MXPA97002038A - Subtilisine mutants that lack from a primary site of union to cal - Google Patents
Subtilisine mutants that lack from a primary site of union to calInfo
- Publication number
- MXPA97002038A MXPA97002038A MXPA/A/1997/002038A MX9702038A MXPA97002038A MX PA97002038 A MXPA97002038 A MX PA97002038A MX 9702038 A MX9702038 A MX 9702038A MX PA97002038 A MXPA97002038 A MX PA97002038A
- Authority
- MX
- Mexico
- Prior art keywords
- subtilisin
- mutant
- amino acids
- calcium
- mutations
- Prior art date
Links
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- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Abstract
The present invention teach novel calcium-free subtilisin mutants, in particular subtilisins which have been subjected to mutations to eliminate amino acids 75-83 and which retain their activity and enzymatic stability. Recombinant methods for producing the same and the recombinant DNA encoding such subtilis mutants are also provided.
Description
CALTIUM FREE SUBTILISIN MUTANTS
GENERAL DATA OF THE INVENTION
A general object of the invention is to provide subtilisin nutants that have been subjected to mutations so that they do not bind calcium. Another object of the invention is to provide the DNA sequences that after their expression provide subtilisin utants which do not bind calcium. Another object of the invention is to provide subtilisin mutants, which comprise specific combinations of mutations that provide improved thermal stability. Another object of the invention is to provide a method for the synthesis of a subtilisin mutant, which is not bound to calcium by the expression of a = ubtilisin DNA, which comprises one or more substitution, deletion or addition mutations in a suitable recombinant host cell. A more specific object of the invention is to provide subtilisin mutants of class I, in particular BPN mutants, which have been subjected to mutation so that they do not bind to calcium.
REF: 24345 Specific object of the invention is to provide DNA sequences, which after their expression aan as hiding subtilisin mutants of class I, y = '? particlir BPN mutants, which do not bind calcium. Ocre specific aspect of the invention is to provide a method for producing mutants I-SI or IS-2, and in particular the BPN 'mutants, which do not bind to calcium, by expression of a subtilisin mutant DNA sequence of class I, and more specifically a DNA coding sequence of BPN ', which comprises one or more mutations of substitution, addition or deletion in a suitable recombinant host cell. The most specific object of the invention is to provide mutant subtilisin I-SI or I-S2, and more specifically BPN ', which do not bind to calcium and also comprise particular combinations of mutations which provide thermal stability improved, or that cooperatively restore the folding reaction. The subtilisin mutants of the present invention: > n to be used in applications where subtiii inas find common use. Since these mutants do not bind calcium they may be particularly well suited for use in industrial environments comprising chelating agents, for example detergent compositions, which substantially results in the activity of the natural subtilisins c "3 to bind to calcium.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to the proteins of subtilisin, which have been modified to eliminate the binding of calcium. More particularly, the present invention relates to the mutants of novel subtilisin I-SI and I-S2, specifically the B? V slayers where the calcium binding cycle A has been deleted, specifically where the amino acids 75-83 have been deleted, and may additionally comprise one or more other routes, for example, modifications of subtilisin, which are provided to improve thermal stability and / or mutations that cooperatively restore the folding reaction.
(2) Description of Related Art
Subtilisin is an unusual example of a m.ci.omeric protein with a substantial kinetic barrier for folding and unfolding. A well-known example of it, the subtiline BPN1 is a senna protease of 275 amino acids -recipitated by p.r? Aci ll us amyl olí quefaci ens. This enzyme is from; Consider 1 industrial importance and has been the subject of numerous protein design cues (Siezen et al., Rotem E ^ T.eering 4: 719-737 (1991): Bryan, Pharmaceutical ¿echnoio / 3 ( B): 147-181 (1992); Wells et al., Trends
- - ^^, em, S i. 13: 291-297 (1988)). The amino acid sequence for s '-tilysin BPN' is known in the art and can be found: in Vasantha et al., J. Bacteriol. 159: 811-819 f 198). The amino acid sequence as found there and therefore is incorporated herein by reference [SEQUENCE ID M0: 1]. After the application, when the applicants refer to the amino acid sequence of the subtilisin BPN 'or the mutants / 1: is, they will refer to the sequence of amino acids at 1: s, a there. or . Subtilisin is a serine protease produced by onecerías ^ ram positive or by fungi. The amino acid sequence of numerous subtilisins are known. (Siezen et al., Prctein Engineering 4: 719-737 (1991)). Those include five subtilisins of Bacillus strains, for example, subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, and mesenticopeptidase. (Vasantha et al., "The alkaline protease gene and the neutral protease of Bacill us amyl oliquefaciens contains a large open reading frame between the coding regions for the sequence: signal and mature protein", J. Bacteriol, lc9: 511-olJ (1984); Jacobs et al., "Cloning, expression and expression of Carlsberg subtilisin from? A ll us -:: '. ErZfor is", Nucleic Acids Res. 13: 8913-8926 1985); K '": ov et al.," Determination of the complete amino acid sequence of subtilisin DY and its comparison c: n the primary structures of the subtilisin BPN', Carlsberg and amylosacchariticus ", Biol. Chem. Hoppe-Seyler 266: 421-430 (1985), Kurihara et al., "Subtilisin a ylosaccha-iticus" J. Biol. Chem. 247: 5619-5631 (1972), and Svendsen al., "Complete amino acid sequence of alkaline mesentericop-ptidase. ", FEBS Lett 196: 228-232 (198 o)). S know the amino acid sequences of the subtilisin of two fungal proteases: proteinase K from Tritirac? Ujam (Jany et al.," Proteinasa K from TptirachiUíi "). albam Limber ", Biol. Chem. Hoppe-Seyler 366: 485-492 (1985)) and thermophilic thermophilic fungi, Malbranchea pulchella (Gaucher et al.," Endopeptidases: Termomicolina ", Metods Enzymol. 45: 415-433 (1976 )). These enzymes have been shown to be related to subtilisin BPN ', not only through their primary sequences and enzymatic properties, but also by comparison of the X-ray crystallographic data. (McPhalen et al., "Molecular crystalline structure of the -hioidor jlin of the leeches in complex with Carlsberg lism" FEBS Lett., 188: 55-58 (1985) and Pahler c: al. "Three-dimensional structure of the proteinase K of gonads:, n '.", Similarity with bacterial subtilisin ", EMBO Z _, 3: 1311- ^ 314 (1984)). Subtilisin BPN 'is an example of a particular ubtilisin gene secreted by Bacill us arp.yloliquef > riens This gene has been cloned, sequenced and expressed c. high levels from their natural promoter sequences in Bacill us subtilis. The structure of the subtilis ^ na BPN 'has been highly refined (R «0.14) at a resolution of 1.3 Á and has revealed structural details for two ion binding sites (Finzel et al., J. Cell. Eiochem. 10A: 272 (1986), Pantoliano et al., Niochemistr 27: 8311-8317 (1988), MacPhalen et al., Biochemistry 27: 6582-6598 (1988)). One of those (site A) binds to Ca "" with high affinity and is located near the N-ter inal, while the other (site B) binds to calcium and other cations much weaker and is located approximately 32 Á further (Figure 1). Structural evidence of two calcium binding sites was also reported by Bode et al., Eur. J. Biochem. 166-673-692 (1987) for the homologous enzyme, Carlsberg subtilisin. In addition, in this regard, the primary calcium binding site in all subtilisins in the groups I-SI-I-S2 [SZ ^ -I et al., 1991, Table 7) are formed from z ^ z ^ zs laugh ... it will almost identical waste in a position -dér. ica d- the helix C. The X-ray structures of the 3PN 'and Carlberg I-SI ptilism, as well as the subtilisin Cavmasa i-2, have been determined at high resolution. A comparison of these structures shows that all three have almost identical calcium A sites. L- X-ray structure of subtyla of class I, t '. causa of Thermoactinomyces vulgaris, is also known. Although the total homology of the BPN 'with the termitase is much less than the homology of the BPN' with the subtilisins I-SI and I-S2, termitase has been shown to have an analogous calcium A site. In the case of termitase, the cycle is interruption of residue ten at the identical site in helix C. The calcium binding sites are common characteristics: - the extracellular microbial proteases probably due to their large contribution to the thermodynamic thermodynamic stability and (Matthews et al., J. Biol. Chem. 249: 8030-8044 (1974); Voordouw et al., Biochemistry 15: 3716-3724 (1976); Betzel et al., Protf Engineering 3: 161 -172 (1990), Gros et al., J. Biol. 266: 2953-2951 (1991)). It is believed that the thermodynamic and kinetic staging of subtilisin is ne 'due to the rigor of the extracellular environment in the secrete, by virtue of which its sole -ready filled with the protease. In consecuense, the activation keys for their unfolding can be e = ensi! . - to stop the native conformation and to prevent transient doubling and proteolysis. Fortunately, the principal industrial uses of the subtilisins are in an environment containing high levels of chelating metals, which separate calcium from subtilisin and compromise its stability. Therefore, it could be of practical significance to create a highly stable subtilisin that is independent. J of calcium. Let us inventors have previously used several strategies to increase the stability of subtilisin to thermal denaturation by adopting simple thermodynamic models to approximate the transition without splitting (Pantoliano et al., Biochemistry 20: 2077-2031 (1987)).; Pantoliano et al., Biochemistry 27: 8311-3317 (1988); Pantoliano et al., Biochemistry 28: 7105-7213 (1989); Rollance et al., CRC Crit. Rev. Biotechnol. 8: 217-224 (1983). However, improved subtilisin mutants that are stable in industrial environments, for example comprising chelating metals, and that do not bind to calcium, are not currently available.
