猪轮状病毒Gottfried株和SB-1A株截短VP8基因的原核表达、蛋白纯化及免疫原性分析

目的原核表达猪轮状病毒(porcine rotavirus,PRV)Gottfried株和SB-1A株截短的VP8*基因,纯化重组蛋白,并检测其免疫原性。方法经PCR从含有全长VP4基因片段的重组质粒pCR4-Gottfried VP4和pCR4-SB-1A VP4中分别扩增截短的VP8*基因(△VP8*,64～223位氨基酸),分别插入原核表达载体pET-28a(+)中,构建重组表达质粒pET-28a-Gottfried-△VP8*和pET-28a-SB-1A-△VP8*,分别转化大肠埃希菌BL21(DE3),IPTG诱导表达,SDS-PAGE和Western blot分析表达产物。应用ProBondTMResin纯化两种重组蛋白,重组蛋白与A(lOH)3佐剂混合后,经肌肉注射免疫BALB/c小鼠,ELISA法检测免疫小鼠的血清抗体水平。结果两种重组表达质粒经双酶切和测序证实构建正确;表达的两种目的蛋白相对分子质量约25 000,均以包涵体和可溶性两种形式存在,均可与鼠源抗Histag单克隆抗体特异性结合;纯化的两种重组蛋白纯度均在95%以上,免疫BALB/c小鼠可产生较高的血清抗体水平。结论成功在大肠埃希菌中表达了PRV Gottfried株和SB-1A株截短的VP8*基因,纯化的两种蛋白免疫原性较好,为进一步研制安全、高效的PRV亚单位疫苗奠定了基础。     

Engineering of the Escherichia coli Im7 immunity protein as a loop display scaffold

Protein scaffolds derived from non-immunoglobulin sources are increasingly being adapted and engineered to provide unique binding molecules with a diverse range of targeting specificities. The ColE7 immunity protein (Im7) from Escherichia coli is potentially one such molecule, as it combines the advantages of (i) small size, (ii) stability conferred by a conserved four anti-parallel alpha-helical framework and (iii) availability of variable surface loops evolved to inactivate members of the DNase family of bacterial toxins, forming one of the tightest known protein-protein interactions. Here we describe initial cloning and protein expression of Im7 and its cognate partner the 15 kDa DNase domain of the colicin E7. Both proteins were produced efficiently in E.coli, and their in vitro binding interactions were validated using ELISA and biosensor. In order to assess the capacity of the Im7 protein to accommodate extensive loop region modifications, we performed extensive molecular modelling and constructed a series of loop graft variants, based on transfer of the extended CDR3 loop from the IgG1b12 antibody, which targets the gp120 antigen from HIV-1. Loop grafting in various configurations resulted in chimeric proteins exhibiting retention of the underlying framework conformation, as measured using far-UV circular dichroism spectroscopy. Importantly, there was low but measurable transfer of antigen-specific affinity. Finally, to validate Im7 as a selectable scaffold for the generation of molecular libraries, we displayed Im7 as a gene 3 fusion protein on the surface of fd bacteriophages, the most common library display format. The fusion was successfully detected using an anti-Im7 rabbit polyclonal antibody, and the recombinant phage specifically recognized the immobilized DNase. Thus, Im7 scaffold is an ideal protein display scaffold for the future generation and for the selection of libraries of novel binding proteins.     

Instability of the NS1 Glycoprotein from La Reunion 2018 Dengue 2 Virus (Cosmopolitan-1 Genotype) in Huh7 Cells Is Due to Lysine Residues on Positions 272 and 324

