteines. Yeast cells, for example S. cerevisiae, have much greater ability to support the formation of disulfide bonds, and may be better able to accommodate disulfide bonds in peroxidase enzymes. In theory, these well-conserved disulfides in HRP
(and other plant peroxidases) are likely to be important for the structural integrity of the 20 protein, and may not be replaceable by mutations elsewhere. Thus, yeast can be used as suitable expression host, in place ofE. coli, particularly if it is desired to relieve the apparent limitation on the folding of HRP imposed by any constraints on disulfide formation in E.
col i.
Accordingly, S. cerevisiae was chosen as an alternative host for the expression of 25 HRP. S. cerevisiae is both a micro-organism and a eukaryote, and possesses much of the eukaryotic protein post-translational and secretory machinery, such as ER and Golgi that catalyze the formation of disulfide bonds and glycosylate polypeptides.
Genetic manipulation techniques (in particular gene transformation) are also readily available. A
drawback is that yeast naturally secrete few proteins. Moreover, yeast glycosylation differs 30 significantly from that in higher eukaryotic organisms, which might present problems for secretion of glycoproteins (4). Nonetheless, several protein~have been efficiently secreted from yeast (4). Stategically, the experiments of this example take advantage of the capacity SUBSTITUTE SHEET (RULE 26) of yeast to catalyze the formation of disulfide bonds while fine-tuning the glycosylation factor through the process of directed evolution. " -Cons~ruc~ion o,~,i~ensl expression systen: for NRP.
The HRP mutant i~RPIAG from Example i was cloned into the yeast secretion vector pl'EX-S 1 obtained from Clontech (Palo Alto, CA} (35), yielding pYEXS 1-HRP
(FIC. 8). This vector utilizes the constitutive phosphoglycerate kinase promoter and a secretion signal peptide from K. lacri.s. pYEX-S 1 was digested with Scrcl, and then blunt-cnded with T4 DNA polymcrasc. TI1C Illallll'C HRP I AG gene was cloned from pETpeIBHRPI AG by PCR techniques using the proofreading polymcrase pfic (Stratagenc, CA) that generate blunt-end products. The forward and reverse primers used were 5'-CAGTTAACCCCTACATTC-3' (SEQ ID No. 25J and 5'-TCATTAAGAGTTGCTGTTGAC-3' (SEQ ID No. 26), respectively. The PCR fragments were then ligated into the restricted and blunt-ended pYEX-S 1, and transformed iMo E. coli DHSa cells. A number of colonies were picked and screened for the presence of the HRP
gene by colony PCR reactions 18 with these two primers: 5'-CGTAGTTTTTCAAGTTCTTAG-3 (SEQIDNo.27[ and 5'-TCCTTACCTTCCAATAATTC-3 [SEQ ID No. 28]. The correct orientation of the HRP
gene was further confirmed by sequencing. This yeast expression vector is generally referred to hereinafter as pYEXSl-HRP (FIG. 8). In this construct, the HRP
gene was placed directly downstream of the secretion signal peptide from K. IacriS, and the expression is under the control of the constitutive phosphoglycerate kinase promoter. The vector also carries the E. coli Amp resistance gene as well as the yeast selectable markers leu2-d and tJRA3 (47).
For expression experiments, the plasmid was first propagated in E. coli strain DHSa, and then transformed into S. cerevisiae strain BJ5465, obtained from the Yeast Genetic Stock Center (YGSC; University ofCalifomia, Berkeley), using a LiAc method that utilizes single strand DNA as described by Gietz et al. (48). BJ5465 is protease deficient and generally suitable for secretion (4). Following transformation, cells were plated on YNB
selective medium supplemented with 20 Pg/ml leucine, 20 ~tg/ml histidine, 20 llg/ml adenine and 20 ug/ml tryptophan. Colonies were picked, and grown in 96-well microplates in YEPD medium at 30°C in an air-circulating incubator for 2 days and 16 hours. HRP

