The C-terminal region controls correct folding of genus Trametes pyranose 2-oxidases

Journal of Biotechnology 130 (2007) 229–235

The C-terminal region controls correct folding of genus Trametes pyranose 2-oxidases Helena Mareˇsov´a, Andrea Palyzov´a, Pavel Kysl´ık ∗ Laboratory of Enzyme Technology, Institute of Microbiology v.v.i., Academy of Sciences of the Czech Republic, V´ıdeˇnsk´a 1083, 142 20 Prague, Czech Republic Received 24 January 2007; received in revised form 13 April 2007; accepted 24 April 2007

Abstract The pyranose 2-oxidases from Trametes ochracea and Trametes pubescens share markedly similar amino acid sequences with identity of 93.4%. When expressed from the recombinant plasmids based on the same vector in the Escherichia coli host strain BL21(DE3) at higher growth temperatures, they differ strikingly in the formation of the inclusion bodies. Upon overexpression in the cultures performed at 28 ◦ C, the specific activity of pyranose 2-oxidase from T. pubescens was eight times higher than that from T. ochracea: 93% of pyranose 2-oxidase from T. ochracea and only 15% of that from T. pubescens was present in the form of inclusion bodies. To ascertain the cause of this difference, both cloned genes were shuffled. Site-directed recombination of p2o cDNAs revealed that DNA constructs ending with 3 end of p2o cDNA from T. pubescens code for proteins that are folded into an active form to the greater extent, regardless of the gene expression level. “In silicio” analysis of physico-chemical properties of the protein sequences of pyranose 2-oxidases revealed that the sequence of amino acid residues 368–430, constituting the small, head domain of pyranose 2-oxidase from T. pubescens, affects positively the enzyme folding at higher cultivation temperatures. The domain differs in six amino acid residues from that of T. ochracea. © 2007 Elsevier B.V. All rights reserved. Keywords: Pyranose 2-oxidase; Recombinant protein; Inclusion body; Site-directed recombination

1. Introduction Bacterial inclusion bodies (IBs) are refractile aggregates of misfolded proteins. Their formation occurs frequently in recombinant bacteria upon overexpression of cloned genes. In biotechnology, the formation of IBs represents a major obstacle in the production of recombinant proteins in a soluble, functional form. Considerable attention is being directed toward this problem as is evident from an enormous volume of published data (Schlieker et al., 2002; Carri´o and Villaverde, 2002; Baneyx and Mujacic, 2004). The main factors that contribute to the aggregation of recombinant proteins are: (1) limiting amount of chaperones when recombinant gene expression is set up to high levels (Rinas and Bailey, 1993; Lorimer, 1996) and (2) environmental conditions such as high temperature of cultivation, medium acidification, etc. (Chalmers et al., 1990;

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0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.04.023

Strandberg and Enfors, 1991). The following successful ways of increasing proper folding of recombinant proteins have been described: (1) chaperone-assisted folding of an over-expressed enzyme (Sareen et al., 2001), (2) optimization of a medium composition and of overall control of a culture- and gene-expression conditions, (3) fusions of cloned genes to nucleotide sequences coding for soluble protein domains such as staphylococcal protein A (Samuelsson et al., 1994) or thioredoxin (LaVallie et al., 1993) and (4) methods of protein engineering used to remove hydrophobic amino acid residues (Murby et al., 1995; Melissard and Berger, 2001; Hoffmann et al., 2004). Pyranose 2-oxidase (glucose 2-oxidase, pyranose: oxygen 2oxidoreductase, EC 1.1.3.10; P2O) catalyzes the oxidation of carbohydrates on carbon-2 in the presence of oxygen to yield corresponding 2-keto derivatives and hydrogen peroxide. This enzyme is widespread among lignin-degrading white-rot fungi and it has been hypothesized that its physiological role is to provide the lignin peroxidases with hydrogen peroxide (Daniel et al., 1994). In addition, P2O is involved in a secondary metabolite pathway leading from d-glucose via 2-keto-d-glucose to the

