SE468480B - Modified thioredoxin and its use - Google Patents

Abstract

Thioredoxin comprising a redox-active Cys32-X-Pro34-Cys35 site (E.coli numbering) or a functional derivative thereof in which the amino residue Pro in position 34 has been replaced with His and in which the amino acid residue X in position 33 is selected from Gly and Ala; processes for preparing folded biologically active disulphide-crosslinked proteins in vitro using such thioredoxin; and these processes carried out in the presence of a catalytic quantity of a selenite.

Description

468 480 10 15 20 25 30 35 2 of denatured, unfolded reduced proteins such as models. The classic model system is ribonuclease A (RNase). Studies of ribonuclease have led to the discovery of PDI as a luminal, endoplasmic reticulum protein in eukaryotic cells catalyzing disulfide exchange and imaging native disulfides. The in vivo oxidant in formation of disulfides from dithiols is, however, unknown.

Recent developments in recombinant technology enable construction and expression in bacteria, yeast or others system of any given sequence of DNA encoding for a known or modified protein. This procedure is of great biomedical significance, which can be exemplified by recombinant insulin production, insulin-like growth factors, growth hormone, tissue plasminogen activity vectors, interferons, factor VIII and many other iner. Since the majority of these biomedical interests santa proteins contain innumerable disulfide bonds seriously complicates the general production of in bacteria, which is economically favorable, by imaging the development of non-native, often aggregated and insoluble pots. The process therefore requires either a fold-in in vitro or production must be performed in eukaryotic systems with much higher costs. Ideally, the production of a recombinant protein in a bacterial-like manner Escherichia coli involve a construct in which protein creasing and native disulfide formation would occur. Al- alternatively, an in vitro production process would be efficient of high yields of native pleated disulfide-linked protein apply.

Protein disulfide isomerase.

The mechanism by which the formation of native disulfide is a network oxidation, where two thiol groups form a di- sulfide and emits two protons and two electrons (Reaction 1). + 2 H + + 2 e (1) R- (SH) 2 ----- -> RS 2 10 15 20 25 30 35 _t> O\ 03 hrs O) C) 3 The in vivo oxidant in this reaction is unknown. In in vitro systems use either oxygen in air as a terminal oxidant resulting in a slow process or utilizing a redox buffer composed of, for example, glutathione and glutathione disulfide (GSH-GSSG). The principal reactions in this process, the thiol disulfide yield and the available knowledge shows that native disul- fider in a protein is formed by an exchange process via non-native disulfides during folding (Creighton, 1990).

PDI catalyzes disulfide isomerization to form a final structure with correct disulfides. The enzyme is a 57 kDa acidic protein located in the lumen of the endoplasmic reticulum (ER). Cloning of PDIs has revealed that it holds two domains of about 100 residues with strong similarity to thioredoxin (Edman et al., 1985). This suggests that active site in the PDI and the mechanism of the protein was similar with thioredoxin. This was proved by the discovery of the PDI is a substrate for mammalian thioredoxin reductase (Lund- stream and Holmgren, 1990). Thioredoxin has far-reaching characterized over the course of 25 years, and its primary structure and three-dimensional structure are known from gene crystallography, 2D NMR och and a comprehensive protein con- structural work (for references see Holmgren, 1985, 1989 and Dyson et al., 1990; Eklund et al., 1991). IN a series of previous publications has shown that thioredoxin is a powerful protein disulfide reductase at coupling to NADPH and thioredoxin reductase (Holmgren, 1984). Thioredoxin contains an active redox disulfide in its oxidized form with the amino acid sequence Trp-Cys-Gly-yro-Cys.