OBC? TOS AND BRIEF DESCRIPTION OF THE INVENTION
As a consequence, an object of the invention is mutant or modified subtilisin enzymes, for example, class I tylases, which have been modified to eliminate the binding of calcium.As described in this invention, the term " Modified mutant subtilisin "means that it includes any enzyme serine protease cm that has been modified to eliminate the binding of calcium 0. This includes, in particular, subtilisin BPN 'and serine proteases that are homologous to subtilisin BPN', in particular subtilases of class I. However, as used herein, and under the definition of mutant or modified subtilisin enzyme, the mutations of this invention may
, 5 be introduced into any serine protease having at least 502, and preferably 80% identity of the amino acid sequence with the sequences referred to above with the subtilisin BPN '. Subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, 0 mesenticopeptidase, termitase, or Savinase, can therefore be considered as homologous. The mutant subtilisin enzymes of this invention are more stable in the presence of metal chelators and may also comprise improved thermal stability in
: 5 comparison as the native or natural subtilisin. The thermal stabilization is a good indicator of the total strength of anaprotein. The high thermal stability proteins are stable in the presence of chaotropic agents, detergents, and under other conditions, which
: r: ritalítent they are used to inactivate proteins. Therefore it is expected; e the thermally stable proteins are useful. There are many industrial and therapeutic applications in which resistance to high temperatures, strong solvent conditions or prolonged shelf life is required. _ has further discovered that the combination of individual c-stabilizing mutations in subtilisin frequently results in an approximately additive increase in free energy stabilization. Thermodynamic stability has also been shown to be related to resistance to irreversible inactivation at high temperature and high pH. Changes in a single site of this invention individually do not exceed a contribution of 1.5 Kcal / mol to free energy
) of folding. However, that very small increase is added to the free energy of stabilization resulting in a dramatic increase in total stability when the mutations are combined, since the total free energy of folding for most proteins is
in the int.rvalo of 5-15 Kcals / mol (Creihton, Proteins: bioreactors and shelf life.
As noted above, the subtilisin mutants, which many deletion mutations replace, are provided for elimination. Preferably, this substitution or insertion of amino acids is carried out in the A-site of the ..aleic, which in the case of the subtilases of the class I e: m? Rende rut and residual amino acids in the helix C. In the taso of i. subtilisin BPN ', the subtilisin mutants will preferably comprise one or more addition mutations, deletion or substitution of the amino acids at positions 75-33, and more preferably will comprise the deletion of amino acids 75-83, of SEQUENCE ID N0 :1. It has been found that the deletion of amino acids 75-83 effectively eliminates calcium binding of the resulting subtilisin mutant by providing the following: even 3PN 'subtilisin proteins having enzymatic activity. Subtilisin mutants lacking amino acids 75-83 of SEQUENCE ID NO: 1 may also include one or more additional amino acid mutations in the sequence, eg, mutations that provide reduced proteolysis. Another object of the invention is to produce subtilisin mutants that lack calcium binding activity that have been subjected to additional mutations to cooperatively restore the folding reaction and thereby increase proteolytic stability. Another object of the invention is to provide thermostable subtilisin mutants that do not bind in addition to calcium and comprise specific combinations of mutations that provide substantially improved thermal stability.
d, in particular, the subtilisin mutants of the saty mm-ention will include the subtilisins of the strains of
:3-?' . _i -l ..; Such as subtilisin BPN ', subtilisin larísber, -... -dtilisina DY, subtilisin amylosacchariticus and what nbtiiisina mesenticopeptidasa, which comprise one or more mutation "is deletion, substitution or addition or present invention further provides mutants subtil- sita lacking the 75-83 amino acid sequence or NO:.. 1, which have protein-interactions prcteina ncr edosas designed in regions around suppression leading to substantial improvements in stability to be more specific, they are provided mutations in ten specific sites in the subtilisin BPN 'and its homologs, seven of which individually and in combination, have been found to increase the stability of subtilisin prottlna. subtilisins cry calcium improved are thus provided by the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 . Crystal structure of subtilisin SI 5 determined with X-rays A. Carbon graph showing the positions of the mutations as noted. The nmt? Racitr .. e natural subtilisin was conserved. The spheres nnteadas. -show the position of calcium at the site of u. ion of m: month, weak (site B) and the first position of the high affinity ... n site (site A). The cycle of site 1 (dotted line) is absent in this mutant. The N- and Z-terminal were indicated. The N-terminal is messy (dotted line). 3 Amplified view of site A deletion. Cycle d: subtilisin S12 is shown as a dotted line 0 in the continuous helix of S15. Superimposed is the difference of the electronic density sigma 3 * (F012-F015, phases of S15) showing the cycle of site A suppressed. - -.gura 2. The crystalline structure of the region of the
.5 site A d ^ 1 calcium of subtilisin S12 determined with X-rays. Calcium is shown as a dotted sphere with half the radius of van der Waals. Stripe lines are coordinating links, while dotted lines represent hydrogen bonds under 3.2 Á. 2 J Figure 3. Differential Scan Calorimetry.
Ce shows the calorimetric scans of apo-S12 (T = 63.5'JC) and S15 (Tm = 63.0 ° C). The measurements were made with a Hart 7707 DSC thermal conduction scan microcalorimeter (scanning calorimetry).
-.3 differential) as described (Pantoliano et al., 1 C Cn otl I "'(3: 7205- ^ 213 (1939)).
: estra was 50 mM glycine, at pH 9.63, a scanning speed of 0.5 ° C / min. The excess thermal capacity was measured in μ3 / °. Calorimetric ampules tcntenian 1" '3 mg protein: .gura 4. Titration calorimetry eubtilism sil Heat calcium binding for successive additions -.?.' Graphic calcium vs the ratio of [Ca] /.. [P] The data is better measured by a calculated junction curve assuming a constant union of 7 x 10 ° and H equal to 11.3 l: eal / mol using equation (I) of the text. show the curves calculated assuming K, = 1 x 10 and 1 x 109. In this titration, [P] = lOOμM and the temperature was 25 ° C Fig. 5. Kinetics of calcium dissociation of subtilisin Sil as function temperature. lum Sil was added to lOμM of Quin2 at time = 0. calcium dissociates from subtilisin and binds to Quin2 until is a new equilibrium is reached. the calcium dissociation rate follows the increase in flowering of Quin2 when it binds to calcium A. The log of percent protein bound to ca lcio se -traficó vs. time. The kinetics of dissociation at four temperatures are shown. Dissociation follows a first-order emetic for the first 25% of the reaction. 1: this is how it fits before the equilibrium is reached, the calcium reactance can be neglected. 3 .. Dependence on the temperature of the speed
I dissociates you from the calcium of subtilisin S15 in the presence of urn2 in excess, pH 7.4 and over a temperature range of 25-45 ° C. The natural log of the equilibrium constant .ata the transition state (calculated from 1- Eyring's equation (see chart vs. the reciprocal of the
0 absolute temperature. The line is adjusted according to the equation (3 'in the text with Tj = 298 K. Figure 6. Analysis of the refill of the subtilisin verified with circular dichroism (CD) A. The CD spectra for S15 are shown as 5 s -.gue: (1) S15 in 25 mM H3P04 at pH 1.85, (2) S15 denatured to pH 1.85 and then neutralized to pH 7.5 by the addition of NaOH, (3) S15 denatured to pH 1.85 and neutralized to pH 7.5, 30 minutes after the addition of 0.6 M KCl, and (4) native S15 subtilisin.The 0 protein concentrations of all the samples were
1 μM. B. Folding kinetics of S15. The samples were denatured at pH 1.85 and then the pH was adjusted to 7.5. At time 0, KCl was added to the denatured protein m. The recovery of the native structure was sealed at 222 n at a KCl concentration of 0.3 M and 0.6 M. The m.scra 0. 0. • M 30 minutes after the refolding was then used to record the corresponding spectrum in the art. F i Jura 7. Kinetics of the refolding of S15 as a function of ionic force. A. The log of percent of refolded protein is recorded. weather. The kinetics of the refolding are shown by four ionic forces. The amount of refolding was determined by circular dichroism (DC) from the increase in negative electity at 222 nm. A 100% folding of the signal at 222 nm was determined for the native S15 at the same concentration and a 10% folding of the signal for acid denatured SI 3 was determined. The refolding follows approximately a first order kinetics for the first 90? of the reaction. The refolding was carried out at 25 ° C. B. The log of the first-order velocity constant for the refolding obtained by CD or fluorescence measurements at 25 ° C was plotted as a function of the log of the ionic strength. The ionic strength was varied from 1 = 0.25 to 1 = 1.5. The refolding speed increases linearly with log I. A ten-fold increase in I results in an approximately 90-fold increase in refolding speed.
Figure 8. Dependence of the refolding viscosity temperature of subtilisin S15 in KCL 0.6M, K? 0: 23 nl-I pH 7.3. The natural log of the equilibrium constant for the transition state (calculated from the Eyring equation) vs. the reciprocal of the absolute temperature. The line is adjusted according to equation 3 in the text with T., = 298K. Figure 9. The crystal structure determined with X-ray of the weak ionic binding region of subtilisin S15. Coordination links are shown as dashed lines. Note the preponderance of charged amino acids.
DESCRIPTION OF THE PREFERRED MODALITIES
As discussed above, calcium binding contributes substantially to the thermodynamics and kinetic stability of extracellular microbial proteases. In addition, with respect to subtilisins, barriers to high activation upon deployment may be essential to retain the native conformation and to prevent transient unfolding and proteolysis given the protease-filled environment when subtilisin is secreted and as a result of self-degradation. The unfolding reaction of subtilisin can be divided into two parts as follows: N (Ca) N or U in where MiCa) is the native form with subtilisin with calcium attached to the high affinity calcium binding site A (Fmzel et al. al., JJ Cell., Biochem., Suppl 10A: 272 (1986), Pantolian et al., Biochemistry 27: 8311-8317 (1988), McPhalen et al., BJochemistry 27: 6582-6598 (1988)); N is the folded protein without bound calcium; and U is the developed protein. Subtilisin is a relatively stable protein whose stability is largely mediated by the high affinity calcium site (Voordouw et al., Biochemistry 15: 3716-3724 (1976)).; Pantoliano et al., Biochemistry 27: 8311-8317 (1988)). The melting temperature of subtilisin at pH 8.0 in the presence of μmolar concentrations of calcium is about 75 ° C and about 56 ° C in the presence of excess EDTA (Takehashi et al., B; che istry 20: 6185-6190 (1981 ); Bryan et al., Proc. Nati, Acad. Sci. USA, 83: 3743-3745 (1986b)). The previous calorimetric studies of the form of calcium-free subtilisin (apoenzyme, ie, protein portion of the enzyme) indicated that it has a marginal stability at 25 ° C with a? G of unfolding of < 5 kcalories / mol (Pantoliano et al., Biochemistry 28: 7205-7213 (1989)). Because free calcium is as such an integral part of subtyiisin, it is thought that the apoenzyme is an intermediary of subtilisin folding. To independently examine the two phases of the folding process, the present invention constructs a series of mutant subtilisins. First, all proteolytic activity was eliminated to prevent self-degradation that occurs during the unfolding and refolding reactions. This can be achieved, for example by converting serine 221 from active site to cysteine.1 This mutation has little effect on the temperature of thermal denaturation of subtilisin, but reduced the peptidase activity of subtilisin by a factor of about 3 x 104
(Abrahmsen et al., Biochemistry 30: 4151-4159 (1991)). This mutant, therefore, allows the folding of subtilisin to be studied without the complications of proteolysis. In the present specification, a stenography to denote amino acid substitutions employs the single-letter amino acid code of the amino acid to be substituted, followed by the number designating where substitution will be made in the amino acid sequence, and followed by single-letter code of the amino acid to be inserted there. For example, S221C denotes the substitution of serine 221 for cysteine. The mutant of subtilisin with this unique
"The S221A mutant was originally achieved for this purpose.The natural form of this mutant was heterogeneous on its N-terminus, however, presumably due to some incorrect processing of the proenzyme.