La Reunion island in the South West Indian Ocean is now endemic for dengue following the introduction of dengue virus serotype 2 (DENV-2) cosmopolitan-I genotype in 2017. DENV-2 infection causes a wide spectrum of clinical manifestations ranging from flu-like disease to severe dengue. The nonstructural glycoprotein 1 (NS1) has been identified as playing a key role in dengue disease severity. The intracellular NS1 exists as a homodimer, whereas a fraction is driven towards the plasma membrane or released as a soluble hexameric protein. Here, we characterized the NS1 glycoproteins from clinical isolates DES-14 and RUN-18 that were collected during the DENV-2 epidemics in Tanzania in 2014 and La Reunion island in 2018, respectively. In relation to hepatotropism of the DENV, expression of recombinant DES-14 NS1 and RUN-18 NS1 glycoproteins was compared in human hepatoma Huh7 cells. We observed that RUN-18 NS1 was poorly stable in Huh7 cells compared to DES-14 NS1. The instability of RUN-18 NS1 leading to a low level of NS1 secretion mostly relates to lysine residues on positions 272 and 324. Our data raise the issue of the consequences of a defect in NS1 stability in human hepatocytes in relation to the major role of NS1 in the pathogenesis of the DENV-2 infection.     

HIV跨膜蛋白gp36和gp41的截短及融合表达

目的将HIV-1的跨膜蛋白gp41和HIV-2跨膜蛋白gp36进行截短,并在大肠杆菌中进行融合表达。方法用PCR将gp41和gp36的编码基因进行截短,回收的PCR产物纯化后克隆到连接载体pGEM-T上,然后用BamHⅠ、EcoRⅠ和SalⅠ切下目的基因,并构建到表达载体pGEX-4T-3,导入宿主细胞BL21,用IPTG诱导表达。结果酶切鉴定显示,截短的HIV-1gp41和HIV-2gp36跨膜蛋白基因大小与预期的一致,表达产物经SDS-PAGE分析显示在相对分子质量66000处出现融合表达条带,Westernblot分析显示,与相应抗体出现特异性反应。结论已成功对gp41和gp36跨膜蛋白进行截短,并构建表达载体进行表达,为跨膜蛋白的进一步应用研究奠定基础。     

Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification

The Strep-tag, an artificial peptide ligand of streptavidin, has gained use as an affinity handle for the purification and detection of recombinant fusion proteins. In an attempt to achieve tighter complexation of the peptide, streptavidin was engineered and the amino acid residues 44-47 in the flexible loop from 44 to 53, which is close to the binding site, were subjected to random mutagenesis. A fusion between alkaline phosphatase and the Strep-tag II sequence, an improved version of the Strep-tag, was constructed as a molecular probe for peptide binding. By means of a filter-sandwich assay, two streptavidin mutants with significantly stronger binding activity for the Strep-tag II were thus identified from a library of Escherichia coli colonies. Both in an ELISA with the alkaline phosphatase fusion and in a fluorescence titration experiment with the synthetic Strep-tag II peptide, which carried an anthraniloyl group as chromophore, their affinities were found to be higher by more than one order of magnitude compared with wild-type streptavidin. The nature of the amino acid exchanges and an enhanced electrophoretic mobility of the streptavidin tetramers suggest an altered loop conformation to be part of the optimized binding mechanism. When one of the streptavidin mutants was immobilized on a chromatographic column it exhibited clearly improved performance in the purification of Strep-tag II fusion proteins, and desthiobiotin turned out to be a suitable reagent for mild competitive elution.      

Processing of N-terminal unnatural amino acids in recombinant human interferon-beta in Escherichia coli

Incorporation of unnatural amino acids into recombinant proteins represents a powerful tool for protein engineering and protein therapeutic development. While the processing of the N-terminal methionine (Met) residues in proteins is well studied, the processing of unnatural amino acids used for replacing the N-terminal Met remains largely unknown. Here we report the effects of the penultimate residue (the residue after the initiator Met) on the processing of two unnatural amino acids, L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG), at the N terminus of recombinant human interferon-beta in E. coli. We have identified specific amino acids at the penultimate position that can be used to efficiently retain or remove N-terminal AHA or HPG. Retention of N-terminal AHA or HPG can be achieved by choosing amino acids with large side chains (such as Gln, Glu, and His) at the penultimate position, while Ala can be selected for the removal of N-terminal AHA or HPG. Incomplete processing of N-terminal AHA and HPG (in terms of both deformylation and cleavage) was observed with Gly or Ser at the penultimate position.     