SUBSTITUTE SHEET (RULE 26) activity tests were performed with a classical peroxidase assay, ABTS and hydrogen peroxide (26). The activity obtained from yeast for HRPlA6 was only about 1/10 of tha,( _ from G. coli, and actually slightly lower than obtained for the wild-type iv this construct.
Gerrerario» n» d Scr-ee»irrQ of HRP A~mants Libraries of HRP mutants were constructed by error-prone PCR (20) as described (53) except that the following two primers flanking the HRP gene were used in the mutagenic PCR reactions: 5'-CAGTTAACCCCTACATTC-3' [SEQ ID No. 25] and 5'-'fGA'fGCTGTCGCCGAAGAAG-3' [SfQ tD No. 29[. Also, the thcnnal cycling lU parameters were: 95 °C for 2 min, (~)4°C for 1 111111, 50 °C for 1 min, and 72 °C for 1 min, 30 cycles).
The PCR products were purified with a Promega Wizard PCR kit (Madison, WI), digested with Sac l and Bam HI (the first 27 amino acid residues of HRP were left unmodified). The digestion products were then subjected to gel-purification with a QIAEX
II gel extraction kit (QIAGEN, Valencia, CA), and the HRP fragments were ligated back into the similarly digested and gel-purified pYEXSI-HRP1A6. Ligation mixtures were transformed in E. coli HB 1 O1 cells by electroporation with a Gene Pluser II
(Bio-Rad), and selected on LB medium supplemented with 100 mg/ml ampiciilin. Colonies were directly harvested from LB plates. This plasmid DNA was subsequently used for transformation into yeast BJ5465 as described above.
Single colonies were picked from yeast nitrogen base (YNB) plates, and grown at °C for 64 h in 96-well microplates containing YEPD medium (I% yeast extract, I%
peptone, 2% glucose) in an incubator. Microplates were then centrifuged at 1,500 g for 10 min, and l Oml of the supernatant in each well was transferred to a new microplate with a 25 Beckman 96-channel pipetting station (Multimek, Beckman, Fulerton, CA), and assayed for total HRP activity. Overall standard deviations of this measurement {including pipetting errors, which was about 2%) did not exceed 10%. Improved mutants (showing the highest total HRP activity) were directly retrieved from the microplates, washed three times with sterile HZOz, and re-grown in YNB selective medium. Plasmids containing the HRP mutants 30 were first extracted from the yeast cells with a Zymo yeast plasmid miniprep kit (Zymo Research, Orange, CA), and then returned to E. coli X 10-Gold for further propagation and preparative isolation.