The molecule is so folded that a protruding part is formed containing the active site within a hydrophobic surface involving Trp-31, Cys-32, Pro-34, Pro-76, Gly-92, Ala- 93. This surface is preserved in thioredoxins from all species ter (Eklund et al., 1991). The mechanism of thioredoxin has proposed to involve a transcently mixed disulfide intermediate lan Cys-32 and the protein disulfide. An essential element of this mechanism is the formation of a complex between 468 480 10 15 20 25 30 35 4 the tin substrate and the hydrophobic surface of thioredoxin. This makes possible for thioredoxin to catalyze thiol disulfide exchange reactions with rate constants within the embankments 1o4-1o5 M * s * in batch to low molecular weight thiol such as glutathione, mercaptoethanol or DTT, which react at physiological.pH with rate constants of 1 s_l (Holmgren, 1985). Tiore- on the order of 1-10 M- doxin thus generally shows a very fast reaction time. kinetics. In other reactions, thioredoxin may have one uniquely higher activity against specific disulfides, which significantly exceeds that of low molecular weight thiols. IN In this case, it can be assumed that thioredoxin, by the formation of a complex with a protein, produces a conformational change that makes an otherwise buried disulfide reactive. This is a key idea for thioredoxin catalyzing thiol disul- fid exchange processes with proteins, namely that the way for protein folding and concomitant disulfide formation can be directed through the catalyst. One can assume that a similar mechanism is relevant in protein disulfide isomerase.

An earlier report (Pigiet and Schuster, 1986) writes folding of ribonuclease RNase in the presence of high concentrations of thioredoxin from E.coli. A speed increase was observed, as well as a better yield of finally RNase. The quantity of thioredoxin used in however, this process was chemical rather than catalytic. (25 pM RNase-SH2 and 500 pM Trx-S2). No comparison else was done with PDI and the process required very long times and high concentrations of thioredoxin.

Due to better availability of thioredoxins compared to, for example, the enzyme protein disulfide isomerase (PDI) it would be an advantage if thioredoxins could be effectively used when properly folding or refolding biologically active proteins containing disulfide bridges.

The main object of the present invention is therefore that provide new thioredoxins, which are redox active and show an improved redox potential compared to national and thioredoxins. 10 15 20 25 30 35 -ß ox oo |> oo O 5 Another object of the invention is to achieve procedures for the correct folding or refolding of sulfide crosslinked proteins using such a mode differentiated thioredoxin.

Another object of the invention is to achieve procedures for the correct folding or refolding of sulfide crosslinked proteins with improved efficiency of such procedures.

Other objects and advantages of the invention will come to be fully apparent from the following description.

For purposes identified above and other purposes the invention provides new thioredoxins and functions derivatives thereof comprising a redox-active Cys- Gly-Pro-Cys site, wherein the amino acid residue Pro is in the 34-position has been replaced by His, and wherein amino acid residues with 33-position tion is selected from Gly and Ala. In this redox active place or sequence refers to positions 32 to 35 corresponding amino acid residues Cys, Gly, Pro and Cys in E.coli numbering.

The substitution performed as defined above redox active site results in an unexpected increase of the redox potential in addition to the fact that they modified the thioredoxins are obtained in very high yields and beyond that they are stable. The findings associated with the present present invention is all the more unexpected as other Scientists have expressed the view that it is relatively larger the effectiveness of a PDI compared to thioredoxin rather due to the presence of catalytic multiple domains down in PDI than in differences in their active-place sequences.

In this regard, reference is made to the article in Bio.Chem.J. (1991) 275, 349-353, by Hilary C. Hawkins et al., Cf. its initial summary.

In a preferred embodiment of the invention thioredoxins or their functional derivatives in 33- position amino acid residue Gly. The thioredoxin of the present invention The present invention is preferably obtained from natural occurring redoxins by site-directed mutagenesis. 468 480 10 15 20 25 30 'ss 6 The invention also provides a method for the in vitro production of a folded biological biological active, disulfide-linked protein, which process consists in turnover of a correspondingly unfolded and reduced in with a thioredoxin or functional derivative thereof, wherein the Pro in 34 position is replaced by His, and wherein the amino the acid residue in the 33-position is selected from Gly and Ala, resulting in the formation of native disulfide. Also in in this method, it is preferred that position 33 be occupied of the amino acid residue Gly.

According to a further improvement with regard to In the present invention, such a reaction is carried out in be a catalytic amount of a selenite. How to categorize lytic amount may be within the concentration range from about 0.1 pM to about 100 μm, especially from about 0.5 to about 10 pM.

According to another option for carrying out this the reaction of the protein may take place in the presence of a redox buffer, such as a buffer of reduced and oxidized glutathione, ß-mercaptoethanol or dithiothreitol.