人细小病毒B19-VP1u保守区外C端氨基酸对sPLA2活性的影响

为探寻人细小病毒B19-VP1u保守区外氨基酸序列对sPLA2活性的影响,依次截短B19-VP1u保守区外C端的氨基酸序列,表达纯化截短突变的蛋白后分别检测其sPLA2活性。结果显示,截短7个氨基酸时,酶活性没有明显变化;截短15个氨基酸时,酶活性显著降低;截短23个氨基酸时,几乎没有酶活性。表明保守区外C端的第8～24个氨基酸之间的序列对酶活性有显著影响,推测其在维持酶的正确三维结构中起重要作用。     

Lyophyllin,a Mushroom Protein from the Peptidase M35 Superfamily Is an RNA N-Glycosidase

Ribosome-inactivating proteins (RIPs) hydrolyze the N-glycosidic bond and depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. In this study, we have purified and characterized lyophyllin, an unconventional RIP from Lyophyllum shimeji, an edible mushroom. The protein resembles peptidase M35 domain of peptidyl-Lys metalloendopeptidases. Nevertheless, protein either from the mushroom or in recombinant form possessed N-glycosidase and protein synthesis inhibitory activities. A homology model of lyophyllin was constructed. It was found that the zinc binding pocket of this protein resembles the catalytic cleft of a classical RIP, with key amino acids that interact with the adenine substrate in the appropriate positions. Mutational studies showed that E122 may play a role in stabilizing the positively charged oxocarbenium ion and H121 for protonating N-3 of adenine. The tyrosine residues Y137 and Y104 may be used for stacking the target adenine ring. This work first shows a protein in the peptidase M35 superfamily based on conserved domain search possessing N-glycosidase activity.     

Functional roles of the N-terminal amino acid residues in the Mn(II)-L- malate binding and subunit interactions of pigeon liver malic enzyme

Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met- Lys-Lys-Gly-Tyr-Glu-. In this work, various mutants of the enzyme with individual or combinational deletion (delta) or substitution at these amino acids were constructed and functionally expressed in Escherichia coli cells. A major protein band corresponding to an Mr of approximately 65000 was observed for all recombinant enzymes in sodium dodecyl sulfate polyacrylamide gel electrophoresis. However, when examining by polyacrylamide gel electrophoresis under native conditions, the recombinant enzymes were found to possess a tetrameric structure with Mr approximately 260000 or a mixture of tetramers and dimers with the exception of delta(K2K3G4) and delta(1-16) mutants, which existed exclusively as dimers at the protein concentration we employed. K3A and K3E also dissociated substantially. K(2,3)A was a tetramer but K(2,3)E essentially existed as dimers. All tetramers and dimers were enzymatically active in the gels. All mutants displayed a similar apparent Km value for NADP+. The apparent Km for L-malate and Mn(II), on the other hand, was increased by 4-27-fold for the delta(K2/K3) and the delta(1-16) mutants. The small binding affinity of delta(K2/K3) with Mn(II)-L-malate was specific. With additional deletion at positions 3 and/or 4, the delta(K2K3), delta(K2G4/K3G4) or delta(K2K3G4) mutants exhibited similar kinetic properties for the wild type. The lysine residues at the positions 2 or 3 seem to be crucial for the correct active site conformation. The results indicate that the N-terminus of malic enzyme is located at the Mn(II)-L-malate binding domain of the active center and is also near the subunit's interface. These results were interpreted with our asymmetric double-dimer model for the enzyme in which the N-terminus was involved in the head-to-tail monomer-monomer interactions but not the dimer-dimer interactions.      

The amino acid sequences in the C-terminal region of glucose-1-phosphate thymidylyltransferases determine their soluble expression in Escherichia coli

Two similar genes, dnmL and rmbA in Streptomyces peucetius, which encode for glucose-1-phosphate (G-1-P) thymidylyltransferases were expressed in Escherichia coli under similar conditions. While RmbA was expressed in soluble form, DnmL was found as insoluble aggregates in inclusion bodies. The difference in expression of these similar proteins led to investigate into the amino acid sequences of these proteins by sequence alignment, hydrophobicity scale and homology modeling. These analyses showed that the two proteins are different only in the C-terminal sequences. Deletion of C-terminal sequence of DnmL increased the expression level of truncated DnmL. Substitution of C-terminal sequence of DnmL with RmbA also expressed the recombinant protein in soluble form. Finally, mutation of six amino acids in DnmL rendered the protein expressed in soluble form. These results suggested that the soluble expression of the thymidylyltransferases lies in the C-terminal sequences. In conclusion, these methods of protein engineering will be a rational tool for enhancing solubility of proteins expressed in E.coli.     