Amino acid ststitueion is denoted as subtilisin S221C. II mutant of subtilisin S221C resulting was designated as
Suotilisin can be subjected to additional mutations to make the production and purification of relatively unstable apoptin easier. SI versions with three or four additional mutations, eg, M50F, Q206I, Y217K and N218S, can also be employed in the method of the present invention. Such additional mutations cumulatively increase the free energy of deployment by 3.0 kcalories / mole and increase the thermal denaturation temperature of the apoenzyme by 11.5 ° C (Par.toliano et al., Biochemistry 28: 7205-7213 (1989)). The mutant containing the mutations M50F, Q206I, Y217K, N218S and S221C is designated as Sil and the mutant containing M50F, Y217K, N218S and S221C is designated S12.2 to produce a subtilisin protein BPN 'which lacks the activity of calcium binding, the inventors of the present chose to suppress the binding cycle at site A of calcium to design a novel calcium-free subtilisin protein. This cycle comprises an interruption in the a-helix of the subtilisin BPN 'which involves amino acids 63-85 of SEQUENCE ID NO: l (McPhalen and James
The specific activities of Sil, S12 and S15 against the 3AAP substrate are not the same (SA = 0.0024 U / mg at 25 ° C, pH 8.0). These measurements were carried out on freshly purified protein on a column of : mercury inity.
13331. Residues 75-83 of the subtilisin protein BPN 'of tn cycle, which interrupts the last turn of the a-helix of 14 residues involving amino acids 63-85 [SEQUENCE ID N0: 1]. Four of the carbonyl oxygen ligands for calcium are provided by a cycle composed of amino acids 75-83 [SEQUENCE ID NO: 1]. The geometry of the ligands is that of a pentagonal pyramid whose axis runs through the carbonyls of amino acids 75 and 79. On one side of the cycle is the bidentate carboxylate (D41), while on the other side is the N -terminal of the protein in the side chain of Q2. The seven coordination distances range from 2.3 to 2.6 A, the shortest is that of the aspartyl carboxylate. Three hydrogen bonds link the N-terminal segment to residues "'8-82 of the cycle in the parallel beta arrangement.A high-affinity calcium binding site is a common feature of subtilisins that make great contributions to its stability. By binding at specific sites in the tertiary structure of subtilisin, calcium contributes its binding-free energy to the stability of the native state.The present invention was used in site-directed mutagenesis to suppress amino acids 75-83 of the
This set of nine residues was chosen for suppression, as opposed to 74-82 (those currently belonging to the cycle), preferably by Ala 74 instead of Gly 83 in the resulting continuous helix. Alanine is more likely to be found in helix a because of the wider glycine range of accessible skeletal conformers.
SEQUENCE ID NO: 1, which creates an uninterrupted helix and suppresses the potential calcium binding at site A (Figure IA
The inventors of the present believe that the stabilization strategy based on the binding of calcium cedar allow survival in an extracellular environment. Since the main industrial uses of subtilisins are in environments that contain high concentrations of metal chelators, it was
1 f practical significance for the inventors herein to produce a stable subtilisin which is independent of calcium and, therefore, is not affected by the presence of metal chelating agents. Av that the inventors of the present chose
To eliminate the binding of calcium by the removal of these amino acids, it would be possible to eliminate the binding of calcium with other mutations, for example, the substitution of one or more of the amino acids at positions 75-83 with alternative amino acids and by insertion, substitution
/ or suppression of amino acids close to positions 75-33. This can also be achieved by site-specific mutagenesis. Additionally, because this cycle is a common feature of subtilisins, it is expected that
Equivalent mutations for other subtilisins, in particular subtilases of class I, for example, by site-specific mutagenesis, probably eliminate calcium binding and provide enzymatically active mutants. In particular, the present inventors synthesized by site-specific mutagenesis is subtilisin BPN 'DNA, which had been subjected to mutations to eliminate amino acids 75-83 involved in the binding of calcium and which also comprise mutations of
additional substitution. These mutant subtilisin BPN 'DNA, upon expression of the DNA, provide subtilisin proteins have improved thermal stability and / or are resistant to proteolysis. The specific subtilisin BPN mutants
1 '' synthesized by the inventors herein are designated in this application as S15, S39, S46, S47, S68, S73, S79 and S86. The specific point mutations set out in the present application identify particular amino acids in the amino acid sequence of subtilisin BPN ', as
'.'0 are set forth in SEQUENCE ID NO: 1, which are mutants according to the present invention. For example, mutant S15 comprises a deletion of amino acids 75-83 and additionally comprises the following substitution mutations: S221C, N218S, M50F and Y217K. Mutant S39 3.5 similarly comprises a deletion of amino acids 75-83 additionally comprises the following substitution mutations: S221C, PSA, N218S, M50F and Y217K. The S46 mutant has a deletion of amino acids 75-83 and t ttt_tnalr -? c =. it comprises the following substitution mutations: M50F, Y217K and N218S. The mutant S47 if ilarment comprises a deletion of amino acids 75-83 and additionally comprises the following substitution mutations: Δ5A, N213S, M50F and Y217K. The S68 mutant comprises a deletion of amino acids 75-83 and
. J additionally comprises the following substitution mutations: P5S, N218S, M50F and Y217K. The mutant S73 comprises a deletion of amino acids 75-83 as well as the following substitution nutations: Q2K, M50F, A73L, Q206V, Y21 K and N213S. The S79 mutant comprises a deletion of the
. : < amino acids 75-83 and additionally comprises the following substitution mutations: Q2K, M50F, A73L, Q206V, Y217K and N218S. Finally, mutant S86 comprises a deletion of amino acids 75-83 as well as the following replacement mutations: Q2K, S3C, M50F, A73L, Q206C Y217K and N218S. HE
: 0 has found that the specific activities of the subtilisins S46, S47, S68, S73, S79 and S86 proteolicamente active is similar or better in relation to the natural enzyme. The different subtilisins? 75-83 that were synthesized by the inventors in Table I below.
' c The particular points of mutation in the? Or amino acid sequence of the amino acid sequence of the sctctycin BPN ', as set forth in SEQUENCE ID NO: l. The intesis of these mutants was described in greater detail
The plus sign shows that subtilisin that contains a particular mutation. It has been determined that the crystalline structures determined with X-rays of S12 and S15 of natural type is 1.8Á. * S1, Sil, S12, S15 and S39 are low activity mutants constructed to aid in the evaluation of structural and conformational stability.
To understand the contribution of celtium binding to the stability of subtilisin, the kinetics of the binding of calcium to site A was measured, the high affinity cale.o by microcalorimetry and fluorescence spectroscopy. . The binding of calcium is a process directed entálpicamente with a constant of association (K,) equal to 7 x 10 ° M "1. The kinetic barrier for calcium, the removal of site A (23 kcalor / mol) is substantially higher than standard free energy binding> 3.3 kcalorias / mol) The calcium dissociation kinetics of subtilisin (for example, excess EDTA) are consequently very slow, for example, the half-life (t? 2) of the dissociation of calcium from subtilisin, that is, the time for half of the calcium to be dissociated with subtilisin, is 1.3 hours at 25 ° C. The X-ray crystallography shows that except for the region of the site of the binding of the suppressed calcium, the structure of the mutants of the subtilisin and the natural protein are very similar.The N-terminus of the natural protein is located on the side of the site of site A and provides a coordinating ligand for calcium, the oxygen of the side chain of Q2. ilysin? 75-33, the cycle disappears, leaving the residues 1-4 untidy. Those first four residues are disordered in the structure determined with X-rays since all their interactions are with the calcium cycle. N-terminal sequencing confirms that the first four amino acids are present, confirming that processing occurs at the normal site. The helix is shown as cinterrumoid and shows a normal helical geometry along its entire length. X-ray crystallography further shows that the structures of subtilisin with and without suppression overlap with a mean square root difference (rms) between the 261 carbon positions of 0.17A, and are remarkably similar considering the size of the suppression . Diffuse difference and high temperature density factors, however, indicate some disorder in the newly exposed residues adjacent to the deletion. Although the removal of calcium binding is advantageous since it produces proteins that are more stable in the presence of metal chelators, this is disadvantageous in at least one aspect. Specifically, the elimination of the calcium cycle without any other mutation that compensates it results in the destabilization of the native state in relation to the partially folded state, and therefore, a loss of the ability to cooperate in the folding. The present inventors, in this way, also sought to genetically engineer the subtilisin protein S15 BPN 'lacking amino acids 75-83 to subtract the capacity of; _;! to. ritn have the folding reaction. In most of the
; As designed, all the parts of the molecule are
Ucellents, making the reaction highly deployed
-a * - The cooperative capacity of the "folding" reaction allows the protein to reach those sufficient stabilities of the native state for proper functioning since the total stability of the native conformation is approximately the sum of all local interactions. Even more, although subtilisin 75-83 is an example of a designed subtilisin, which is active and stable in the absence of calcium, the present inventors seek to improve this protein by additional mutations. highly stable calcium
.3 particular depends on an interactive design cycle. The inventors of the present found that the first necessary step in the cycle was to greatly decrease the proteolytic activity of subtilisin. This is necessary because calcium contributes greatly to the
.C conformational stability of subtilisin and the first versions of calcium-free subtilisin are susceptible to proteolysis. After reducing the susceptibility to proteolysis, the next step in the cycle was to eliminate the essential sequences for the union
Calcium, that is, site A. Although subtilisin S15? 3-33 is much less stable than subtilisin of the natural type in the presence of calcium, this mutant is more stable s_.e subtilisin of type In the presence of the metal chelator EDTA, the third step was to improve the stability of the calcium-free subtilisin protein.In order to improve the stability of the calcium-free subtilisin, the present inventors then tried to create a place to the disordered N-terminal residues.To create a highly stable calcium-free subtilisin, the
N-terminal protein that is destabilized by the
- suppression of calcium A cycle can be modified. For example, the N-terminal that is disordered can be deleted or extended. In this way, however, it is problematic because the requirements to process the propeptide from the mature protein are not known. It is known, however, that the processing site is not determined by the amino acid sequence since the mutants Y1A (the C-terminal of the propeptide) AlC and Q2R do not alter the cleavage site. It is also known that the native structure of the N-terminal in the subtilisin does not determine the breaking point because the variants? 75-83 are processed correctly. Since it is not yet known how to alter the processing site, interactions with existing N-terminals can be optimized.