SUBSTITUTE SHEET (RULE 26) WOliO/06718 PCT/US99/17127 -Where indicated, pre-screening of HRP-expressing yeast clones were carried out as follows. Colonies on YNB plates were replicated onto MSI supported pure nitrocellulose" -membranes ( Micron Separations Inc., Wcstboro, MA), which were grown on fresh YEPD
agar at 30 °C for 34 hr. Membranes were then immersed in 100 ml of TMB
membrane substrate (O.S mM TMB, 2.9 mM HzOr, and 0.12% (W/V) dextran sulfate as cnhancer) for 5 min to allow colored product to develop. Those yeast clones that exhibited bright green color were traced back to the master YNB selective plates, and picked and grown in YEPD
for further screening as described above.
Firsr ~eu~rmion HXP r»«rnQC»esis in a~ensl jon it»nrovi«Q exnressio».
A first generation of error-prone PCR of HRP1A6 in yeast was aimed at improving the expression level. An error-prone PCR protocol incorporating both unbalanced nucleotide concentrations and manganese ions as described previously (20, 21 ) was used. This protocol was shown to generate roughly random mutations, allowing for sampling of a broader spectrum of amino acid residue changes. The manganese ion concentration used was 100 ~tM, which generated an error rate of approximately mutations per gene on average (22). The PCR products were purified with a Promega Wizard PCR kit, digested with Sac I and Bam H1 (thus the first 27 amino acid residues of HRP were left unmodified). The digestion products were then subjected to gel-purification with a QIAEX II gel extraction kit, and the HRP fragments were ligated back into the similarly digested and gel-purified PEXSI-HRP1A6. Ligation mixtures were transformed in HB 1 O 1 cells by electroporation with a Gene Pluser II (Bio-Rad). Colonies were scratched from the E. coli plates and resuspended in LB medium, from which plasmids were prepared.
Then the plasmids were transformed into yeast and yeast colonies were obtained and grown as described above.
A total of about 14,000 colonies were picked and screened for this generation, which represented an exhaustive search of all accessible single mutants, and a probability of 95%
for any mutant to be sampled at least once (25). Of these colonies, a number of mutants showed significantly higher activity than the parent (HRP1A6) in yeast. Two exemplary improved mutants arc designated HRP1-11764 ~SEQ. ID NO. 12 and SEQ. ID NO. 13]
and HRP1-7 7E2 (SEQ. ID NO. 5 and SEQ. ID NO. 6~. HRP1-11764 gave a 16-fold higher activity than the parent, or a total activity of about 220 units/L
(FIG. 9). HRP 1-77E2 SUBSTITUTE SHEET (RULE 26) showed a total activity of about 147 units/L. Both of these were higher than the highest w level obtained from E. coli. Sce also FIC 12 (HRP1-77E2) and FIG. 16 (HRPI-1 1764). ~ -Second Qeneraiion ojNRP rnrnaQenesis in yeast for ironrovirrQ ernression.
The second generation of error-prone PCR used HRP 1-1 1764 as the parent. For this generation, a higherconccntration ofmanganesc ion was used to increase the mutation rate.
This change was made based on the following considerations. Since screening can only handle a library of about 10, to 10' mutants at the present time, the rate of mutagencsis has been conservatively limited to creating predominately single mutants in the last (1S). In this example, the fraction of clones more active than the parent for a given generation remains relativeiy constant with the error-rate up to G mutations per gene.
The advantage of using higher error rates is that it would allow neutral mutations to exist along with beneficial mutations isolated through screening. These accrued neutral mutations may become useful in subsequent generations by either providing a bridge for generating new 1 S types of mutations, or by synergetic interactions with newly created mutations. The manganese ion concentration used in this generation was 3SO lM, which generated an error rate of approximately 4-5 mutations per gene on average (22).
Additionally, a prescreening of the colonies using nitrocellulose membranes was performed. This was possible because the higher error-rate significantly reduce the number of colonies that showed similar or higher activity than the parent. The procedures were as follows. Colonies were first replicated from the master plates onto nitrocellulose membranes and grown on YEPD plates at 30°C for one day and 6 hours. The membranes were then retrieved from the plates and immersed in a mixture of TMB
(tetramethylbenzidine) and Hz02. The colonies with the brightest color were identified, and 2S corresponding mother colonies were picked and grown from the master plates.
For this generation, about 120,000 colonies were screened (about 5,000 were actually picked and grown), and the mutant HRP2-28D6 was obtained. It showed an activity 85%
higher than its parent, HRPI-11764, or a total activity of 410 units/L (FIG. 9).
Third Qerrera~ion ojHRP mutaQenesis in .Least for imnrovirrQ expression.
The third round of random mutagenesis was carried out under similar conditions with HRP2-28D6 as the parent. For this generation, a total of 90,000 colonies were pre-SUBSTITUTE SHEET (RULE 26) screened, and 3,000 picked and grown. The best mutant, HRP3-17E12, gives an expression level of 1080 units/L, an increase of 1 GO% over the parent HRP2-28DG, or 85 fold over the, _ starting mutant, 1-IRPIAG.
I-'irsr ~c~uermion o~HHP mmnQmresis in vens~ (or inrnr'oainQ stnhilitt~
Onc ~,encration of random mutagencsis of HRP for improving thern~ostability and resistance towards 1~,0: ~~~as can-led out using: HRP1-77):2 as the parent.
The random muta~cncsis (with 100 pM manganese) and cell growth was essentially performed as described above (with no prescreening). Thennostability tests were performed with a MJ
I O fTC-200 cycler (MJ Research, MA) at 73 °C with an incubation time of 1 U min. I~zOZ
resistance tests were separately perforn~ed in 25 mM HzOi at room temperature and a pre-incubation tin~c of 30 min., followed by ABTS screening in 25 mM HiOZ. Mutants that were more thennostable or chemically stable (H,O; resistant) than the parent were further characterized at various temperatures (far thennostability) or H~02 concer;trations (for HzOi stability).
Out of 3,000 colonies screened, one thermostable mutant (HRP I -4BG) showed a T"~
of over G°C higher than that of the parent (T"z is the transition midpoint of the HRP
inactivation curve as a function of temperature) (FIG. 10). Another mutant, also showed some improvement in thermostability. The mutant HRPI-24D11 was not markedly more thennostable than its parent HRP1-77E2, but was more resistant to HiOZ
degradation. (A feedback mechanism common to HRP enzymes is that they are degraded by HIOZ, which is a reactant in the enzymatic reactions that HRP facilitates.) The HRP1-24D 11 mutant retained about 60% of activity after incubation with 25 mM HiOi for 30 min, while the parent exhibited a 42% residual activity under the same conditions (FIG. 11).
1%ctor Constrrrctiorr for HRP expression irr Pichia nastoris.
The improved HRP mutants were further cloned into the Piehia expression vector pPIZaB (Invitrogen Cotp., Carlsbad, CA) to facilitate production of the mutants for biochemical characterization (54). This vector contains the a-factor signal peptide including a spacer sequence of four residues Glu-Ala-GLu-Ala at the C-terminus of the secretion signal, and the methanol-inducible PAOX 1 promoter. pPIZaB was restricted with Pst I first, blunt-ended with T4 DNA polymerase, and then further digested with EcoR I. The sample SUBSTITUTE SHEET (RULE 26) was purified with a Promega DNA purification kit. The coding sequences for the HRP
- variants were obtained from the corresponding pYEXS 1-HRP plasmids by PCR
techniques ~ _ using the proofreading polvmcrasc Pfu. The following two primers were used in the PCR
reactions: 5'-TCAGTTAACCCCTACATTC-3' (forward) ESEQ ID No. 30] and 5' S CCACCACCAGTAGAGACATGG-3' (reverse) ]SEQ 1 D No. 31 ). The PCR products were restricted with Eco Rl, and iigated into digested and purified pPIZaB, yieldingpPlZaB-HRP
(Fig. lb) in which the HRP genes were placed immediately downstream of the a-factor signal. The ligation products were first transfonned into E. ~oli strain ~L10-Gold and selected on low salt LB IllCdll1111 ( 1 % tryptophan, 0.5% yeast extract, 0.5°/, NaCI, pl-1 adjusted to 7.5) supplemented with 25 mg/ml Zeocin (Cayla, Toulouse codex, France).
Colonies were screened for the presence of the HRP genes by colony PCR
reactions (55) with these two primers: 5'- GAGAAAAGAGAGGCTGAAG TC-3' (fonvard) ~SEQ ID
No.3Z) and 5'-TCCTTACCTTCCAATAATTC-3 (reverse) )SEQ 1D No. 33). The forward primer contained the last three nucleotides of the signal sequence and the first nucleotide of the HRP sequence (as underlined), which ensured that the positive colonies carried the fuli-length HRP genes in the correct orientation. Plasmids were isolated with a QIAgen miniprep kit from liquid cultures of positive transformants, and used for further transformation into Pichia for the expression of HRP.
Transformation of Pichia was performed with electroporation according to the manufacturer's instructions (Invitrogen). Before transformation, plasmids were linearized with Pme I, purified with a Promega DNA purification kit, and further treated with Princeton Centri-Sep columns equilibrated in d.d. HBO to remove any residue impurities.
The linearized vectors were integrated into the Pichia genome upon transformation via homologous recombination between the transforming DNA and the Pichia genome.
The transformed cells were plated on YPDS medium (1% yeast extract, 2% peptone, 2%
glucose, 1 M sorbitol) supplemented with 100 mg/m1 Zeocin. For each construct containing a distinct HRP mutant, typically 4-6 transformants were picked, and purified on new YPDS
plates (supplemented with 100 mg/ml Zeocin) to isolate single colonies, which were then screened to identify the clones that conferred the highest expression levels.
The Pichia strain X-33 was used in all expression experiments. It was determined in initial tests that X-33 (Mut+) afforded significantly better HR.P expression than KMI7 (MutS).