Trypsin-like Inhibitor Domain (TIL)-Harboring Protein Is Essential for Aedes aegypti Reproduction

Cysteine-rich trypsin inhibitor-like domain (TIL)-harboring proteins are broadly distributed in nature but remain understudied in vector mosquitoes. Here we have explored the biology of a TIL domain-containing protein of the arbovirus vector Aedes aegypti, cysteine-rich venom protein 379 (CRVP379). CRVP379 was previously shown to be essential for dengue virus infection in Ae. aegypti mosquitoes. Gene expression analysis showed CRVP379 to be highly expressed in pupal stages, male testes, and female ovaries. CRVP379 expression is also increased in the ovaries at 48 h post-blood feeding. We used CRISPR-Cas9 genome editing to generate two mutant lines of CRVP379 with mutations inside or outside the TIL domain. Female mosquitoes from both mutant lines showed severe defects in their reproductive capability; mutant females also showed differences in their follicular cell morphology. However, the CRVP379 line with a mutation outside the TIL domain did not affect male reproductive performance, suggesting that some CRVP379 residues may have sexually dimorphic functions. In contrast to previous reports, we did not observe a noticeable difference in dengue virus infection between the wild-type and any of the mutant lines. The importance of CRVP379 in Ae. aegypti reproductive biology makes it an interesting candidate for the development of Ae. aegypti population control methods.     

Residue 75 of Interleukin‐8 is Crucial for its Interactions with Glycosaminoglycans

The interactions between regulatory proteins such as interleukin‐8 (IL‐8) and glycosaminoglycans are of great interest both for the general understanding of regulatory processes in biology and for the development of implant coatings and innovative materials that suppress undesired immune responses and improve wound healing. In previous work, a number of residues of IL‐8 that interact strongly with several glycosaminoglycans (GAGs) have been identified. In particular, the negatively charged Glu75 was reported to be involved in interactions with charged GAGs. To improve understanding of the role of this residue, we generated a selectively ¹⁵N‐labeled E75K variant of IL‐8(1–77) by expressed protein ligation. NMR and fluorescence spectroscopy in combination with molecular modeling were applied to evaluate the particular role of residue 75 in interactions with GAGs. Remarkably, more residues in the variant responded to GAG binding than in the wild‐type. For the first time, we identified amino acids 34 to 36 as additional residues in the loop region of IL‐8(1–77) that participate in the interactions with GAGs. These findings indicate that the N terminus of the E75K variant is more important as a second binding site for GAGs than that of the wild‐type IL‐8(1–77).     

A strategy for high-level expression of soluble and functional human interferon alpha as a GST-fusion protein in E. coli

Escherichia coli is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. To achieve high-level expression of soluble recombinant human interferon alpha (rhIFNalpha) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFNalpha2b), in which we merged the hIFNalpha2b cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac-inducible promoter. Using this plasmid, we have achieved 70% expression of soluble rhIFNalpha2b as a GST fusion protein using E. coli BL21 strain, under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E. coli preferred codon within the thrombin cleavage site. We have used the engineered plasmid (pGEX-Delta-hIFNalpha2b) and the modified E. coli trxB(-)/gor(-) (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNalpha2b. Our results show the production of soluble and functional rhIFNalpha2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFNalpha2b was equal to 2.0 x 10(8) IU/mg when compared with the WHO IFNalpha standard. Our data are the first to show that high yield production of soluble and functional rhIFNalpha2b tagged with GST can be achieved in E. coli.     