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␤-pyrone-antibiotic cortalcerone (Baute et al., 1987). The high potential of P2O application in bioprocesses, clinical chemistry analytics and in synthetic carbohydrate chemistry has recently been reviewed (Giffhorn, 2000). In spite of the application potential, no data have been published so far on the construction of a high-expression system for P2O. The data on P2O engineering are scarce. P2O from Coriolus versicolor was subject to random mutagenesis and a single amino acid substitution Lys for Glu at position 542 generated an enzyme with higher thermal stability and lower Km values for d-glucose and 1,5-anhydro-d-glucitol (Masuda-Nishimura et al., 1999). P2O from Peniophora gigantea was also engineered to improve the thermo- and pH-stability by molecular enzyme evolution. Replacement of Lys-312 by Glu improved catalytic efficiency of the enzyme (Bastian et al., 2005). Protein engineering of P2O from Trametes ochracea (Kujawa et al., 2006) yielded the data on the structural basis for the substrate binding and enzyme regioselectivity. We have described previously (Veˇcerek et al., 2004; Mareˇsov´a et al., 2005) that the recombinant P2Os from two basidiomycetes, T. ochracea and T. pubescens share 93.4% identical the amino acid sequences. The two enzymes are known to differ in formation of inclusion bodies when expressed in recombinant cultures at the growth temperature of 28 ◦ C. This temperature is convenient for the industrial processes but it may frequently be non-permissive for the proper folding of recombinant proteins. To ascertain the cause of the difference in formation of inclusion bodies, the cDNAs of the two P2Os of genus Trametes were shuffled. A site-directed recombination experiment and computational analysis of physico-chemical parameters of protein domains of the two related P2Os make it possible to identify the enzyme domain responsible for aggregation into IBs. 2. Materials and methods 2.1. Plasmids and strains Host strains of Escherichia coli used in experiments: BL21/DE3 (Invitrogen, Carlsbad, CA, USA), TOP10 (Invitrogen) and K12-W3110 (␭− , F− ) (Bachmann, 1972) containing plasmid pRQ5 (based on the vector pSE420, Invitrogen) bearing cDNA encoding P2O from basidiomycete T. pubescens (Mareˇsov´a et al., 2005) or the plasmid pSE33 (based on the vector pSE420) bearing cDNA encoding P2O from basidiomycete T. ochracea (synonymum multicolor) Veˇcerek et al., 2004). 2.2. Growth conditions E. coli hosts were routinely cultured at 37 ◦ C in LB medium (Sambrook et al., 1989). If required, the medium was supplemented with agar (15 g/l) and ampicillin (150 ␮g/ml). To evaluate the production of P2O, the recombinant strains were cultured in 500 ml flasks with 100 ml of LB or M9 medium (in g/l: Na2 HPO4 ·12H2 O 14.6, KH2 PO4 3, NaCl 0.5, NH4 Cl 1, MgSO4 ·7H2 O 0.12, pH 7.2) containing casein hydrolysate NZ-Amine A (Quest International, Norwich, NY, USA) 10 g/l and

glycerol 10 g/l (MCHGly medium). Flask cultures were grown for 24 h at 25 or 28 ◦ C on an orbital shaker (200 rpm). To induce the expression, IPTG was added to exponentially growing culture at OD600 of 0.6 at final concentration of 0.02 mM or 1 mM. One millililiter of the culture grown in LB medium supplemented with antibiotics for 10 h at 28 ◦ C was used to inoculate the production culture. 2.3. DNA and RNA manipulation techniques Recombinant DNA techniques including agarose gel electrophoresis, digestion of DNA with restriction endonucleases, ligation with T4 DNA ligase (Gibco BRL, Inchinnan, Scotland), transformation of competent cells with plasmid DNA, and smallscale plasmid DNA preparations were performed by standard procedures (Sambrook et al., 1989). 2.4. Construction of chimeric plasmids DNAs of plasmids pSE33 and pRQ5 were cut with the enzyme HindIII at the same restriction sites located 395 bps behind ATG codon and 181 bps behind the stop codon for the plasmid pSE33 and 180 bps for pRQ5. The cut-out fragments were crosswise exchanged between open plasmids so that the p2o start region in the plasmid pSE33 was ligated to the terminator-adjacent region of p2o cloned on pRQ5 (construct pSE:pR) and “vice versa” (construct pRQ:pS). The same host strain E. coli BL21/DE3 was transformed with each construct to get recombinant strains. 2.5. Detection of P2O-positive clones Grown up colonies bearing chimeric plasmids (solid medium MCHGly supplemented with inducer IPTG) were transferred on membrane filter. The filter was placed in a closed jar and exposed to toluene vapours for 5 min. The filter with permeabilized cells was removed and over-laid with detection solution used for qualitative assay of P2O (100 mM phosphate buffer, pH 6.5, 100 mM ABTS, 100 mM d-glucose and peroxidase) (3 U in 1 ml of detection solution). After 15 min, violet halo forms around colonies producing P2O. 2.6. Enzyme assay and quantitative densitometry of protein bands Biomass from 1 ml of the culture was separated by centrifugation and kept at −20 ◦ C. Frozen biomass was re-suspended in 500 ␮l of sodium phosphate buffer (0.1 M, pH 6.5) and ultrasonicated three times for 30 s (samples were kept on ice during disintegration). Cell debris was pelleted by centrifugation and the supernatant used for the assay of activity. The activity of P2O was determined using chromogenic substrate ABTS (Danneel et al., 1993). The activity of 1 U is defined as the amount of P2O required for the oxidation of 2 ␮mol of ABTS/min. The specific activity of P2O is expressed in U/mg of the total soluble protein of the cell. Protein concentration was determined by means of BCA Protein assay