SUBSTITUTE SHEET (RULE 26) NRP ernressio» i» Pichia nasroris.
Pichia cell growth was carried out at 30oC in a shaker. pPIZaB-HRP-harboring cells ~ -were first grown overnight in BMGI' ( 1% yeast extract, 2% peptone, 100 111M
polaSSlunl phosphate, pH G.O, 1.34% YNB, 4X 10-5% biotin, 1 % glycerol) supplemented with casamino acids to an OD G00 of 1.2-1.G. The cells were then pelleted and resuspended to an ODD of 1.0 in BMMY medium (identical to BMG>' except 0.5% methanol in lieu of 1 glycerol) supplemented with 1 % casamino acids. Growth was continued for another 54-72 h. Sterile methanol was added every 24 h to maintain induction conditions. HRP
Icveis in the supernatants peaked around 54-GO h post-induction (at whlCh llllle the OD~,«, reached about 8.0-10.0). Where applicable, at the point of induction, 1.0 mM vitamin B
I, 1.0 mM
d-ALA, and 0.5 ml/L trace element mix (0.5 g/L MgCli, 30 g/L FeCIz.GHZO, 1 gIL
ZnCIZ.4H:0, 0.2 g/L CoCI,.GHZO, 1 g/L Na,Mo0,.2H20, O.Sg/L CaCl,.2H,0, 1 g/L
CuClz, and 0.2 g/L HZB03) were added to the growth medium.
I S Peroxidase Activity Assay.
Peroxidase activity tests for HRP were performed with a classical peroxidase assay, ABTS and hydrogen peroxide (2G). 10 ul (or 15 pl) of cell suspension were mixed with 140 ~tl (or 150 ~I1) of ABTS/H,Oz (the concentrations of ABTS and H=O, are 0.5 mM
and 2.9 mM respectively, pH 4.5) in a microplate, and the increase of absorbance at 405nm (e of oxidized ARTS is 34.700 cm''M-~) was determined with a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA) at 25°C. A unit of HRP is defined as the amount of enzyme that oxidizes 1 ~t mole of ABTS per min under the assay conditions.
Guaiaco! assay.
The assay is perfonrted with 1 mM HZOZ and SmM Guajacol in SOmM
phosphate buffer pH 7.0 and an increase of absorbance at 470nm is followed (t:
of oxidized product at 470nm is 26.000 cm~'M~') after adding the yeast supernatant.
The stability of mutants was assessed using assays for initial activity (A;) and residual activity (A,~s;~, perfotzrted as described above with ABTS as substrate. A,K;d is measured after incubation of HRP mutants in NaOAc buffer pH 4.5 containing no H~Oz or 1 mM HiO~ and incubating at 50°C for 10 min.