结核分枝杆菌TB10.4与呼吸道合胞病毒F1蛋白的融合表达

目的在大肠杆菌中融合表达结核分枝杆菌(Mycobacterium tuberculosis,MTB)TB10.4蛋白与呼吸道合胞病毒(Respiratory syncytial virus,RSV)F1蛋白。方法从MTB标准菌株H37Rv全基因组中扩增TB10.4(N/X)和TB10.4(N/B)基因,分别插入原核表达载体pET-28a和pET-30a中,构建重组表达质粒TB10.4(N/X)/pET-28a和TB10.4(N/B)/pET-30a;从F/18T/DH5α菌株中扩增F1(B/X)基因,与TB10.4(N/B)/pET-30a质粒连接,构建重组表达质粒TB10.4-F1/pET-30a;将质粒TB10.4(N/X)/pET-28a和TB10.4-F1/pET-30a分别转化感受态E.coliBL21(DE3),IPTG诱导表达;表达的重组蛋白进行Western blot鉴定。结果重组表达质粒TB10.4(N/X)/pET-28a和TB10.4-F1/pET-30a经双酶切及测序证明构建正确;表达的重组TB10.4和TB10.4-F1蛋白相对分子质量约为13 000和59 000,可与鼠抗His单抗特异性结合。结论成功在大肠杆菌中表达了重组融合蛋白TB10.4-F1,为进一步研究其免疫原性及保护性奠定了基础。     

Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin

Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides mimicking defined epitopes of the bone modulator protein sclerostin, which has been identified as a negative regulator of the Wnt pathway. For a fast exploration of activity defining epitopes, we produced a set of synthetic peptide constructs mimicking native sclerostin, in which intervening loops from the cystine-knot protein sclerostin were truncated and whose sequences were optimized for fast and productive refolding. We found that the second loop within the cystine knot could be replaced by unnatural sequences, both speeding up folding, and increasing yield. Subsequently, we used these constructs to pan the HuCAL phage display library for antibodies capable of binding the native protein, thereby restricting recognition to the desired epitope regions. It is shown that the antibodies that were obtained recognize a complex epitope in the protein that cannot be mimicked with linear peptides. Antibodies selected against peptides show similar recognition specificity and potency as compared with antibodies obtained from full-length recombinant protein.     

Nucleobase amino acids incorporated into the HIV-1 nucleocapsid protein increased the binding affinity and specificity for a hairpin RNA

L-alpha-amino acids with a nucleobase in the side chain (nucleobase amino acids; NBAs) were used to enhance the function of RNA-binding proteins that recognize structured RNA. These NBAs were utilized in the three-dimensional structure of the protein to enhance RNA binding affinity and specificity as a result of selective recognition of NBAs by RNA bases. NBA units were incorporated at various positions into the HIV-1 nucleocapsid protein NCp7 (residues 1-55), which contains two CCHC-type (Cys-X(2)-Cys-X(4)-His-X(4)-Cys-type; X=an amino acid residue) zinc knuckle domains. The binding ability was evaluated by using the stem-loop (SL)3 region of HIV-1 Psi-RNA. Visible light absorption measurements revealed that two zinc ions bound strongly and quantitatively to the NBA-NCp7 molecule and to the wild-type NCp7 protein. This result indicates that the incorporation of NBA units composed of L-alpha-amino acids did not influence the formation of the specific structure of NCp7. Binding analysis with fluorescein-labeled SL3 RNA revealed that incorporation of NBA units into the NCp7 protein at appropriate positions increased its RNA binding affinity and specificity. An NBA-NCp7 protein that possessed cytosine and guanine NBA units at positions 13 and 46, respectively, showed a binding affinity for SL3 RNA ninefold higher than that of wild-type NCp7 as a result of the specific and cooperative interaction of the NBA units with RNA bases. These results clearly demonstrate that inclusion of NBA units in the three-dimensional structure of an RNA-binding protein is a useful strategy for enhancing the function of the protein.     