The examination of the structure of subtilisin S15 numerous possibilities of improvement of the mutant enzyme. The regions of the ras structure affected by the deletion are the amino acids: id-terminals 1-8, the cycle co 36-45, the helix at 70-74, the turn of the helix 84-89 and the strip ß 202-219 . As stated previously, the first four residues in the subtilisin? 75-33 are disordered in the structure determined by X-rays since all their D interactions have been with the calcium cycle. N-terminal sequencing shows, however, that the first four amino acids are present, confirming that processing occurs at the normal site. In addition to the N-terminus, there are three other residues whose side chain 3 conformations are substantially different from the wild type ones. Y6 rotates out of a surface niche into a position more exposed to the solvent, as an indirect effect of the N-terminal destabilization. D41, a precursor calcium ligand, and Y214 undergo coordinated rearrangement, 0 forming a new hydrogen bond. The B factors of the three residues are significantly increased due to the suppression of amino acids 75-83. In addition, S87 and A88 do not change conformation but exhibit significantly increased B-factors. P86 determines the helix at which the calcium cycle was suppressed. In view of 3. above, other mutations in one or more of the aforementioned sites, or in the amino acids close to them, will epitomize subtilisin BPN 'mutants comprising greater enzymatic activity or greater ability. There are several logical strategies for reshaping this region of the protein to produce subtilisin BPN 'mutants that comprise greater enzymatic activity or greater stability. Since the four N-terminal amino acids are disordered in the determined structure of the X-rays, a possible method could be to suppress them from the protein. The requirements to process the propeptide of the mature protein are not understandable, Nevertheless. The insertion or deletion of amino acids from the N-terminal region is, therefore, problematic. For this reason, insertions and deletions in the N-terminal region in favor of amino acid substitutions were avoided. It can be assumed that many of the original amino acids in the regions described above of subtilisin that interacted with the amino acid cycle 75-83 are not optimal. Therefore, it was possible to increase the stability of the molecule by substituting, deleting or adding at least one amino acid in positions whose environment was changed by deletion 75-83.
The first attempt was to make the proline in the alanine phase 3 to create more flexibility in the
^. -icion 3. This greater flexibility allowed the N-terminal r ~ ^ e to find a unique position along the n eva surface of the protein, created by the suppression of the calcium cycle. Once the N-terminal assumes a single place its local interactions can then be optimized. The P5A mutation was made to try to create more flexibility for the N-terminus and allow it to find a unique position along the new surface of the protein that was created by a suppression of the calcium cycle. In the native structure, the first five amino acids are in an extended conformation and form a pair of β-hydrogen bonds with the calcium cycle, as well as the interaction of the Q2 side chain with calcium. The proline at position 5, which was conserved among seven bacterial subtilisins having a homologous calcium A site, may help to stabilize the extended conformation. The P5A mutation in subtilisin A75-83 could thus result in an increase in the cooperative capacity of the unfolding reaction. The structure determined with X-rays of this variant has been determined at 1.3 A.
In toto, the inventors of the present selected amino acids in ten different positions whose nabia emitter was substantially changed by substitution. 3e dismantled iin mutagenesis procedure and selection: wax select all possible substitutions at a particular site. The technique for generating and selecting subtilisin variants involves mutagenesis in vi tro of the cloned subtilisin gene, the expression of mutant genes in B. s useful, and select the connected stability. D For example, site-directed mutagenesis was performed on the subtilisin S46 gene using oligonucleotides which degenerated into a codon. The degenerate codon contained all the combinations of the NNB sequence, where N is any of the four nucleotides and B is T, C or 3 3. The 48 codons represented in this population code for the 20 amino acids but exclude termination codons tere yu ber. The mutant genes were used to transform 3. s ubtili s. Examples of particular mutations are shown in Table II as follows:
r-? n o p Table II - Site-directed mutagenesis
Mutations Oligonucleotide Protein regions Mutants N-terminal mutant site: Q2 K, W, L AC GCG TAC GCG NNB TCC GTG CCT TAC S3 C * GCG TAC GCG AAG NNB GTG CCT TAC GG V4 none C GCG AAG TCC NNB CCT TAC GGC G P5 S CAG TCC GTG NNB TAC GGC GTA TC cycle or ega 36-44: D41 A GAT TCT TCT CAT CCT NNB TTA AAG GTA GC K43 R, N CAT CCT GAT TTA NNB GTA GCA GGC GG helix a 63-85: A73 L, Q GGC AC GTT NNB GCT GTT GCG A7 no C AC GTT GCG NNB GTT GCG CCA AG strip ß 202-220: Q206 I, v, W, CC GTA TCT ATC NNB AGC ACG CTT CC Y214 no CCT GGA AAC AAA NTN GGG GCG AAA TC
It has been found that double cysteine mutations at positions 3 and 206 are stabilized as a disulfide bond.
To have a 98% chance of finding tryptophan, glutamine, glutamate or methionine in the mutant population, approximately 200 mutant clones should be selected. Each of these codons is represented only by one of the 48 codons contained in the population of NNB sequences. The codons for all other amino acids are represented by at least two codons in the population and could require the selection of approximately 100 mutant clones to have a 98% probability of being represented in the mutant population. To define the optimal amino acid at a position, mutants were selected to detect retention of enzymatic activity at high temperature. 100 μl of media were dispersed in each of the 96 wells of a microtiter disk. Each well was inoculated with a Bacillus transformant and incubated at 37 ° with shaking. After 18 hours of growth, 20 μl of culture was diluted in 80 μl of 100 mM Tris-HCl, pH 8.0 in a second microtiter disc. This disk was then incubated for one hour at 65 ° C. The disk was allowed to cool to room temperature after incubation at high temperature and 100 μl of 1 mM SAAPF-pNA was added to each well. It was estimated that the wells from which the pNA was cleaved (which turned yellow) faster contained the most heat-resistant subtilisin mutant. Once the preliminary identification of a stable mutant of the second microtitre disk was made, the Bacill clone us in the corresponding well in the first microtiter disc was grown for further analysis. The selection procedure identified stabilizing mutations in seven of the ten positions examined. As noted, those amino acid positions were selected at positions of the protein whose environment had changed substantially by virtue of the suppression of the calcium domain. No mutations were identified in positions 4, 74 and 214 that by themselves significantly increased the half-life of the mutant relative to undifferentiated subtilisin. However, only the effect of the hydrophobic amino acids at position 214 was selected. No mutations were found at positions 5, 41 and 43 resulting in measurable but moderate increases in stability. In addition, several mutations were found at positions 2, 3, 73 and 206 that significantly increased the half-life of the mutant relative to the undifferentiated subtilisin. Those stabilizing mutations are shown in Table III as follows:
Table III - Stabilizing Mutations Protein Region Increment Site
N-terminal: Q2K 2.0 times Helix at 63-85: A73L 2.6 times
Strip ß 202-220: Q206V 4.5 times
N-terminal - strip ß: S3C-Q206C (disulfide) 14 times
Modifications of stabilizing amino acids at positions 2 (K), 73 (L) and 206 (V) were then combined to
create subtilisin S73. The properties of subtilisin S73 as well as S46, S79 and S86 are summarized in Table IV.
Table IV Activity Average life Increment
_ O Mutant Mutations1 Specific2 (60 ° C) 3 S46 100 U / mg 2.3 min
S73 Q2K 160 U / mg 25 min 11 times A73L Q206V S79 Q2K N.D. 18 min 8 times A73L Q206C S86 Q2K 85 U / mg iO min 35 times S3C5 A73L A206C5 25 1) All subtilisins S46, S73, S79 and S86 contain the mutations M50F, Y217K and N218S and Q271E. 2) The specific activity was measured against succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (SAAPF-pNA) in 10 mM Tris-HCl, pH 8.0 at 25 ° C. 3) The half-life was measured at 60 ° C in 10 mM Tris-HCl, pH 8.0, 50 mM NaCl and 10 mM EDTA. 4) Not determined 5) The disulfide bond was formed between the cysteine at positions 3 and 206. The formation of a disulfide bond was confirmed by measuring the radius of gyration of the denatured protein by gel electrophoresis. In many cases, the choice of amino acid in a particular position will be influenced by amino acids in nearby positions. Therefore, in order to find the best combination of stabilizing amino acids, it may be necessary in some cases to vary the amino acids in two or more positions simultaneously. In particular, this was done at positions 3 and 206 with the amino acids whose side chains can potentially interact. It was determined that the best combination of modifications was the modification with cysteine in positions 3 and 206. Because the close proximity and proper geometry between the cysteines in those two positions forms a cross-disulfide bond spontaneously between these two residues.