SUBSTITUTE SHEET (RULE 26) The assay for stability in organic solvent/buffer (NaOAc buffer SOmM pH4.5) mixture was done with 1 mM H202 and 2mM ABTS using supernatant of HRP mutants -expressed in yeast ( l0ul) in dioxanc/buffcr (20/SO).
l'ro~locrion o~llXP 111111111115 lI1 Piclrin.
To obtain sufficient quantities of purified enzymes, l'iclliel was used in an further effort to increase production of 1-iRP mutants. 1-iRP-C (wild-type) , HRP 2-13A 10 (FIG. 19, [Sh.Q ID No. 21 and SEQ ID No. 22[) and HRP 3-17E12 (hlG. 20, ~SEQ ID No. 23 end SEQ ID No. 24[) were cloned into the l'icllicr secretion vector pPICZaB.
In this construct (pPICZaB-HRP, FIG. 1 b), HRP was fused to the a-factor signal peptide, and the expression was induced with methanol. A typical expression curve is shown in F1G. 25.
For HRP3-17E12, after SS h of cultivation, about 6,500 units/L of HRP activity was detected in the supernatant (F1G. 25, open squares), or G.5 fold of that obtained from yeast.
The work from others as well as from our laboratory found that the addition of trace metal elements, heme synthesis intenrediate aminolevulinic acid, and vitamin supplements to growth medium (such as thiamine) resulted in substantial improvement in the yields of holoenzymes of heme-containing prtoeins in E. coli (59-62). Addition of these additives to the Pichia growth medium in our experiements led to a 32% increase in HRP3-activity detected in the supernatant (FIG.25, solid squares).
Se4~g Data Sequencing revealed that HRPI-77E2, the parent used for thennostability and HzO~
stability studies carries a reverent D255 to N255 (GAC to AAC), and a second mutation L37I (TTA to ATA). This residue is part of the helix 2, and is near the heme pocket (34).
See, FIG. 12, (SEQ. ID. NO. 5) and [SEQ. ID. NO. 6[.
The mutant HRPI-4B6 carries K232M (AAG to ATG) in addition to L37I. This residue is part of the helix 14, and is exposed to solvent on the surface.
See, FIG.13, (SEQ.
ID. NO. 7[ and (SEQ. ID. NO. 8].
HRP I -2SB 11, the mutant with thennostability between HRP 1-77E2 and HRP 1-has the mutation F221 L (TTT to TTA) in addition to L37I. This residue is in a structural SUBSTITUTE SHEET (RULE 2b) loop and pare of the substrate access channel (34). Sec, FIG. 14, [SEQ. ID.
NO. 9] and [SEQ. ID. NO. 10]. -1'he mutant HRP1-24D1 1 contains the mutation L131P (CTA to CCA) in addition to L371. This residue is at the tip of the helix 7, and is on the surface.
See, F1G. 15, [SEQ.
1D. NO. 11] and [SEQ. 1D. NO. 12[.
The mutant HRPI-11764, a preferred mutant from the first generation in tcm~s of total activity, contains five mutations with respect to its parent: ( i ) a reversion of D255 to N255 (GAC to AAC) (the wild-type sequence); (2) L131 P (CTA to CCA); (3) L223Q
(C'fG
to CAG); ~~~itli silent mutations (4) at N135 (AAC to AAT) and (5) T257 (ACT
to ACA).
For the mutation L223Q, this amino acid residue is in a loop and is exposed to solvent. See, FIG. 1G, [SEQ. ID. NO. 13[ and [SEQ. 1D. NO. 14).
Strikingly, the improved HRP mutants, HRP1-80C i 2 (FIG. 17, [SEQ. ID. NO. 17[
and (SEQ. ID. NO. 18)). and HRP1-77E2 (FIG. 20, [SEQ. 1D. NO. 23[ and [SEQ.
ID.
NO. 24]) also carry the revenant D255 to N255 (GAC to AAC). In addition, HRP 1-contains L131P (CTA->CCA), found in HRP1-7764. On the other hand, HRP-77E2 has a second mutation L37I (TTA --> ATA) which is part of the helix B, and is in the heme pocket, presumably accessible to solvent as well.
HRP2-28D6 (FIG. 18, [SEQ. ID. NO. 19] and [SEQ. ID. NO. 20]) contains two additional mutations with respect to HRP1-11764: T102A (ACT --> GCT) and P226Q
(CCA --> CAA). T102A is pan of the helix D, and is the only mutation found to be buried inside the structure. P226Q is located in the same loop as L223Q. HRP2-13A10, on the other hand, contains four more mutations with respect to HRP1-I 1764: R93L
(CGA -->
CTA); T102A (ACT --> GCT); K241T (AAA --> ACA); and V303E (GTG --> GAG).
R93L, which is solvent accessible, is in the structure loop connecting helices C and D.
K241T is in the structural loop connecting helices G and H. This residue is again exposed to the solvent. Finally, V303E is pan of the long strand extending from helix J at the C-terminus of the protein. These three mutations seem to contribute to the increased stability of this mutant compared to the others.
HRP3-I7E12 contains three more mutations with respect to the parent HRP2-28D6:
N47S (AAT --> AGT); K241 T (AAA --> ACA), and one silent mutation at G 121 (GGT -->
GGC). It is noteworthy that K241T was also found in HRP2-13A10. N47S is located in a structural loop which connects helix B and a 3-helix, and is also solvent accessible.