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kit (Pierce, Rockford, IL, USA) using bovine serum albumin as a standard. The density of bands of soluble proteins and inclusion bodies in sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) was evaluated using quantitative densitometry and image analysis according to Veˇcerek et al. (2004). 3. Results 3.1. Induction of synthesis of P2O Recombinant strains of E. coli BL21/DE3(pRQ5) and BL21/DE3(pSE33) were grown in MCHGly or LB medium in the presence of inducer at 25 and 28 ◦ C. In extracts prepared from cells grown in MCHGly medium, the specific activity of the strain BL21/DE3(pSE33) was inversely related to temperature (Table 1). After induction with 1mM IPTG at 25 ◦ C, the specific activity of P2O was six times higher than that at 28 ◦ C and soluble, active enzyme represented about 11% of the total soluble cell protein. SDS-PAGE analysis showed that the cells of the strain BL21/DE3(pSE33) grown at 28 ◦ C contained a large amount of the enzyme in the form of IBs (Fig. 1A). Using quantitative densitometry we were able to determine the proportion of P2O: the amount of IBs was 93% of the total amount of P2O. Induction with 0.02 mM IPTG at 25 ◦ C resulted in expression level about two times higher than that at 28 ◦ C. At the latter temperature, inclusion bodies represented 28% of the total amount of P2O. Following induction with 1 mM IPTG, the activity of T. pubescens P2O in E. coli BL21/DE3(pRQ5) grown at 25 ◦ C was similar to that of BL21/DE3(pSE33). At 28 ◦ C, the specific activity was higher by about 25% than at 25 ◦ C and 15% of the enzyme was in the form of IBs (Fig. 1B). Active enzyme represented 16% of soluble proteins. Compared to the MCHGly medium, the expression level of P2O in cultures of both strains in the LB medium with or without IPTG was markedly lower: a value of specific activ-

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ity of P2O synthesized from plasmid pSE33 without induction was 0.009 and 0.022 U/mg, respectively, on induction at 28 ◦ C. Under the latter conditions, 30% of the enzyme formed inclusion bodies. The specific activity of P2O from plasmid pRQ5 without induction was 0.27 and 0.57 U/mg, respectively, in the presense of 1 mM IPTG at 28 ◦ C. Only 7% of P2O formed inclusion bodies in the medium with an inducer (data not given). To exclude the possibility that the different extent of IBs formation is specific for the host BL21(DE3), we have also transformed other E. coli hosts, namely the strains TOP10 and W3110 with the recombinant plasmids pSE33 and pRQ5. The expression of P2Os was followed in minimal medium at the same growth conditions (Table 2). We found the enzyme expression from plasmid pSE33 to be low in both hosts, while that from the plasmid pRQ5 was much higher. 3.2. Site directed recombination The results of the above experiment suggest that the formation of IBs of P2O is not affected by the host strain. Formation of inactive enzyme upon expression from the p2o cDNA of T. ochracea could be rather explained at the level of the nucleotide sequence. Both cDNAs share 93.4% identity and differ in 39 amino acids. We decided to shuffle genes by a simple site directed recombination and to follow inclusion bodies formation of P2Os expressed from chimeric DNAs. Both p2o cDNA genes were cut with the restriction enzyme HindIII, specific fragments of DNA were exchanged and the host BL21/DE3 was transformed with each chimeric construct. We isolated 10 positive clones bearing each type of chimeric plasmid and cultivated them in medium MCHGly with or without 1 mM IPTG at 28 ◦ C. The specific activity of P2O and the amount of inclusion bodies determined in the culture samples are shown in Table 3. The data suggest that the N-terminal sequence of 131 amino acids does not positively affect the process of protein folding: pRQ:pS construct has a higher content of IBs than the wild-type plasmid pRQ5.