The present invention is also applicable to folding misfolded proteins, such as scramble pro- to form the correct fold with di- sulfide bonds. Such a process is characterized by loading of a scramble protein with a thioredoxin or functional derivative thereof, in which Pro in 34-position has been replaced with His, and wherein the amino acid residue in the 33-position is selected among Gly and Ala, in the presence of a reductant or a doxbuffert, glutathione, ß-mercaptoethanol or dithiothreitol. This one too such as a buffer of reduced and oxidized reaction can take place in the presence of a selenite at the concentration conditions similar to those set out above.

According to yet another aspect of the invention, there is provided method for forming the correct folding fold in vitro of an unfolded and reduced protein to form a di- sulfide crosslinked protein. In said process it is reacted unfolded and reduced the protein with thioredoxin, a thio- 10 15 20 25 30 35 7 redoxin, wherein Pro in the 34-position is replaced with His, and wherein the amino acid residue in the 33-position is selected from Gly and Ala or a functional derivative thereof, or PDI in the presence of a catalytic amount of a selenite, resulting in native disulfide.

According to that aspect, an alternative procedure those in the correct folding of an incorrectly folded protein by reaction with a thioredoxin, a thioredoxin, wherein Pro in the 34-position has been replaced by His, and wherein the amino acid the rest in the 33-position are selected from Gly and Ala, or one functional derivative thereof, or PDI in the presence of a NADPH and a thioredoxin reductase and a catalytic amount of a selenite resulting in the formation of native disulfide.

Using the equilibrium between Trx and NADPH in the thioredoxin reductase (TR) -catalyzed reaction (Rea- 2), it has been found that the redox potential of P34H- Trx at pH 7.0 was -235mV compared to -270 mV for the then type (wt) Trx.

TR E> Trx-suz + NADP * (2) Trxz + NADPH + H * The difference in E0 'of 35 mV corresponds to approximately one factor of 20 in Keq for Reaction 3.

Keq (3) TIK-S2 + Pr0tGiH-SH2 1g ==== r> Trx-SH2 + protein-S2 The higher EQ 'value also made P34H-Trx more similar. PDI and contributed to significant changes in the Trx namely improved activity with TR but lower production of protein disulfides. Compared to wt-Trx, P34H Trx-S2 about twice as good as substrate for TR from E.coli and four times as effective with calf thymus TR. One newly developed fluorometric acid allowed direct registration of the reaction between insulin disulfides and Trx-SH2. pH 8 and 15 ° C, second order constant for wild typtioredoxin or 2 x 104 M_1 s-1 and for P34H-Trx of 3 x 103 M'1 s'1 was obtained and another equilibrium was observed which was 10 15 20 25 30 35 468 480 8 consistent with the differences in EQ 'values.

The present invention is generally applicable to production of biologically active disulfide-crosslinked proteins and proteins of interest include insulin, proinsulin, Igf-I Igf-II, t-PA, growth hormone factor VIIIF and coagulation factors, interleucins, pancreatic mammals malieribonucleases, lysozymes, pancreatic mammalian tryps sinin inhibitors, interferons, rennin, prolactin, human a- l-trypsin inhibitor, etc. Common to all of these pro- teiner is the fact, that they in properly pleated form contains disulfide bridges that provide proper con- figuration.

With respect to the side of the invention at which using a selenite in the folding reaction is the ion or cation which was not used in a significant as long as it is inert in relation to the reaction but it is preferred to use alkali metals or alkaline earth metals, wherein sodium and potassium are especially preferred.

The invention will in the following further described in more detail by non-restrictive concrete example. This illustration is made in connection with laid drawings.