SUBSTITUTE SHEET (RULE 2b) Analysis o~Mutations.
All three improved mutant forms of the first generation of evolution carry they _ revenant D255N with respect to the parent t~RPIAb. 'Plus appears to suggest that the giycosylation sites on HRP are beneficial for folding and expression. The function of glycosylation in proteins has been an intriguing matter, but its role in protein folding, processing and secretion is being gradually recognized (SG-58).
The three synonymous mutations can not be easily explained by changes in codon usage (G3). Two of them, N 135 (AAC --> AAT) and ~f257 (ACT --> ACA), resulted in few changes in the frequencies of used, while for G 121 (GG'r --> GGC) , a more frequently used 1 Q codon (GCT, G 1 %) was replaced by a Icss frequent ot~e (GGC, 16°io). However, it is unclear how this substitution would significantly affect the translation of HRP mRNA.
Char ucteri°atiorr o~rrrrrtants reear-dine reaclivify and stabilil.
Besides the ABTS assay also as a second independent activity assay for HRP
mutants the guajacol system is used (FIG. 21 ). Both assays show a good correlation regarding activity of the mutants.
Figures 22a and 22b show the correlation between reactivity and stability after incubation at 50°C without H~O~ (a) and with ImM H20~ (b). In both cases mutant shows the highest stability in combination with a good reactivity. As revealed by sequencing three amino acid changes seem to be responsible for this stability.
A similiar pattern of stability is observed in organic solvent system (Figure 23) where mutant HRP 2-13A10 shows the best ratios of activity in dioxane/buffer system versus those in buffer only.