The stability of subtilisin S86 was studied in relation to S73. It was found that the half-life of S86 is 80 minutes at 60 ° C in 10 mM Tris-HCl, pH 8.0, 50 mM NaCl and 10 mM EDTA, an increase of 3.2 times in relation to subtilisin S73. In contrast, a 3-206 disulfide cross-link may not be able to be formed in native subtilisins containing calcium A site because the 75-83 binding cycle separates the N-terminal amino acids from the β-strip 202 -219. Therefore, the increase that occurs in stability in the target S86 mutants lacking the 75-83 binding cycle will probably not be observed with the native subtilisins equally modified with cysteine at those positions. The similar increase in stability is expected to be inherent in other subtilisins of group I-Sl and I-S2 if the calcium cycles were suppressed (see Siezen et al, Protein Engineering, 4, precipitation, 719-737 in Figure 7). ). This is a reasonable speculation based on the fact that the main calcium site in these different subdivisions forms from cycles of almost identical 9 residues comprised in the identical position of the helix C. The structures determined by X-rays of the subtilisins BPN and Carlsberg I-SI, as well as subtilisin I-S2 (savinase), have been determined at high resolution. The comparison of these structures shows that all three have almost identical calcium A sites. The X-ray structure of subtyla of class I, the terminase of Thermoactinomyces vulgaris, is also known. Although the total homology of the BPN 'with the termitase is much less than the homology of the BPN' with the subtilisins I-SI and I-S2, termitase has been shown to have an analogous calcium A site. In the case of termitase, the cycle is an interruption of ten residues in the identical site in helix C. In this way, it is expected that stabilizing modifications will be quantified here, imparting similar beneficial effects on the stability of the versions with cycles of calcium suppressed from other subtilases of class I. The stability of subtilisins S73, S76 and S86 in relation to subtilisin S46 was compared by measuring its resistance to thermal inactivation at 60 ° C in 10 mM Tris-HCl, pH 8.0, 50 mM NaCl and 10 mM EDTA. Aliquots were removed at intervals and the remaining activity in each aliquot was determined. Under these conditions, the half-life of subtilisin S46 is 2.3 minutes and the half-life of S73 is 25 minutes (Table IV). To identify the other mutants that have increased stability, any mutagenesis technique known to those skilled in the art can be used. An example of such techniques for generating and selecting subtilisin variants involves three steps: 1) mutagenesis in vi tro of the cloned subtilisin gene; 2) expression of the mutant genes in B. subtili s, and 3) select stability set. The key element in the random mutagenesis method is being able to select large numbers of variants. Although random mutagenesis may be employed, the mutagenesis procedure described above allows the mutations to be directed to localized regions of the protein (e.g., the N-terminal region). As noted above, it was found that mutants S46, S47, S68, S73, S79 and S86 (which comprise the active site S221) were enzymatically active. It is expected that other substitutions that provide stability and equivalent or greater activity can be identified. The activities of the examples of calcium-free subtilisin mutants of the present invention against the substrate sAAPF-pNA in Tris-HCl, pH 8.0 and 25 ° C are given in Table V as follows:
Table V
Subtilisin Specific activity Average life (55 ° C)
BPN '80 U / mg 2 min S12 0.0025 U / mg ND1 S15 0.0025 U / mg ND1 S39 0.0025 U / mg ND1 S46 125 U / mg 22 min S47 90 U / mg 4.7 min S68 -100 U / mg 25 min
1) Half-lives were not determined for inactive subtilisins. As shown above, the subtilisin mutants S46, S47, C68, C73, S79 and S86 have increased catalytic activity compared to the subtilisin BPN '. Changes in catalytic efficiency due to suppression are not expected due to the fact that the active site of subtilisin is partially distant from site A of calcium. The stability of these subtilisin mutants was compared with natural subtilisin BPN 'by measuring its resistance to thermal inactivation. Since the stability of the calcium-free subtilisin mutants could not be affected by metal chelating agents, the experiments were carried out in 10 mM Tris-HCl, pH 8.0, 50 mM NaCl and mM EDTA (the association constant of EDTA for calcium is 2 x 10> JM "1) Proteins were dissolved in this buffer and heated to 55 ° C. Aliquots were removed at intervals and the remaining activity in each aliquot was determined. of inactivation in Figure 10. Under these conditions, the half-life of the subtilisin mutants improved much better than that of the subtilisin BPN ', which indicates that subtilisins that had undergone mutations to eliminate calcium binding in site A have complete catalytic activity and better stability in EDTA relative to subtilisin BPN 'A reasonable level of stability was achieved in S46 even without additional mutations that compensated for the loss a of the interactions resulting from the deletion of amino acids 75-83. Thus, the present inventors have provided convincing evidence that mutants of subtilisin that remain active can be obtained that do not bind to calcium. It is therefore expected that these mutants can be used in industrial environments comprising chelating agents. Although this has been shown only specifically with the subtilisin BPN ', the equivalent mutations could work with other serine proteases as well, more particularly other subtilisins I-SI or I-S2 since those subtilisins possess substantial sequence similarity, especially in the calcium binding site. Such strategies, for example, may involve comparing the subtilisin BPN 'sequence with other serine proteases to identify the amino acids that are suspected to be necessary for calcium binding and then making the appropriate modifications, for example, by specific mutagenesis for the site. Since many subtilisins are related to subtilisin BPN 'not only through their primary sequences and enzymatic properties, but also by crystallographic X-ray data, other active subtilisin mutants lacking calcium binding are expected. can be produced by site-specific mutagenesis. For example, there is structural evidence that enzymes homologous to Carlsberg subtilisin also comprise two calcium binding sites. Similarly, the structure of the termitase determined by X-rays is known and this subtilisin has a calcium A binding site analogous to that of the subtilisin BPN '. For termitase, the calcium binding cycle is an interruption of ten residues at the identical site in helix C. Consequently, those enzymes will also be susceptible to the mutations described here, which remove the calcium binding site and produce a stable, active enzyme. Furthermore, as discussed supra, Siezen et al, have shown that the primary calcium binding site in all subtilisins in the I-SI and I-S2 groups are formed from nearly identical residue cycles in the identical position of the helix C. In this way, in view of the almost identical structures of calcium A sites, the methods described here could be applied to most if not all of the subtilisins of groups I-SI and I-S2 discussed in Siezen et al. Alternatively, if the amino acids comprising the calcium binding sites are already known for a particular subtilisin, the corresponding DNA could be subjected to mutations by site-specific mutagenesis to suppress one or more such amino acids, or to provide mutations of substitution, suppression or adhesion that eliminate the binding of calcium. The subject mutant subtilisins will generally be produced by the recombinant methods, in particular by the expression of a subtilisin DNA that has been subjected to mutations so that upon expression of this, a subtilisin protein is obtained which is enzymatically active and It does not bind to calcium. Preferably, the subtilisin DNAs will be expressed in microbial host cells, in particular Bacillus subtilis, because these bacteria that naturally produce subtilisin are efficient protein secretors, and are capable of producing the protein in an active conformation. However, the invention is not restricted to the "expression of the subtilisin mutant in: '>; Bacill us, but encompasses expression in any host cell that provides expression of the desired subtilisin mutants. Suitable host cells for expression are well known in the art and include, for example, bacterial host cells such as Escherichia coli, Bacillus, Salmonella r Pseudomonas; yeast cells such as Saccharomyces cerevisiae Pichia pastoris, Kl uveromyces, Candida, Schizosacchaomyces; and mammalian host cells such as CHO cells. Bacterial host cells, however, are the preferred host cells for expression. Subtilisin DNA expression will be provided using available vectors and regulatory sequences. The actual selection will depend largely on the particular host cells that are used for the expression. For example, if the DNA of the subtilisin 0 mutant is expressed in Bacillus, a producer of Bacillus as well as a vector derived from Bacillus will generally be used. The inventors of the present invention in particular used the expression vector based on pUBUO and the native promoter of the subtilisin gene BPN 'to control the expression in Bacillus subtilis.
It should be further noted that once the amino acid sequence of the particular subtilisin mutant that does not bind calcium has been elucidated, it may also be possible to produce the subtilisin mutant by protein synthesis, for example, by Merrifield synthesis. However, the expression of the subtilisin mutants in microbial host cells will generally be preferred since this will allow the microbial host cell to produce subtilisin protein in a conformation suitable for the enzymatic activity. However, since the present inventors also teach here a method for obtaining refolding in vi tro of the subtilisin mutant, it might be possible to convert subtilisin mutants inappropriately folded to an active conformation. To better illustrate the present invention and the advantages thereof, the following specific examples are provided, it should be understood that they are intended only to be illustrative and not limiting.
EXAMPLES
Example 1
Cloning and Expression. The subtilisin gene of Bacill us amyloliquefaciens (subtilisin BPN ') has been cloned, sequenced and expressed at high levels of its natural promoter sequences in Bacillus subtilis (Wells et al., Nucleic Acids Res. 11: 7911-7925 (1983 ), Vasantha et al., J. Bacteriol 159: 811-819 (1984)). All mutant genes were cloned back into a pUBUO-based expression plasmid and used to transform B. subtilis. The B. subtilis strain was used as the host containing a chromosomal deletion of its subtilisin gene and therefore does not produce basal wild-type (wt) activity (Fahnestock et al., Appl. Microbial Environ., 53: 379 -384 (1987)). The oligonucleotide mutagenesis was carried out as described previously (Zoller et al., Methods Enzymol 100: 468-500 (1983); Bryan et al., Proc. Nati Acad. Sci. 83: 3743-3745 (1986b)) . The S221C was expressed in a New Brunswick fermenter of 1.5 1 at a level of approximately 100 mg of the mature form processed correctly per liter. The addition of natural subtilisin to promote production of the mature form of subtilisin S221C was not required in our bacillus host strain as in the case of previous strains (Abrahmsen et al., Biochemistry 30: 4151-4159 (1991)) . Purification and Characterization of Protein. The wild-type subtilisin and the variant enzymes were purified and verified by homogeneity essentially as described in Bryan et al., Proc. Nati Acad. Sci. 83: 3743-3745 (1986b); Pantoliano et al., Biochemistry 26: 2077-2082 (1987): and Biochemistry 27: 8311-8317 (1988). In some cases the C221 mutant subtilisins were repurified on a sulfhydryl specific mercury affinity column (Affi-gel 501, Biorad). The assays of peptidase activity were carried out by verifying the hydrolysis of succinyl- (L) -Ala (L) -Ala- (L) -Pro- (L) -Phe-p-nitroanilide, and subsequently sAAFna, according to to that described by DelMar et al., Anal. Biochem. 99: 316-320 (1979). The concentration of the protein, [P], was determined using P ° '1% = 1.17 at 280 nm (Pantoliano et al, Biochemistry 28: 7205-7213 (1989)). For variants which contain the Y217K change, the P0-1 * at 280 was calculated to be 1.15 (or 0.96 X wt), based on the loss of a Tyr residue (Pantoliano et al., Biochemistry 28: 7205-7213 ( 1989)). N-terminal analysis. The first five amino acids of subtilisin S15 were determined by sequential Edman degradation and analysis by CLAP. This revealed that 100% of the material had the amino acid sequence expressed from the DNA sequence of the gene and that the processing of the propeptide was in the same position in the sequence for the mutant as for the wild-type enzyme.