Explanation of drawings Figure 1. Kinetics for recovery of RNase activity from completely reduced RNase catalyzed by PDI or thioredoxins in the presence of 100 μM GSSG. RNase, 25 μM, in- was incubated at 37 ° in 100 mM potassium phosphate, pH 7.0, 1 mM EDTA in the absence of thioredoxin or PDI X --- X or in the presence of 10 pM ~ ---- ~ and 100 pM ° ---- ° E.coli w.t. Trx, 10 pM A ---- A and 100 pM A ---- A p34H Trx, and l uM I ---- I and 10 pM D ---- U PDI. Aliquot amounts were removed from the reaction medium. mixtures and were examined for RNase activity with 160 pg / ml 2'3'cCMP in 50 mM Tris-Cl, pH 7.5, 25 mM KCl, 5 mM MgCl 2. Hydrolysis of the nucleotide was followed 10 15 20 25 30 35 468 480 9 spectrophotometrically at 288 nm, and the recovery is calculated from a standard curve for native RNase.

Figure 2. Refolding of fully reduced RNase in the presence of catalytic amount of selenite. The experimental conditions were as in Fig. 1 except for the addition of 1 μM Se032- instead for GSSG: In the absence of PDI or thioredoxin X ---- X, i presence of 10 μM ~ ---- ~ and 100 μM ° ---- ° E.coli w.t. Trx, 10 μM A ---- A and 100 μM A ---- A P34H thioredoxin, and 1 μM I ---- I and 10 μM D ---- D PDI.

Figure 3. Comparison of PDI, w.t. E.coli Trx and P34H Trx when refolding sRNase in the presence of 100 μM GSH. Randomly unoxidized RNase (25 pM) was incubated in 100 mM potassium phosphate pH 7.0, 1 mM EDTA at 37 ° in the absence of thioredoxin or PDI X ---- X and in the presence of 10 pM ~ ---- ~ and 100 pM ° ---- ° E.coli w.t. Trx, 10 pM A ---- A and 100 pM A ---- A P34H Trx, and 1 μM I ---- I and 10 μM D ---- U PDI. Aliquot quantities of was removed and examined for RNase activity as in Fig. 1.

Figure 4. Comparison of PDI, w.t. E.co1i Trx and P34H Trx by refolding sRNase in the presence of 100 pM NADPH and 10 nM bovine thioredoxin reductase. Without thioredoxin or PDI X ---- X or in the presence of 10 μM 0 ---- ~ and 100 μM ° ---- ° E.coli w.t. Trx, 10 pM A ---- A and 100 pM A ---- A P34H thio- redoxin, and 1 μM I ---- I and 10 μM D ---- D PDI. Experimental- the conditions were as in Fig. 1.

Figure 5. Effects of different concentrations of PDI, w.t.

Trx or P34H Trx on recovery of RNase activity from fully reduced RNase after 24 hours incubation at 37 °. 5. air only §. in the presence of 100 μM GSSG and Q. _. The values are averages of 2 to 5 in the near varo of l pM SeO3 different experiments. w.t.

Figure 6. Effects of different concentrations of PDI, 468 480 10 15 20 25 30 '35 10 Trx or P34H Trx on recovery of RNase activity from sRNase after 24 hours incubation at 37 ”. 5. in absence of catalytic thiol §. in the presence of 100 pM GSH and Q. i presence of 10 nM bovine thioredoxin reductase and 100 pM NADPH. The values are averages from 3 different experiments.

EXAMPLE 1 EXPERIMENTAL PROCEDURES.

Material - E.coli strain JF52l (A (lac-proAB), thi. supE, metE46, srl 300:: Tn 10 trxA2 (7004), recA.

(F '(raD36, proAB +, lac ZAM15)) (Langsetmo, K., Fuchs, J., and Woodward, (1989) Biochemistry 28, 3221-3220) phage M13K07, and plasmid pUC118, Vieira, J., and Brass, J. (1987) Methods Enzymol 153, 3-11) were gifts from Dr. J.

Fuchs, St Paul, Mn. The mutagenesis host strain E.coli CU 9276 (hsdR17, merAB, recA1, A (lac-proAB), (F'traD36, proAB +, 1ac1qZAM15)) and the test template and primer for mutagenesis the system of Vandeyar, M.A., Weiner, M.P., Hutton, C.J. and Batt, C.A. (1988) Gene (Amst) 65, 129-133) held by Dr. E. Holmgren, KabiGen, Stockholm. Nucleotide positions in the pUC118-trxA-P34H map under Scheme 2 to Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene (Amst) 33, 103-119).