Expression and Secretion of CCP in E. coli Corrsrrrrction of expression vector for CCP.
The S. cerevisiae cytochrome c peroxidase (CCP) gene from pT7CCP (16,17), donated by Dr. Dave Goodin, The Scripps Research Institute, La Jolla, CA) was recloned by PCR techniques to introduce an Msc I site at the start codon and a Hind III
site immediately downstream from the stop codon. The PCR product was restricted with Msc SUBSTITUTE SHEET (RULE 26) i and Hind III, and then iigatcd into similarly digested pET-22b(+), yielding pETCCP (Fig.
26). The pT7CCP carries a gene for CCP in which the N-terminal sequence has bee ~ -modified to code for amino acids Met-Lys-Thr, as described in Goodin et al.
(17) and Fitzgerald et al. ( 1 G). Thus, in this construct, the CCP gene was placed under the control of the T7 promoter, and was fused in-frame to the pelB signal sequence for periplasmic Vocalization.
Frnrcs.sio~r ojCC'l'.
Eaprcssion experiments of CCP in E roll BL21 (DE3) were carried out in LB
I U medium containing 100 pg/ml ampicillin. Cells were grown at 37°C to an A~ of 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. Growth was continued at 30°C for an additional 20 hours, and cells and supenlatant were harvested by centrifugation.
CCP is known to fold correctly inside E. coli. Surprisingly, greater than 95%
of the 1 S CCP protein was found in the LB culture medium at high levels (approximately 100 mg/liter, as assessed by SDS-PAGE). The protein was active towards ABTS, showing that the secreted CCP is folded and contains the required ferric heme.
Having thus described exemplary embodiments of the invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various 20 other alternatives, adaptations, and modifications may be made within the scope of the invention. For example, it will be understood by practitioners that the steps of any method of the invention can generally be performed in any order, including simultaneously or contemporaneously, unless a particular order is expressly required, or is necessarily inherent or implicit in order to practice the invention. Accordingly, the invention is not limited to 25 any specific embodiments or illustrations herein. The invention is defined according to the appended claims, and is limited only according to the claims.
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SUBSTITUTE SHEET (RULE 26)