Example 2
structure of the Si or A of the calcium of subtilisin S12. The calcium A site is coordinated by five carbonyl oxygen ligands and an aspartic acid. Four of the carbonyl oxygen ligands for calcium are provided by a cycle composed of amino acids 75-33 (Figure 2). The geometry of the ligands is that of a pentagonal bipyramid whose axis runs through carbonyls 77 and 79. One side of the cycle is bidentate carboxylate (D41), while the other side is the N-terminus of the protein and the side chain of Q2. The seven coordination distances range from 2.3 to 2.6 Á, the shortest distance to the aspartyl carboxylate. Three hydrogen bonds link the N-terminal segment to residues 78-82 of the cycle in a parallel beta arrangement. Preparation of aposubtilisin. The subtilisins Sil and S12 contain an equilibrium amount of strongly bound calcium after purification. X-ray crystallography has shown that this calcium is bound to site A (Finzel et al., J. Cell. Biochem. Suppl 10A: 272 (1986); Pantoliano et al., Biochemistry 27: 8311-8317 (1988); McPhalen et al., Biochemistry 27: 6582-6598 (1988)). The complete removal of calcium from subtilisin is very slow, 24 hours of dialysis against EDTA at 25 ° C is required to remove all calcium from the protein and then 48 hours more dialysis at high salt content (Brown et al., Biochemistry 16: 3883-3896 (1977)) at 4 ° C to remove all EDTA from the protein.To prepare the calcium-free form of the Sil and S12 subtilisins, 20 mg of lyophilized protein was dissolved in 5 ml of EDTA 10 mM, tris (hydroxymethyl) amino-methane-10 mM hydrochloric acid (here after Tris-HCl) at pH 7.5 and dialyzed against the same buffer for 24 hours at 25 ° C. To remove the EDTA, which binds to the subtilisin at a low ionic strength, the protein was then dialyzed twice against 2 liters of 0.9 M NaCl, 10 mM Tris-HCl at pH 7.5 at 4 ° C for a total of 24 hours and subsequently three times with 2 liters of Tris- 2.5 mM HCl at pH 7.5 at 4 ° C for a total of 24 hours Chelex 100 was added to all the buffers that did not contain EDTA. When subtilisin C221 versions that did not contain substitutions for stabilizing amino acids were used, up to 50% of the protein precipitated during this procedure. It is essential to use pure native apoenzyme in the titration experiments since the spurious heat produced by the precipitation after the addition of calcium does not interfere with the measurement of the binding heat. To ensure that the aposubtilisin preparations were not contaminated with calcium or EDTA, the samples were checked by calcium titration in the presence of Quin2 before performing the calorimetric titration. Calorimetric Measurements by Ti Tulation. The calorimetric titrations were carried out with a Microcal Omega calorimetric titrator according to what is described in detail by Wiseman et al., Analytical Biochemistry 179: 131-137 (1989). The calorimetric titration consists of a reference cell containing the buffer and a solution cell (1374 mL) containing the protein solution. Aliquots of microliters of the ligand solution were added to the solution cell through a rotary agitator syringe operated with a node driven by an alternative motor. After achieving a stable basal level at a given temperature, automated injections were initiated and the accompanying heat change by injection was determined by means of a thermocouple detector between the cells. During each injection, an acute exothermic peak appeared which returned to the basal level before the next injection occurred 4 minutes later. The area of each peak represents the amount of heat that accompanies the binding of the ligand added to the protein. The total heat (Q) was adjusted by means of a non-linear least squares method
(Wiseman et al., Analytical Biochemistry 179: 131-137 (1989)) for the concentration of total ligand, [Cahotai / according to the equation: dQ / d [Caitos =? H [l / 2 + (l- ( l + r) / 2-Xr / 2) / Xr-2Xr (lr) + l + r2) 1 2] (1)
where 1 / r = [P] totaixKa and Xr = [Ca] t -.tai [P] total • The binding of calcium to the subtilisins Sil and S12 was measured by calorimetric titration since this allows to determine both the binding constant as the binding enthalpy (Wiseman et al., Analytical Biochemistry 179: 131-137
(1989); Schwarz et al., J. Biol. Chem. 266: 24344-24350
(1991)). The subtilisin mutants Sil and S12 were used in the titration experiments because the production of the wild type apoenzyme is impossible due to its proteolytic activity and low stability. The titrations of Sil and S12 were carried out at protein concentrations [P] = 30 μM and 100 μM. The titration of the Sil apoenzyme with calcium at 25 ° C is shown in Figure 4. The data points correspond to the relative heat of calcium binding associated with each calcium titration. The calorimetric titrant is sensitive to changes in Ka under conditions in which the product of Ka x [P] is between 1 and 1000 (Wiseman et al., Analytical Biochemistry 179: 131-137 (1989)). Since Ka for subtilisin is approximately lxlO7 M-1, protein concentrations result in values of Ka x [P] = 300 and 1000. At lower protein concentrations the amount of heat produced by titration is difficult. measure exactly.
The results of matching the titrations of Sil and S12 to a calculated curve are summarized in Table 2. The parameters in the table include the binding parameters for the stoichiometric ratio (N), the binding constant (Ka) and enthalpy of union (? H). Those parameters were determined from deconvolution using a non-linear least squares minimization (Wiseman et al., Analytical Biochemistry 179: 131-137 (1989)). The measurements of each experimental condition were made in duplicate at 25 ° C. The protein concentrations ranged from 30 to 100 μM while the concentration of the calcium solutions were approximately 20 times the protein concentrations. The binding enthalpy and constant were based on several titration assays at different concentrations. The titration tests were carried out until the titration peaks were close to the basal level.
TABLE 2: Calorimetric Titration of Site A of Calcium in Mutants of Subtilisin Sil and S12.
Mutant [P] Calculated parameters of the adjuster n Ka? H
Sil 100 μM 0.98 + 0.01 7.8 + 0.2 x 10b -11.3 + 0.1 Sil 33 μM 0.9 + 0.3 6.8 + 1.5 x 106 -10.9 + 0.2
S12 100 μM 0.99 + 0.01 6.4 + 0.2 x 106 -11.8 ± 0.5 The average values obtained are similar for
Sil and S12:? H = -11 kcal / mol; Ka = 7 x 106 M "1 and a binding stoichiometry of 1 calcium site per molecule.The weak binding cycle B does not bind calcium at concentrations lower than a millimolar range, and therefore does not interfere with the measurement of the binding to the binding site A.
The standard free binding energy at 25 ° C is 9.3 kcal / mol. The binding of calcium is thus primarily directed enthalpically with only a small net loss in entropy (? Sun? Ín = -6.7 cal / mol).
Ex «ampio 3
Refolded in vi tro of subtilisina S15. For refolding studies, subtilisin was maintained as a standard solution in 2.5 mM Tris-HCl at pH 7.5 and 50 M KCl at a concentration of approximately 100 μM. The protein was denatured by diluting the standard solution in 5M guanidine hydrochloride (Gu-HCl) at pH 7.5 or in most cases in 25 mM H3PO4 or HCl at pH 1.8-2.0. The final protein concentration was from 0.1 to 5 μM. S15 completely denatured in less than 30 seconds under these conditions. S12 required approximately 60 minutes to be completely denatured. The acid denatured protein was then neutralized to pH 7.5 by the addition of Tris-base (if denatured in HCl) or 5M NaOH (if denatured in H3P04). The refolding was initiated by the addition of KCl, "NaCl or CaCl2 at the desired concentration, For example, KCl was added from a 4M standard solution at a final concentration of 0.1 to 1.5 M with rapid agitation, in most cases the renaturation was carried out at 25 C. The rate of renaturation was determined spectrophotometrically by absorption of uv from the increase in the extinction a = 286, the increase in the intrinsic fluorescence of tyrosine and tryptophan (excitation = 282, emission - 347), or by circular dichroism from the increase of the negative ellipticity to? = 222 nm.
Example 4
X-ray crystallography. The collection of the single-crystal growth data and X-ray diffraction was performed essentially as reported previously (Bryan et al., Proteins Struct., Funct. Genet., 1: 326-334 (1986a). Pantoliano et al., Biochemistry 27: 8311-8317 (1988); Pantoliano et al., Biochemistry 28: 7205-7213 (1989)) except that it was not necessary to inactivate the S221C variants with diisopropyl fluorophosphate (DFP) to obtain suitable crystals. The starting model for S12 was made from the hyperstable subtilisin mutant 8350 (input to the Protein Data Bank ISOl.pdb). The structure of S12 was refined and then modified to provide the starting model for S15. Data sets were used with approximately 20,000 reflections with a resolution between 8.0 Á and 1.8 Á to refine both models using the least squares constrained techniques (Hendrickson et al., "Calculations in Crystallography" in Diamond et al., Eds., Bangalore: Indian Institute of Science 13.01-13.23 (1980)). The initial difference maps for S15, phased by an S12 version with the A site region omitted, clearly showed a continuous density representing the uninterrupted helix, allowing an initial S15 model to be built and refinement to begin . Each mutant was refined from an R of about 0.30 to an R of about 0.18 in about eight cycles, interspersed with electron density map calculations and manual adjustments using the FRODO graph modeling program (Jones, J. Appl. Crystallogr 11: 268-272 (1978)). Except for the region of the suppressed calcium binding cycle, the S12 and S15 structures are very similar, with a mean square root deviation (r.m.) of 0.18 A among the 262 carbons a. The N-terminus of S12 (as in the wild-type one) is located on the side of the site A cycle, providing a calcium coordination ligand, oxygen from the Q2 side chain. In S15 the cycle is absent, leaving the residues 1-4 untidy. The S12 (as in the wild type) the cycle of site A occurs as an interruption in the last turn of an alpha helix of 14 residues; in S15 this propeller is not interrupted and shows the normal helical geometry in its entire length. Diffuse difference and higher temperature density factors indicate some disorder in the recently exposed residues adjacent to the deletion.