Restriction and DNA modifying enzymes, M13 vector DNA, 1a-35S] dATP, and other molecular biologicals materials were purchased from Boehringer Mannheim, Pharmacia LKB Biotechnology Inc., and Amersham Corp. For elution of DNA from agarose, the GeneCl1ean kit of Bio101 was used. DNA sequencing equipment and T7 DNA polymerase was from Pharmacia. 5-Bromo-4-chloro-3-indoyl-β-D-galactoside and isopropyl-1-thio- ß-D-galactopyranoside, DTNB, and DTT were purchased from Sigma.

Wild-type thioredoxin purified from E.coli SK3981 (Dyson, H.J., Holmgren, A. and Wright, P.E. (1989) Bio- chemistry 28, 7074-7087), PDI from calf liver (Hillson, D.A., Lambert, N. and Freedman, R.B. (1984) Methods Enzymol. 107, 281-294, Lundström, J. and Holmgren, A. (1990) J.Biol.Chem. 265, 9114-9120), and thioredoxin ”L 10 15 20 25 30 35 11 reduced from E. coli BH 215 / pMR13 (Russel, M. and Model, P. (1985) J.Bacterio1. 163, 238-242) and calf thymus (Luthman, M. and Holmgren, A. (1982) Biochemistry 21, 6628-6633) homogeneous preparations were available in the spring laboratory.

Mutant construct - A 0.5-kilobase HindII fragment containing the trxA gene was transferred from pBHK8 (Wallace, B.J. and Kushner, S.R. (1984) Gene (Amst) 32, 399-408) to M13 mp18 using the SmaI site of its polylinker region. Ml3 clones with the right insert orientation was identified by dideoxy sequencing.

A 19-mer oligonucleotide with the sequence 5 'CAT TTT GLA x GLA CTG ACC GCA C 3 'was synthesized by Dr. Sune Kvist, Ludwig Institute for Cancer Research, Stockholm branch, and fused to M13 mp 18-trxA (Scheme 2). Details be- affecting the oligonucleotide-directed mutagenesis according to Vandeyar, M.A., Weiner, M.P., Hutton, C.J. and Batt, C.A. (1988) Gene (Amst) 65, 129-133 and screening with respect to mutant directly by sequencing, as well as subcloning procedures resulting from the expression plasmid pUC118 trxA-P34H was as previously described (Krause, G. and Holmgren, A. (1991) J.Biol.Chem. 266, 4056-4066). Simple- stranded DNA obtained from the hybrid phagemid present in Scheme 2 served as a template for another sequence confirmation to exclude secondary mutations. Over-express mutation thioredoxin in JF521, a trxA background, and Purification of the mutant protein was performed as previously reported. terats (Krause, G. and Holmgren, A. (1991) J.Biol.Chem. 266, 4056-4066).

For comparison, Scheme 1 shows the domain structure of liver PDI and active site homology to thioredoxin from E.co1i.

EXAMPLE 2 EFFICIENT FOLDING CATALYLE WITH P34H TRX AND A NEW OXIDIZATION SYSTEM USING CATALYTIC QUANTITIES OF SELENITE IN AIR. 468 480 10 15 20 25 30 35 12 Results Oxidative folding_of reduced RNase Fully reduced inactive RNase with 8 sulfhydryl groups was used as a substrate in refolding experiments at a concentration of 25 pM. These conditions were used previously by Pigiet and Schuster, 1986, who found that 500 μM E.co1i Trx gave maximum reactivation of RNase activity after 30- 50 hours incubation. A concentration of 25 μM is also 3- 5 times higher than the apparent Km value of RNase for PDI as recently reported by Hawkins et al., 1991, and Lyles and Gilbert, 1991. Incubations were performed at 37 ° C at pH 7.0 in the presence of 1 mM EDTA in air. During these no significant recovery was observed RNase activity after 6 hours of incubation with 1 or 10 μM PDI, 1, 10 or 100 pM wt Trx or 1 or 10 pM P34H Trx, all added in its oxidized state (data not shown). With 100 pM P34H Trx, significant re- activation (15%). It has previously been shown that a disulfide low molecular weight required for reactivation: Creighton and Freedman, 1980, used 100 pM GSSG as an oxidant at their RNase refinement studies. As shown in Fig. 1 with 100 μM GSSG, recovery of RNase activity was now essential. when the reactions were monitored for 6 hours. Two concluded looks can be deduced from this data. First, the PDI was it best catalyst and was about 100-fold more active than wt Trx on a molar basis. Secondly and most importantly was P34H Trx more active than wt Trx; in fact, 10 appeared pM P34H Trx be more active than 100 pM wt Trx. The relationship between PDI and P34H Trx was also a factor of 10 in activity on molbas. The yield of Pro-34 to His in Trx thus increased disulfide isomerase activity very dramatically.