Claims (35)

What is claimed is:

1. A method of obtaining and improving the production of an expression-resistant polypeptide comprising the steps of:
providing at least one parent polynucleotide encoding a parent polypeptide that is resistant to functional expression in selected host cells, altering the nucleotide sequence of the parent polynucleotide to product a population of mutant polypeptides;
transforming the host cells to express the mutant polypeptides; and screening for functional mutants produced by the host cells and having at least one of improved folding or expression without inclusion bodies.

2. A method of claim 1 wherein the parent polypeptide forms inclusion bodies when over-expressed in the host cells.

3. A method of claim 1 wherein the parent polypeptide has at least one of a disulfide bridge structure and a glycosylated structure.

4. A method of claim 1 wherein the parent polypeptide has or is associated with at least one heme group.

5. A method of claim 1 wherein the parent polypeptide is produced in a non-functional form when over-expressed in the host cells and is produced in a functional form when under-expressed in the host cells.

6. A method of claim 5, wherein the parent polypeptide is over-expressed under the control of an inducible promoter in the presence of an inducer, and is under-expressed under the control of an inducible promoter in the absence of an inducer.

7.~The method of claim 1 comprising repeating the method for a number of cycles wherein the parent polynucleotide in each cycle is a mutant polynucleotide from a previous cycle.

8. The method of claim 1, wherein the step of altering the nucleotide sequence is performed by at least one of random mutagenesis, site-specific mutagenesis and DNA
shuffling.

9. The method of claim 7 wherein the step of altering the nucleotide sequence is performed by at least one of random mutagenesis, site-specific mutagenesis and DNA
shuffling.

10. A polynucleotide evolved according to the method of claim 1.

11. A polynucleotide evolved according to the method of claim 7.

12. A polynucleotide evolved according to the method of claim 9.

13. A method of claim 1, wherein the host cells are transformed by a vector having a signal sequence that directs secretion of polypeptides encoded by mutant polynucleotide.

14. The method of claim 13, wherein the signal sequence is the PeIB signal sequence.

15. A method of claim 1 wherein the host cells are facile host cells.

16. A method of claim 1 wherein the host cells are selected from yeast and bacteria.

17. A method of claim 7 wherein the host cells are selected from yeast and bacteria.

18. A method of claim 9 wherein the host cells are selected from yeast and bacteria.

19. The method of claim 1 wherein the host cells are E. coli cells.

20. The method of claim 1 wherein the host cells are S. cerevisiae cells.

21. The method of claim 1 wherein the host cells are P. pastoris cells.

22. The method of claim 9 wherein the host cells are E. coli cells.

23. The method of claim 9 wherein the host cells are S. cerevisiae cells.

24. The method of claim 1 wherein the host cells are P. pastoris cells.

25. The method of claim 7 wherein the polypeptide is a heme-containing protein.

26. The method of claim 9 wherein the polypeptide is a heme-containing protein.

27. The method of claim 18 wherein the polypeptide is a heme-containing protein.

28. The method of claim 7 wherein the polypeptide is a peroxidase enzyme.

29. The method of claim 9 wherein the polypeptide is a peroxidase enzyme.

30. The method of claim 18 wherein the polypeptide is a peroxidase enzyme.

31. The method of claim 26 wherein the polypeptide is a horseradish peroxidase enzyme.

32. The method of claim 27 wherein the polypeptide is a horseradish peroxidase enzyme.

33. The method of claim 28 wherein the polypeptide is a horseradish peroxidase enzyme.

34. A polynucleotide encoding for a horseradish peroxidase which has one or more, mutations at an amino acid position selected from 255, 371, 131, and 223, wherein the starting methionine residue is at position 0.

35. A polynucleotide encoding for a horseradish peroxidase which has at least one mutation selected from N255D, L3711 and L131P.