Table 1 Effects of temperature and IPTG concentration on expression of recombinant P2Os in the host strain Escherichia coli BL21/DE3 Plasmid

Temperature (◦ C) 25

pSE33 28

25 pRQ5 28

IPTG (mM)

Specific activity of P2O (U/mg)

Total P2O (%)

Soluble P2O (%)

Inclusion bodies (%)

0 0.02 1 0 0.02 1

0.41 0.75 1.30 0.29 0.41 0.22

± ± ± ± ± ±

0.07 0.04 0.31 0.05 0.03 0.04

3.1 13.8 14.2 3.1 6.2 39.5

2.2 10.4 10.5 1.6 4.5 2.9

22 13 32 29 28 93

0 0.02 1 0 0.02 1

0.47 1.2 1.42 0.78 1.65 1.78

± ± ± ± ± ±

0.08 0.3 0.3 0.3 0.1 0.22

2.6 6.4 7.8 6.3 14.4 18.5

2.2 5.6 7.3 5.6 12.5 15.5

17 12 6 11 13 15

The cell extracts were prepared from flask cultures grown in MCHGly medium with or without inducer (IPTG) for 20 h at 25 or 28 ◦ C. The activity of P2O was determined from five independent flask cultures. The values of soluble P2O and inclusion bodies were determined by image analysis of SDS gels. Total P2O (a sum of soluble P2O and inclusion bodies) is expressed as a percentage of total soluble protein. Soluble P2O is expressed as a percentage of total soluble protein. Inclusion bodies are expressed as a percentage of the total amount of P2O.

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Fig. 1. SDS-PAGE electrophoresis of supernatants (S) and cell pellets (P) of samples of cultures of the strain BL21/DE3 harbouring plasmid pSE33 (A) and pRQ5 (B) grown in MCHGly medium supplemented with 1 mM IPTG at 28 ◦ C.

3.3. Physico-chemical analysis of amino acid sequence of the P2O The site-directed recombination experiment revealed that active P2O is formed from the protein having the C-end of P2O from T. pubescens with 32 differences in the sequence of amino acids. It was concluded from the crystal structure of P2O from T. ochracea that amino acid residues 368–430 constitute a small, head domain (Hallberg et al., 2004) that protrudes from the surface of the central body and forms the ␣␤␤␣ motif. It is Table 2 Production of heterologous P2Os by cultures of different E. coli hosts transformed with plasmid pSE33 or pRQ5 Strain

Plasmid

Specific activity of P2O (U/mg) −IPTG

+IPTG

TOP10 W3110 BL21/DE3

pSE33

0.06 ± 0.02 0.10 ± 0.02 0.18 ± 0.06

0.19 ± 0.04 0.01 ± 0.005 0.24 ± 0.06

TOP10 W3110 BL21/DE3

pRQ5

0.30 ± 0.05 0.78 ± 0.07 0.77 ± 0.15

1.06 ± 0.12 1.23 ± 0.14 1.92 ± 0.28

Flask cultures were grown in MCHGly medium with or without IPTG (1 mM) at 28 ◦ C. Three parallel cultures were performed with each strain.