Experiment using a non-disulfide oxidant led to the discovery that selenite is useful. As As can be seen from Fig. 2, sodium selenite acts at a lytic constellation of 1 μM well as oxidant for PDI also as for thioredoxins; the final yield at 6 hours was similar to the experiments in the presence of 100 pM GSSG. See- * r 10 15 20 25 30 35 480 13 lenite is an effective oxidant of active site dithiol in thio- redoxin and promotes non-stochiometric oxidation by redox cycling with oxygen in air (Holmgren and Kumar, 1989; Kumar, Björnstedt, Holmgren, manuscript under preparation ning). The use of selenite is thus a way of avoid adding a redox buffer to the system. The the relative efficiency of P34H Trx was again equal to or greater than that of 100 pM wt Trx. Even in this system Thus, P34H Trx was about ten-fold more effective than redoxin.

Folding of scramble RNase (sRNase) Recovery of RNase activity from the inactive, the randomly oxidized molecule requires isomerization of di- sulfide bonds by thiol disulfide exchange; This means reduction and oxidation cycles. The process thus requires a thiol, such as GSH or DTT, to initiate reduction (Creighton and Freedman, 1980). We compared PDI, wt Trx and P34H Trx using 100 pM GSH as thiol. Un- where these conditions (Fig. 3) showed wt Trx almost no effect on activity recovery at either 10_ or 100 pM compared to a control without enzyme. Contrary in addition, P34H Trx gave significant activity at 10 μM. The however, the fastest activation was obtained with 1 or 10 pM PDI. Thus, with 100 μM GSH, P34H Trx was more active than wt Trx; this is probably an expression of the higher redox the potential (Eo ') of P34H Trx. It is known that wt Trx S not reduced by 100 pM GSH (unpublished results). 2 Because PDI is a substrate for mammalian thio doxin reductase, we have tried to use NADPH and thiore- doxin reductase as a source of reducing equivalence for thiol disulfide yield. As shown in Fig. 4, 100 operated pM NADPH and a very low concentration of thioredoxin reduced well. In theory, 100 pM NADPH is enough for complete reduction of 25 μM sRNase with 4 disulfides.

With 10 nM thioredoxin, efficient recovery of ribo- nuclease activity. The recovery of the activity was rapid 468 48Û 10 15 20 25 30 35 14 bare and generally higher than with 100 pM GSH. Under these conditions, PDI was the best catalyst accompanied by P34H Trx. The relationship between wt Trx and P34H Trx was also one here factor of 10, as illustrated by the similar activation none with 100 pM Trx and 10 μM P34H (Fig. 4). With all of them these three proteins thus function as NADPH and thioredoxin reduced as an excellent source of reducing equivalence for the disulfide exchange. Oxygen in air then consumes NADPH, but this procedure has not been studied. These results show even though there is no mandatory requirement for gluten or other small disulfide, such as cystamine, in tea disulfide isomerization reactions.

Final replacement of ribonuclease activity In all comparisons, a simple batch of full was used reduced or sRNase. However, different theorems showed different final recovery of activity varying between 60 and 95%. This has been noted by others (see Hawkins and Freedman, 1991). In a separate series of experiments were used several preparations and we decided on the final replacement of active ribonuclease by removal of samples at 2 hours (data not shown) and 24 hours. With reduced RNase only resulted in exposure to air below 24 hours of high activity recovery, especially for 100 pM P34H Trx and 10 pM PDI. It is of special importance that long incubations with 1 μM selenite are generally resulted in better activity recovery than with GSSG.

