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Biomedical subjects

K Takkinen

Publications and source records attributed to K Takkinen.

At least 19 recordsLinked to original sources

Baculoviral display of functional scFv and synthetic IgG-binding domains.

Viral vectors displaying specific ligand binding moities such as scFv fragments or intact antibodies hold promise for the development of targeted gene therapy vectors. In this report we describe baculoviral vectors displaying either functional scFv fragments or the synthetic Z/ZZ IgG binding domain derived from protein A. Display on the baculovirus surface was achieved via fusion of the scFv fragment or Z/ZZ domain to the N-terminus of gp64, the major envelope protein of the Autographa californica nuclear polyhedrosis virus, AcNPV. As examples of scFv fragments we have used a murine scFv specific for the hapten 2-phenyloxazolone and a human scFv specific for carcinoembryonic antigen. In principle, the Z/ZZ IgG binding domain displaying baculoviruses could be targeted to specific cell types via the binding of an appropriate antibody. We envisage applications for scFv and Z/ZZ domain displaying baculoviral vectors in the gene therapy field.

Animals↗

X-ray studies of recombinant anti-testosterone Fab fragments: the use of PEG 3350 in crystallization.

Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 A. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.

Buffers↗

Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs.

BACKGROUND: We have previously reported specificity improvement of an anti-testosterone monoclonal antibody (3-C4F5) by random mutagenesis of the third complementarity determining regions (CDR3s) and by phage display selection. OBJECTIVES: Here we extend the mutagenesis strategy to the other CDRs and select the mutant libraries using two different approaches in order to further fine-tune the binding properties of this recombinant Fab fragment. STUDY DESIGN: To improve the affinity the new mutant libraries were selected by using limiting, decreasing concentrations of biotinylated testosterone (TES) in solution and capturing the binders on streptavidin-coated microtiter plate. The specificity was improved by preincubating the mutant libraries in solution with a high concentration of the most problematic cross-reacting steroid, dehydroepiandrosterone sulfate (DHEAS). RESULTS: In two different light chain CDR1 mutant clones isolated from the affinity pannings, the relative TES affinity was increased over 10-fold while the cross-reactivities to related steroids were preserved at the same level as in the parental combined CDR3 mutant clone. New heavy chain CDR1 and light chain CDR2 mutants showing slightly decreased cross-reactivities were isolated from specificity selections. By combining compatible mutant CDRs together we were able to create a Fab fragment with over 12-fold higher relative TES affinity and significantly lower cross-reactivity to DHEAS when compared to the original monoclonal antibody 3-C4F5. CONCLUSIONS: Our results demonstrate that a high-affinity and selective recombinant Fab fragment working over a wide TES concentration range with clinical samples could be generated by CDR mutagenesis and phage display selection.

Antibody Affinity↗

Specificity improvement of a recombinant anti-testosterone Fab fragment by CDRIII mutagenesis and phage display selection.

The monoclonal antibodies so far developed by hybridoma technology have not had high enough specificity or affinity to distinguish the closely related steroid hormones in routine clinical assays. We have employed random mutagenesis and phage display approaches to improve the specificity of one anti-testosterone monoclonal antibody (3-C4F5). The affinity of the antibody is 0.3 x 10(9) M(-1) and the cross-reactivities with most of the related steroids are low. However, the antibody cross-reacts about 1% with dehydroepiandrosterone sulfate (DHEAS) and owing to the high DHEAS serum concentration this is about 1000-fold too high for clinical immunoassays. The complementarity-determining regions (CDRs) of the heavy and light chains, which were predicted by molecular modelling to be in close contact with the testosterone (TES) ligand, were randomized and mutant Fab libraries were cloned into a phagemid vector. Binders were selected by a competitive panning procedure. By combining the identified light and heavy chain CDRIII mutations the TES affinity was preserved at the wild-type level but DHEAS cross-reactivity was decreased to 0.03%. An important finding was that by the competitive panning procedure the overall binding specificity of the 3-C4F5 antibody was refined, since the cross-reactivities to related steroids were also significantly decreased in the combined mutant.