hypothesized that this head domain can be involved in oligomerization of subunits of P2O. The exposed surface of this domain contains six Thr and two Ser residues all of which can be target sites for post-translational modifications. In the amino acid sequence of P2O from T. pubescens, only two Thr (position 381 and 401) were found with the remaining four residues being replaced by Met, Ser, Gly and Asn residues. To analyse consequences of amino acid sequence on the traits of protein, we used the ProtParam program (Gasteiger et al., 2003). The following computed parameters are shown in Table 4: instability index, aliphatic index and hydropathicity of a protein. The determination of a hydropathicity was performed using the procedure based on a scale of Kyte and Doolittle (1982). The results indicate that there are differences between the two enzymes regarding the instability index for the small domain (region between 368 and 430 AA): instability indexes of 27.13 and 43.77 were computed for T. ochracea and T. pubescens, respectively. The values of the aliphatic index are similar and there is no significant difference even when computed from the whole sequences of T. ochracea and T. pubescens. Once again, there is a difference of the aliphatic index for the small domain alone: 58.73 for T. pubescens and 52.54 for T. ochracea. Values of the protein hydropathicity suggest another difference between P2Os: the enzyme from T. ochracea is more hydrophobic than that from T. pubescens. This is true especially for the

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Table 3 Inclusion body formation and specific activity of P2O in strain E. coli BL21/DE3 harbouring chimeric plasmid constructs Chimeric plasmid

pSE:pR pRQ:pS

Specific activity of P2O (U/mg)

Total P2O (%)

Soluble P2O (%)

Inclusion bodies (%)

−IPTG

+IPTG

+IPTG

+IPTG

+IPTG

0.44 ± 0.05 0.41 ± 0.12

1.23 ± 0.21 0.54 ± 0.15

21 27

16 6.5

23 83

The specific activity of P2O was determined in cultures of 10 clones of each recombinant strain. Flask cultures were grown in MCHGly medium with or without IPTG (1 mM) at 28 ◦ C. The values of soluble P2O and inclusion bodies were determined by image analysis of SDS gels. Total P2O (a sum of soluble P2O and inclusion bodies) is expressed as a percentage of total soluble protein. Soluble P2O is expressed as a percentage of total soluble protein. Inclusion bodies are expressed as a percentage of the total amount of P2O.

small domain. For chimeric molecules, hydropathicity of P2O expressed from the plasmid pSE:pR is close to that determined for P2O from T. pubescens. The same is true for chimeric P2O expressed from plasmid pRQ:pS. We computed the same physico-chemical parameters also for other available amino acid sequences of pyranose 2-oxidases from the strains: C. versicolor, T. hirsuta, P. gigantea and Peniophora sp. SG. These strains share 80.4–93.4% amino acid identity with T. pubescens. The instability indexes of the whole proteins are similar (ranging from 41.90 to 43.73) and this group of proteins can be termed as an unstable one. Aliphatic indexes ranged from 69.33 to 73.35 and hydropathicity from −0.393 to −0.438. The values of these parameters for the small, head domain of all these enzymes are comparable with the values calculated for the small domain of T. ochracea. The instability index of the small, head domain ranged between 18.9 and 28.14, aliphatic index between 52.54 and 58.73 and hydropathicity from −0.819 to −0.898. 4. Discussion 4.1. Expression of the two related P2Os In this study, we focused on the problem of formation of IBs of two related recombinant P2Os of the genus Trametes that are expressed in E. coli host under identical conditions of cultivation and the state of “enzyme overexpression”. This experimental set up makes it possible to compare the process of folding of two structurally close proteins and to verify experimentally predicted role of enzyme domains in the process of folding. We found that in different host strains of E. coli (TOP10, W3110 and BL21/DE3) the process of formation of inclusion bodies of two related P2Os has a similar trend: P2O