P34H Trx and selenite thus constitute an excellent system for a fully reduced protein.

Using similar incubations with sRNase (Fig. 6) essentially no recovery of activity was obtained without the addition of a reductant. The addition of PDI low but significant recovery of activity, possible due to the fact that because isolated PDI contains about one free sulfhydryl group (Lyles and Gilbert, 1991). Using 100 pM GSH was significantly recovered activity without enzyme and low concentrations of wt Trx f) 10 15 20 25 30 35 .P- O"\ OO -PI- OD CD 15 showed no effect compared to P34H Trx and PDI.

Yields after 24 hours using 100 μM NADPH and 10 nM TR showed a gradual increase in relative activity in the order wt Trx, P34H Trx and PDI.

Conclusions Pro-34 to His in thioredoxin is an effective pro- tea disulfide isomerase and catalyzes protein folding and formation of native disulfide. The protein can be obtained in homogeneous gene form in large quantities from strain JF521 / pUCll8 trxA P34H and is a useful easily accessible very stable and suitable catalyst.

Selenite is used aerobically in catalytic amounts as well 1 pM can replace previously used redox buffers of exemplary pelvis GSH and GSSG as an oxidant during refolding and formation of disulfides in proteins catalyzed by PDI or P34H Trx. Sodium selenite and other selenium derivatives are cheap and easily accessible. As a reductant for induction of the thiol disulfide yield involved in disulfide isomerization a low level of thioredoxin reductase and NADPH works well.

E.coli strain JF 521, containing the expression vector pUC118-trxA-P34H, has been deposited with the DSM May 24, 1991 with deposit number 6528.

Claims (16)