Example 5
Differential Scan Calorimetry. The stabilizing properties of S12 and S15 were studied using DSC (differential scanning calorimetry). The mutant of the? 75-83 (S15) is very similar in its fusion temperature to the S12 apoenzyme. The DSC profiles of apoS12 and S15 are shown in Figure 3. The temperature of the maximum heat capacity is 63.0 ° C for S15 and 63.5 ° C for apo-S12 at pH 9.63. The DSC experiments were carried out at high pH to avoid aggregation during the denaturation process. The amount of excess heat assumed by a protein sample increased the temperature through a transition from the folded to the unfolded state at constant pressure, which provided a direct measurement of ΔH of unfolding (Privalov et al., Methods Enzymol. : 4-51 (1986)). The "unfolding Hcai of apo-S12 and S15 is approximately 140 kcal / mol. Above pH 10.0, the unfolding transition for S15 conforms to a reasonably good two-state model, consistent with the equilibrium thermodynamics expressed in the van't equation
Hoff (din K / dT =? HvH / (RT2)) with? HvH (the enthalpy of van't Hoff or apparent enthalpy) approximately equal to? Hcal (the calorimetric or true enthalpy). At pH 9.63, however, the fusion profile for both proteins was asymmetric indicating that the unfolding is not a pure two state process.
Example 6
Kinetic measurements of calcium di sociation.
The dissociation of calcium from subtilisin is a slow process. A Quin 2 fluorescent calcium chelator was used to measure this rate. Quin 2 binds calcium with a Ka of 1.8 x 108 at pH 7.5 (Linse et al., Biochemistry 26: 6723-6735 (1987)). Quin 2 to 495 mm fluorescence increases approximately 6 fold when bound to calcium (Bryant, Biochem J. 226: 613-616 (1985)). The Sil or S12 subtilisins as isolated have a calcium ion per molecule. When mixed with an excess of Quin 2, the kinetics of calcium release from the protein can be followed from the increase in fluorescence at 495 nm. It is assumed that the reaction follows the N (Ca) or N + Ca + Quin 2 or Quin (Ca) pathway.
The dissociation of calcium from subtilisin is relatively very slow at the binding of calcium to Quin 2, so that the change in fluorescence of Quin 2 is equal to the dissociation rate of calcium from subtilisin. As can be seen in Figure 5a, the initial calcium release of Sil follows a simple first order kinetics. Dependence on the temperature of the dissociation of calcium. The first order velocity constant (k) for calcium dissociation was measured from 20 ° to 45 ° C. The graph of ln k vs. 1 / T ° K is approximately linear. The calcium dissociation data were fitted to the curve using the transition state theory according to the Erying equation: ΔG * = -RT ln K * = -RT ln kh / kBT (2) where kB is the Boltzman constant, h is the Planck constant and k is the first order velocity constant for the retracted one. The graph of ln hk / kBT vs. 1 / T is shown in Figure 5b. The data was then fitted to the curve according to the equation (Chen et al., Biochemistry 28: 691-699 (1989)): ln K * = A + B (To / T) + C ln (To / T) (3) where A = [? Cp * +? S * (To)] / R; B = A -? G * (T0) / RTo; C =? CpVR. The obtained data produce the following results:? G = 22.7 kcal / mol; ? Cp '* = -0.2 kcal / ° mol; ? S'F = -10 cal / ° mol; and? H'F = 19.7 kcal / mol at a reference temperature of 25 ° C. A possible slight curvature of the graph could be due to a change in the thermal capacity associated with the formation of the transition state (? Cp '* =
0. 2 kcal / lmol). The? Cp for the folding of the protein has been shown to be closely correlated with a change in the exposure of the hydrophobic groups to water (Privalov et al., Adv. Protein Chem. 39: 191-234 (1988); Livingstone et al. , Biochemistry 30: 4237-4244 (1991)). In terms of heat capacity, the transition state therefore appears to be similar to the native protein. The values for
? S '* and? H' f were obtained from Figure 5b indicating that the transition state is enthalpically less favorable than the calcium bound form with only a small change in entropy.
The other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention described herein. It pretends that the specification and examples are considered exemplary only, with the scope and true spirit of the invention being indicated by the following claims. All the references cited here are incorporated in their entirety, as if they were incorporated individually as a reference.
SEQUENCE LIST
) GENERAL INFORMATION:
(i) APPLICANT: BRYAN, Philip N ALEXANDER, Ptrick STRAUSBERG, Susan L
(ii) TITLE OF THE INVENTION: CALTIUM FREE SUBTILISIN MUTANTS
(iii) NUMBER OF SEQUENCES: 1
iv) DOMICILE FOR CORRESPONDENCE: (A) RECIPIENT: Burns, Doane, Swecker & Mathis
(B) STREET: P. O. Box. 1404 (C) CITY: Alexandria (D) STATE: Virginia (E) COUNTRY: United States (F) C.P .: 22313-1404
! v) LEGIBLE FORM IN COMPUTER: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0, Version # 1.25
(vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION:
(viii) INFORMATION FROM THE MANDATORY / AGENT (A) NAME: Meuth, Donna M (B) REGISTRATION NUMBER: 36, 607 (C) REFERENCE NUMBER / FILE: 028755-021
(ix) INFORMATION BY TELECOMMUNICATION: (A) TELEPHONE: (703) 836-6620 (B) TELEFAX: (703) 836-2021
(2) INFORMATION FOR SEQ ID NO: l:
(i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 1868 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 450..1599
(ix) CHARACTERISTICS: (A) NAME / KEY: mat_peptide (B) LOCATION: 772..1599
(ix) CHARACTERISTICS: (A) NAME / KEY: misc_caracteristica (B) LOCATION: 450 (C) OTHER INFORMATION: / note: "The Amino Acid val in position 450 is fMet".
i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
rrg pt AR- aac? r TCAC? AIXTC ?? O? O OQ &x? rtc? tt? TC? GGAITTT «O tJiz ???? T8 tttcac * ßct rrtctcoatc AAOJUMCCCA Mcaciaarx tcecri? Cßt 120 rrccATa cT crt zct-- t CA? C? AAJ'-QA K? CXXTT? X CCTCTTTTTC OAAO CCCC no
OUUUV? T8? mauu ecoT taixccaao MOC? cea »« tß * -ftocrc cTctctxatf .2-40 ttjkctca.?ct cakxccaaaß erTTrrtc? TOJ? S SX -UMKTTTCC? urtucT? cs * 300 icAxwx? X * ecT ??? Z? ß? > ? T »K > G »» TCTO? MUU? ? nccct a 3ßo ca? xzsrt arrccxact rcu-? Ax-i ?? maauuAX AAICTOTC ?? TTOOGOITC 420? Gauutt? A i? NnfHT'Wff ce? I ?? Jta * OTO M? OOC A? & IM ct? Te XTC 473 Val? Rg Oí iy »y» V »l Trp II * • 107 -103 -100 ACT TTC ero TTT ßcr TTA« seo TTA AT TTT ACÓ ATO OCO TTC OCC AOC 321
Sar Uu Ua Paa Al * Lau Wing LM I le Paa Tur Mat Wing Pha Oly tor -95 -90 -ßS ACÁ TCC TCT ßCC CAC OCO OCA 000 AAA TCA AAC Gßß CAÁ AAO? AA T? T 569 Thr tmx 3 «r Wing OIA Ala Ala ßly Lya Mrnx A *;; Oly ßlu Lyß Lya Ty »-ßO -7S -70 Air CTC eco TTT AAA CAO ACÁ ATO ABC ACO ATO ACC oee ßcr AAO AAO 417
Xla Val Oly Pba Lya Wave Thr Ma Sar? Hr Mac Sar Ala Wing Lya Ly »-43 -40 -IS AA? CAT CTC ATT TCT CAA AAA TQC GOO AAA OTO CA AAO CAÁ TTC AAA 6 «S
Lya Asp val Zla Sar Olu Lyß Oly Cly lyß Val Ola Lya Ola Pba Ly * -30 -43 -40 TAI OTA OAC OCA OCT TCA CCT ACÁ TTA AAC SAA AAA CCT OTA AAA OA? 73J Tyr Val Aap Ala Ala Sar Ala Thr Lau Aan Ola and Ala Ala Lys C to -3S -30 -23 -20 TTß AAA AA? CAC «CO AOC CTC OCt TAC OTT QAA CA CAT CAC OtA CCA 7ßl
Lau Ly * Ly »Aap Pro §t Val Wing Tyr Val Oís OW Aap Wing Val Wing -1S -10 -I CAT OCS TAC 9t? CAO TCC OTO CCT TAC OOC OTA TCA AA ATT AAA CCC 89 »E? A Ala Tyr Ala wave Sar Val Pro Tyr Cly al? OU lia Lya Wing 1 S 10 (XT CCT CTO CM TCT CAA eCC TJW? CT OOA TCA AAT CTT AAA STA CCß S37 Pro Ala Lau lia Sar C a ßly Tyr Tar Oly Sar Aa Val Lya Val 1S 20 2S OTT ATC OAC AOC OOT ATC OAT TCT TCT CAT CCT OAX TTA ?? ß STA 909
Val? The Ar »? Mx oly Zla Aap ßav sar Ala Pra Aap Lev Lya at Wing 30 3S 40 43 OOC OOA« SCC AOC ATC CTT CCT TCT SAA ACÁ AAT CCT TTC CAA SAC AAC 933 ßly ßly Wing S x «a Val Pro S s ßlu Thr Aaa Pro Paa Ola Aap? A to 30 SS 40 AAC TCT CAC OOA ACt CAC CTT OCC OOC AC OTT OC to OCT CTT AAT AAC 1001
Ara sar Bis ßly Tr Sis val Ala oly Thr val Ala Ua m Aaa Asa • 9 70 73 TCA ATC CCT OTA TTA GGC CTT OCß CCA? CC OC? TCA Crt A ƒ CT CT? 1049 My Xlà Oly val Lau ßly Val Ala Pro Sar Ala Sar Laß Tyr Ala Val SO 43 90
CTT ere? oct CAC oct TCC OCC CAA TAC AOC TOO ATC AIT AAC 1097 Lya Val I t ßly Wing Aap Oly x ßly wave Tyr Sar? rp? l?? Ara 93 100 103 GßA ATC ßAß Tßß OCß ATC OCA? ACAATATCC? CßTTATTAACATOAOC 1143