from T. ochracea forms more inclusion bodies in all hosts at the cultivation temperature of 28 ◦ C than P2O from T. pubescens. We concluded, therefore, that the formation of IBs of the two P2Os is not a specific trait of the host strain BL21/DE3. The effect of the state of “enzyme overexpression” on the formation of IBs was studied in the defined MCHGly medium at 25 and 28 ◦ C. The higher formation of inclusion bodies in T. ochracea in comparison to T. pubescens was observed regardless of the level of P2O expression that varied in the order of 10 (Table 1). 4.2. Effect of the primary structure of P2O on protein folding We decided to exploit the sequence alignment of both DNAs to construct the chimeric cDNA and to identify the protein domain taking part in the formation of active enzyme. A sitedirected recombination experiment confirmed the hypothetical predictions that formation of IBs is controlled by the primary structure of the specific domain of P2O. The P2O is a homotetrameric protein with a molecular mass of the subunit of 69.34 and 69.21 kDa for T. ochracea and T. pubescens, respectively. Hallberg et al. (2004) concluded from the structure of P2O of T. ochracea that the S sub-domain (residues 80–158) is involved in oligomerization and formation of the tertiary structure of the mature protein. Different amino acids were detected in this domain of P2O at position 120: Lys (T. pubescens) and Thr (T. ochracea). A site-directed recombination performed between p2o cDNA from both basidiomycetes (nucleotides 399–1869/1866) revealed that the 3 end of p2o cDNA from T. pubescens bears the information for “more correct” folding of the protein in cultures grown at 28 ◦ C. Altogether, 32 differences in amino acids were found in this part of the protein. To identify

Table 4 “In silicio” analysis of the properties of amino acid sequence of the wild-type and chimeric P2Os: a whole protein and small, head domain Strain

P2O sequence

Amino acid length

Instability index

Aliphatic index

Hydropathicity

T. ochracea

Whole protein Small domain Chimeric protein pSE:pR

623 Region 368–430 622

44.55 27.13 45.41

69.33 52.54 70.55

−0.415 −0.898 −0.364

T. pubescens

Whole protein Small domain Chimeric protein pRQ:pS

622 Region 368–430 623

45.16 43.77 44.29

70.55 58.73 69.33

−0.379 −0.749 −0.43

Parameters were calculated by the program ProtParam (Expasy proteomic server).

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substitutions responsible for increased solubility of the enzyme from T. pubescens at 28 ◦ C, we focused only on a small, head domain (residues 368–430) that is expected to be also involved in oligomerization. The exposed surface of this small domain of T. ochracea contains six Thr and two Ser residues. In the sequence of T. pubescens, there are only two Thr in positions 381 and 401, the remaining Thr residues being substituted by Met, Ser, Gly and Asn. 4.3. “In silicio” analysis of the properties of amino acid sequences of the small, head domain Idicula-Thomas and Balaji (2005) observed that: (1) the instability index is higher for soluble proteins than for insoluble proteins and (2) both the solubility and the thermostability of a protein increase with increasing value of aliphatic index. The values of aliphatic indexes for small domains of P2Os support this conclusion. The authors suggested that the lifetime of partially folded intermediates affects the propensity of the protein to aggregate for the following reasons: (1) longerlived partially folded intermediates (less-soluble forms of a protein) have higher probability of interaction with other partially folded intermediates and (2) the same partially folded intermediates would titrate out the available chaperones that otherwise prevent protein aggregation by chaperone-protein interaction. It is known that a low temperature has the advantage of slowing down the rate of transcription and translation, and reducing the strength of hydrophobic interactions that contribute to protein misfolding. Determination of small, head domain hydropathicity revealed the more hydrophobic character of this domain of T. ochracea. This trait can play a role in the formation of IBs at a higher temperature. At a lower temperature (25 ◦ C), the formation of IBs is similar for both enzymes. A comparison of physico-chemical parameters for small, head domains calculated for a group of other pyranose 2oxidases published in the literature with those for the enzyme from T. ochracea shows that the values of a given specific parameter are similar. Parameters calculated for the small, head domain of T. pubescens, however, are different. Massive formation of inclusion bodies of a recombinant P2O in cultures of E. coli was reported for pyranose 2-oxidase from C. versicolor (Nishimura et al., 1996) and P. gigantea (Bastian et al., 2005) which supports our conclusions. Another detected substitution of amino acid residue which could be involved in an interaction between subunits is the amino acid residue at position 513 (Hallberg et al., 2004): P2O of T. pubescens has the residue Gln and T. ochracea a more hydrophobic Lys. While we cannot offer any proof, it is our opinion that this single amino acid difference is unlikely to affect the solubility substantially. Murby et al. (1995) studied the effects of substitution of amino acid residue by a hydrophobic one on the solubility of a recombinant protein from a respiratory syncytial virus. Two substitutions Cys/Ser did not significantly change the solubility but multiple engineering of hydrophobic residues increased the solubility and stability dramatically.

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