468 480 10 15 20 25 30 35 16 PATENT REQUIREMENTS Thioredoxin comprising a redox-active Cys32-X-Pro34-Cys35 site (E. coli numbering) or a functional derivative thereof, wherein the amino acid residue Pro in the 34-position has been replaced by His, and wherein the amino acid residue X in 33 - position is selected from Gly and Ala. Thioredoxin according to claim 1, wherein the 33-position is occupied by the amino acid residue Gly. Thioredoxin according to claim 1 or 2 obtained from a naturally occurring thioredoxin by site-directed mutagenesis. A process for the in vitro production of a folded, biologically active, disulfide-linked protein, characterized in that reacting a corresponding unfolded and reduced protein with a thioredoxin or functional derivative thereof, wherein Pro in the 34-position has is added with His, and wherein the amino acid residue in the 33-position is selected from Gly and Ala, resulting in the formation of native disulfide. The method of claim 3, wherein the 33 position is occupied by the amino acid residue Gly. 6. A process according to claim 4 or 5, characterized in that said protein is reacted in the presence of a catalytic amount of a selenite. The method of claim 6, wherein said selenite is used at a concentration of from about 0.1 pM to about 100 pM, especially from about 0.5 to about 10 pM. A method according to claim 4 or 5, characterized in that said protein is reacted in the presence of a redox buffer, such as a buffer of reduced and oxidized glutathione, β-mercaptoethanol or dithiothreitol. 9. A process for the in vitro preparation of a pleated, disulfide-linked protein, characterized in that an incorrectly plated protein is reacted with a thioredoxin or functional derivative thereof, wherein Pro in the 34-position is replaced by His, and wherein amino acid residue in the 33-position is selected from Gly and Ala in the presence of a reductant or a redox buffer, such as a buffer of reduced and oxidized glutathione, β-mercaptoethanol or dithiothreitol, resulting in the formation of native disulfide. The method of claim 9, wherein the 33 position is occupied by the amino acid residue Gly. 11. A process according to claim 9 or 10, characterized in that said protein is reacted in the presence of a catalytic amount of a selenite. The method of claim 11, wherein said selenite is used at a concentration of from about 0.1 μM to about 100 pM, especially from about 0.5 to about 10 pM. 13. A process for the in vivo production of a pleated, disulfide-linked protein, characterized in that a corresponding unfolded and reduced protein is reacted with thioredoxin, a thioredoxin, wherein Pro in the 34-position is replaced by His, and wherein amino acid the residue in the 33-position is selected from Gly and Ala, or a functional derivative thereof, or PDI in the presence of a catalytic amount of a selenite, resulting in the formation of native disulfide. 14. A process for the in vitro production of a pleated, disulfide-linked protein, characterized in that an incorrectly plated protein is reacted with a thioredoxin, a thioredoxin, wherein Pro in the 34-position is replaced by His, and wherein the amino acid residue in the 33-position is selected from Gly and Ala, or a functional derivative thereof, or PDI in the presence of NADPH and a thioredoxin reductase and a catalytic amount of a selenite, resulting in the formation of native disulfide. A method according to claim 13 or 14, wherein said selenite is used at a concentration of from about 0.1 μM to about 100 pM, especially from about 0.5 to about 10 pM. 10 15 20 25 30 35 468 480 18 A method according to claim 13, 14 or 15, wherein the 22-position is occupied by the amino acid residue Gly. 10 15 20 25 30 35 $> Ch CD / 9 REFERENCES Anfinsen, C.B. (1937) Science 181, 223-230 Creighton, T.E. (1990) Biochem.J. 270, 1-16. Creighton, T.E., Hillson, D.A. and Freedman, R.B. (1980) J.Mol.Bio1. 142, 43-62. Dyson, H.J., Gippert, G.P., Case, D.E., Holmgren, Wright, P.E. (1990) Biochemistry 29, 4129-4136. Edman, J.C., Ellis, L., Blacher, R.W., Roth, R.A. Rutter, W.J. (1985) Nature 317, 267-270. Eklund, H., Gleason, F.K. and Holmgren, A. (1991) Proteins, in press. Hawkins, H.C. and Freedman, R.B. (1991) Biochem.J. 275, 335-339. Holmgren, A. (1984) Methods Enzymol. 107, 295-300. Holmgren, A. (1985) Annu.Rev.Biochem. 54, 237-271. Holmgren, A. (1989) J.Biol.Chem. 264, 13963-13966. Holmgren, A and Kumar, S., Proceedings of the 4th International Symposium on Selenium in Biology and Medicine. Oath. A. Wendels et al., Springer-Verlag, Berlin 1989, 58- 62. Jaenicke, R. (1991) Biochemistry 30, 3147-3161. Lundström, J. and Holmgren, A. (1990) J.Biol.Chem. 265, 9114-9120. Lyles, M.M. and Gilbert, H.F. (1991) Biochemistry 30, 613-619. Pigiet, V.P. and Schuster, B.J. (1986) Proc.Natl.Acad.Sci. USA 83, 7643-7647. A. and and f »20 468 4% scHEMA1 PDI 37 388 (v) PhäïaIGIuPhcTyrMaProTz-pcys fi lyniscyny lnfauklaProïle Domain structure of liver PDI and active-slre homology to thioredoxin from E.coli. In the domain definition and sequence delineation according to Edman et al. (1985) the squares symbolize regions of more than 30% internal sequence homology. The amino acid sequences in the dashed regions (in a and a ') containing the redox active disulfides / dithiols are compared with the corresponding part of E. coli thioredoxin. f) i I É * r fl ï- ”fffï 02 / SCHEME 2 Ä: (2783) Cfr1OI (2255) B: az as: ng wrp ey: sly ars cys nya ua: coon rrx-Pa4a aktíu: ica 5 'TGG TGC GGT CLQ TGC AAA ATG 3 '1220 1210 1200 TTT TAC 5' 350 puciia - ;;; ¿-Psc fl + string- _ map positions mutagenesis primer- 3 'cacc _ _w __' sequencing positions CCÅ GIQ ÅCG 360 Construction of the P34H mutant by E.coli thioredoxin. A physical map of the expression vector pUC118-trxA-P34H. Expression of the mutant thioredoxin was obtained after induction of the lac promoter. The insert orientation is identical to the M13 template used for site-directed mutagenesis. The binding regions of the universal sequencing primer (S) and the mutagenesis primer (M) are indicated. In B, the amino acid and nucleotide sequences of the active site region of E. coli thioredoxin modified at residue 34. The underlined nucleotide positions differ from the wild-type gene.