Mg~(2+)和氨基酸对重组大肠杆菌BL21(DE3)生长及羧肽酶原B表达的影响

目的研究Mg2+和氨基酸对重组菌株生长及表达pCPB的影响。方法通过摇瓶和发酵罐培养,观察不同Mg2+浓度和氨基酸对重组菌的生长和羧肽酶原B表达的影响。结果添加1g/L Mg2+能提高质粒的稳定性;添加氨基酸能使羧肽酶原B表达量由18·7g/L提高到24g/L。结论添加适量的Mg2+和氨基酸能促进重组菌的生长和提高目的蛋白的表达量。     

How to improve nature: study of the electrostatic properties of the surface of alpha-lactalbumin

It was recently shown that alpha-lactalbumin interacts with histones and simple models of histone proteins such as positively charged polyamino acids, suggesting that some fundamental aspects of the protein surface electrostatics may come into play. In the present work, the energies of charge-charge interaction in apo- and Ca(2+)-loaded alpha-lactalbumin were calculated using a Tanford-Kirkwood algorithm with either solvent accessibility correction or using a finite difference Poisson-Boltzmann method. The analysis revealed two major regions of alpha-lactalbumin that possessed highly unfavorable electrostatic potentials: (a) the Ca(2+)-binding loop and its neighboring residues and (b) the N-terminal region of the protein. Several individual mutants were prepared to neutralize specific individual surface acidic amino acids at both the N-terminus and Ca(2+)-binding loop of bovine alpha-lactalbumin. These mutants were characterized by intrinsic fluorescence, differential scanning microcalorimetry and circular dichroism. The structural and thermodynamic data agree in every case with the theoretical predictions, confirming that the N-terminal region is very sensitive to changes in charge. For example, desMet D14N mutation destabilizes protein and decreases its calcium affinity. On the other hand, desMet E1M and desMet D37N substitutions increase the thermal stability and calcium affinity. The Met E1Q is characterized by a marked increase in protein stability, whereas desMet E7Q and desMet E11L display a slight increase in calcium affinity and thermal stability. Examination of the unfavorable energy contributed by Glu1 and the energetically favorable consequences of neutralizing this residue suggests that nature may have made an error with bovine alpha-lactalbumin from the viewpoint of stabilizing structure and conformation.     

Communication pathways between the nucleotide pocket and distal regulatory sites in protein kinases

Protein kinases control many cellular processes via the ATP-dependent phosphorylation of specific amino acids on target proteins. Despite the availability of the three-dimensional structures of a variety of protein kinases, it has been particularly difficult to explain how noncatalytic domains removed from the active site regulate catalytic function. In this review, we describe how solution methodologies complement the available structural data and help explain how protein kinases may utilize medium-to-long-range effects to regulate substrate phosphorylation. For illustration, two protein kinases, cAMP-dependent protein kinase and the C-terminal Src kinase, are presented as paradigms for the serine/threonine- and tyrosine-specific families. While active-site residues provide an optimal environment for fast phosphoryl group transfer in these and other kinases, the overall rate of protein phosphorylation is limited by nucleotide binding and associated structural changes. Hydrogen-deuterium exchange studies reveal that nucleotide binding induces changes that radiate from a central structural assembly composed of the catalytic loop, glycine-rich loop, and helix alpha C to unique peripheral regions inside and outside the kinase core. This collection of conserved and unique elements delivers information from the active site to distal regions and possibly provides information flow back to the active site. This "push-pull" hypothesis offers a means for understanding how protein kinases can be regulated by protein-protein interactions far from the active site.     

Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments

The thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) has been truncated to molecular regions essential for polymerase activity. Two truncated forms of the full-length 832 amino acid Taq polymerase have been constructed according to sequence alignments and the known domain structure of the homologous Escherichia coli DNA polymerase I (E.coli pol I): variant delta288 (lacking the N-terminal 288 amino acid portion) and variant delta413 (lacking the N-terminal 413 amino acid portion). Both protein fragments were stable and showed polymerase activity, albeit specific activity and thermostability of the variant delta413 were significantly decreased compared with the full length Taq polymerase. In order to increase the thermostability of the variant delta413, a three-dimensional model of the polymerase domain of Taq polymerase was built by homology with a model of the Klenow fragment of the E.coli pol I based on the available Calpha coordinates. Consequently two variants were designed and constructed using site-directed mutagenesis. The strategies used were deletion of 10 flexible amino acids and replacement of two hydrophobic amino acids on the surface by more hydrophilic ones. Compared with the initial protein fragment, both variant enzymes showed an increase in polymerase activity and thermostability. After the completion of this work, X-ray coordinates of the Taq polymerase became available from the protein structure data bank. A comparison between the homology model and the experimental three-dimensional structure proved the quality of the model.