Oly Zla Olu Trp Wing Zla Wing Aaa Aaa Hat Aap Val? A Mae? M UO 113 120 US CTC OCC OOA CCT TCT GCT TCT ß «CT OCT TTA AAA OCO C« CA «STT CAZ AAA 1192 Lau ßly ßly Pro Sar Cly Sar Ala Ala Lau Lya Ala Ala val Aap Lya 130 131 140 ßCC ßTT OCA TCC OOC OTC OTC OTC OCC OCC CCC CCT AAC OAA OßC 1241 Wing Val Ala Sar Oly Val Val Val Val Al * A Ala oly Aaa Olu ßly 14S 130 133 ACT TCC OOC AßC TCA AOC OCCAT OOC TAC CCT OOT AAA TAC CCT TCT 12S9 Thr Sar ßly Sar Sar Smx Tor Val ßly Tyr Pro ßly Lya Tyr Pro 9mr ISO 16 * 170 ßTC A O C OTA CCC ßCT CTT CAC AßC ACC AAC CAÁ AO OCA TCT TTC 1337 Val II * Ala Val ßly Ala Val Aap Sar Sar Aaa Ola Ara Wing M? Z »B * 173 180 BS TCA AßC OTA G6A CCT OAO CTT OAT CTC ATO OCA CCT OOC OTA TCT ATC as Sar Sar Val ßly Pro ßlu I ?? Aap Val Mac Wing Pro ßly Val $ mx Zla 190 193 200 20 $ CAA ACC ACC CTT CCT OOA AAC AA? TAC OCßCCC TAC AACCCT ACO TCA 1433 ßla Sar Thr Lau Pro ßly? Aa Ly * Tyr Oly Wing Tyr Asa Oly Thr 3 * r 210 21S 220 ATC CCA TCT CCO CAC ßTT ßCC 66A ßCO -3CT ßCT TTß ATT CTT TCT A? O 14B1 Ma * Ala? * Pro Ai * Val Ala Oly Ala Ala Ala Lau Lau Xla Lau Sar Ly * 223 230 233 CAC CCO AAC TOO ACÁ AAC ACT CAÁ ßTC CSC AßC ACT TTA CAAC AAC ACC 1529 Bis Pro Handle Trp Tar Aaa Tar ßla Val Arg Sar Sar Lau ßlu Aaa Tar 240 24S 210 ACT ACA AAA CTT OCT CAT TCT TTC TAC TAT OCA AAA ßCC CTO ATC? C 1377 Thr Ihr Ly * Leu ßly Aap Ser Pha Tyr Tyr ßly Ly * Oly Lau? The Handle a «or ats A ACATAAA ? AA 1429 CCCGCCCOOT TTTTTATTAT U1 .1 HXT CCTICATWTTC AATCCOCTCC ATAATCCACC "3ATOOCTCCC 14S9 TCTCAAAATT TTAACBAßAA ACßOCßßßTT OALLLU jxu AßTCCCCTAA CCOCCAACTC 1749 CTSAAACCTC TCAATCOCCB CTTCCCßOTT TCCSOTCAßC TCAATCCCCT AACSCTCCCC 1809 QQH.l'l'I'llL TOATACCßaO AßACCCC? XT CßTAATCOßA TCACAAßCAA A? CTOAOC? 1868
It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional particle to the manufacture of the objects to which it refers. Having described the invention as above, property is claimed as contained in the following:
Claims (24)
1. An enzymatically active subtilisin protein, which has been subjected to mutations to eliminate the ability of said subtilisin protein to bind calcium at a high affinity calcium binding site, characterized in that the mutant subtilisin protein comprises one or more deletion mutations, substitution or addition in at least one of the following regions: amino acids 1-8 of the N-terminus, amino acids 36-45 of the cycle?, amino acids 70-74 of the helix a or amino acids 84-89 of the return of the propeller.
2. The subtilisin mutant according to claim 1, characterized in that the amino acids at positions 75-83 were deleted to eliminate the ability of the mutant to bind calcium.
3. The subtilisin mutant according to claim 2, characterized in that the mutant lacking amino acids 75-83 has one or more additional mutations in the amino acid sequence.
4. The subtilisin mutant according to claim 3, characterized in that the mutant has one or more additional mutations at amino acids 202-219 of the β-strip.
5. The subtilisin mutant according to claim 3, characterized in that the subtilisin mutant comprises at least one substitution mutation of N218S, M50F, Y217K, P5S, D41A, K43R, K43N, Q271E, Q2K, Q2, Q2L, A73L, A73Q , Q206C ,. Q206V, Q206I, Q206 or S3C.
6. The subtilisin mutant according to claim 5, characterized in that the subtilisin mutant comprises the substitution mutations of N218S, M50F, Y217K and P5S.
7. The subtilisin mutant according to claim 5, characterized in that the subtilisin mutant comprises the substitution mutations of N218S, M50F, Y217K, Q271E, Q2K, A73L, and Q206C.
8. The subtilisin mutant according to claim 5, characterized in that the subtilisin mutant comprises the substitution mutations of N218S, M50F, Y217K, Q271E, Q2K, A73L and Q206C.
9. The subtilisin mutant according to claim 5, characterized in that the subtilisin mutant comprises the substitution mutations of N218S, M50F, Y217K, Q271E, Q2K, A73I, Q206C and S3C.
10. The subtilisin mutant according to claim 1, characterized in that the subtilisin is from a strain of Bacillus.
11. The subtilisin mutant according to claim 10, characterized in that the subtilisin mutant is a mutant of subtilisin BPN ', a mutant of subtilisin Carlsberg, a mutant of subtilisin DY, a mutant of subtilisin amylosacchariticus or a mutant of subtilisin mesenticopeptidase or a mutant of subtilisin Savinasa.
12. The subtilisin mutant according to claim 11, characterized in that the subtilisin mutant is a mutant of subtilisin BPN '.
13. A recombinant method, which is provided for the expression of an enzymatically active subtilisin protein, which has been subjected to mutations to eliminate the ability of said subtilisin protein to bind calcium at a high affinity calcium binding site, where the mutant subtilisin protein comprises one or more deletion, substitution or addition mutations in at least one of the following regions: amino acids 1-8 of the N-terminus, amino acids 36-45 of the? cycle, amino acids 70-74 of the helix a or amino acids 84-89 of the turn of the helix, the method is characterized in that it comprises: (a) transforming a recombinant host cell with an expression vector, which contains an enzymatically active subtilisin DNA, which upon expression provides the expression of a subtilisin on which calcium binds to the protein; (b) culturing the host cell under the conditions that provide for the expression of the enzymatically active subtilisin mutant; and (c) recovering the mutant of enzymatically active subtilisin from the microbial host.
14. The recombinant method according to claim 13, characterized in that the subtilisin mutant lacks the calcium binding site A.
15. The recombinant method according to claim 14, characterized in that the mutant lacks amino acids 75-83.
16. The recombinant method according to claim 15, characterized in that the mutant has one or more additional mutations in the amino acid sequence.
17. The recombinant method according to claim 16, characterized in that the subtilisin mutant comprises at least one substitution mutation of S221C, N218S, 50F, Y217K, P5S, D41A, K43R, K43N, Q271E, Q2K, Q2, Q2L, A73L, A73Q, Q206C, Q206V, Q206I, Q206W or S3C.
18. The recombinant method according to claim 13, characterized in that the subtilisin mutant is a mutant of subtilisin BPN '.
19. The recombinant method according to claim 18, characterized in that the DNA of the subtilisin BPN 'comprises one or more additional mutations, which are provided for the enhanced or improved thermal stability or which are provided for the restoration of the cooperative capacity of the folding of the protein subtilisin.
20. A recombinant DNA, which codes for a subtilisin protein which has been subjected to mutations to eliminate the ability of said subtilisin protein to bind calcium at a high affinity calcium binding site, characterized in that the mutant subtilisin protein comprises one or more deletion, substitution or addition mutations in at least one of the following regions: amino acids 1-8 of the N-terminus, amino acids 36-45 of the cycle?, amino acids 70-74 of the helix a or amino acids 84-89 of the spin of the helix, and the mutant subtilisin protein which retains the activity and enzymatic stability.
21. The recombinant DNA according to claim 20, characterized in that the subtilisin DNA is a coding sequence of subtilisin BPN 'which lacks the codons coding for amino acids 75-83.
22. The recombinant DNA according to claim 21, characterized in that the DNA of subtilisin is a coding sequence of subtilisin BPN 'comprising a codon encoding one or more additional mutations in the amino acid sequence.
23. The recombinant DNA according to claim 22, characterized in that the subtilisin DNA is a coding sequence of subtilisin BPN 'that 7T it comprises a codon that codes for one or more additional mutations at amino acids 202-219 of the β-strip.
24. The recombinant DNA according to claim 22, characterized in that it further comprises a codon coding for at least one substitution mutation of S221C, N218S, M50F, Y217K, P5S, D41A, K43R, K43N, Q271E, Q2K, A73L, A73Q, Q206C, Q206V, Q206I, Q206W or S3C.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/309,069 US5567601A (en) | 1993-06-01 | 1994-09-20 | Subtilisin mutants lacking a primary calcium binding site |
| US08309069 | 1994-09-20 | ||
| PCT/US1995/005520 WO1996009396A1 (en) | 1994-09-20 | 1995-04-28 | Calcium free subtilisin mutants |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| MX9702038A MX9702038A (en) | 1997-10-31 |
| MXPA97002038A true MXPA97002038A (en) | 1998-07-03 |
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