Amino Acid Sequence↗

Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment.

Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for diagnostic immunoassays. Lysine residues at antibody surfaces are ready targets for labelling by an isothiocyanate derivative of the europium chelate (Eu3+). Here the labelling efficiency of a recombinant anti-human alpha-fetoprotein (hAFP) Fab fragment has been improved by increasing its lysine content by protein engineering. Molecular modelling was used to identify three light chain constant domain surface arginine residues, R154, R187 and R210, which were mutated to lysine residues. The mutations did not influence the affinity of the lysine-enriched Fab fragment and its labelling efficiency was found to be approximately 40% higher than that of the wild-type Fab fragment. With low degree of labelling, the affinities of the two Fab fragments were identical and comparable with that of the original monoclonal anti-hAFP IgG. With a higher degree of labelling the affinities of both Fab fragments decreased more than that of the intact IgG since more lysine residues are available for labelling in the additional heavy chain constant domains of the larger molecule. Electrostatic adsorption and covalent immobilization of the Fab fragments were characterized by BIAcore and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.

Amino Acid Sequence↗

Properties of a single-chain antibody containing different linker peptides.

Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody. Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues. The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues. Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains. The hapten binding properties of different antibody fragments were studied by ELISA and BIAcoreTM. The interdomain linker peptide improved the hapten binding properties of the antibody fragment when compared with Fv fragment, but slightly increased its susceptibility to proteases. Single-chain antibodies with short CBHI linkers of 11, six and two residues had a tendency to form multimers which led to a higher apparent affinity. The fragments with linkers longer than 11 residues remained monomeric.

Amino Acid Sequence↗

Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain.

Fab fragments of IgG1 and IgG3 subclass antibodies which bind to 2-phenyloxazolone (Ox) were produced in Escherichia coli. The signal sequences of the Fd and L chains were correctly processed, the fragments were secreted into the periplasmic space and released into the culture medium upon prolonged cultivations. The yields of active Ox IgG1 and Ox IgG3 Fab fragments after one-step purification from the culture medium by affinity chromatography were 2 micrograms/ml and 0.5 micrograms/ml, respectively. The majority of the purified Ox IgG1 Fab was properly assembled, but in the case of Ox IgG3, the preparation was found to consist of a complete L chain and C-terminally degraded fragments of the Fd chain. A deletion up to the interchain disulfide bond in the first constant domain (CH1) of the Ox IgG3 Fd chain led to proper assembly of the truncated Fab fragment. The production level of the truncated fragment was comparable to that of the Ox IgG1 Fab and its hapten-binding activity similar to that of the idiotype monoclonal antibody. The temperature stability of the Ox IgG1 Fab was similar to that of the intact antibody. However, both of the Ox IgG3 Fab fragments showed reduced stability, suggesting that the CH1 domain contributes significantly to the thermal stability of the Fab fragment.

Animals↗

An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli.

Single-chain antibodies consist of the variable, antigen-binding domains of antibodies joined to a continuous polypeptide by genetically engineered peptide linkers. We have used the flexible interdomain linker region of a fungal cellulase to link together the variable domains of an anti-2-phenyloxazolone IgG1 and show here that the resulting single-chain antibody is efficiently secreted and released to the culture medium of Escherichia coli. The yield of affinity-purified single-chain antibody is 1-2 mg/l of culture medium and its affinity and stability are comparable to those of the corresponding native IgG.

Amino Acid Sequence↗

Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein.

The processing and stability of the non-structural (ns) proteins of Semliki Forest virus were studied in vivo. Virus-specific proteins from infected cells were identified by immunoprecipitation with monospecific antisera. The complete ns precursor, P1234, translated within 7 to 9 min of the start of translation, was processed before the completion of translation into P123 and nsP4. Pulse-chase experiments showed that the mature ns proteins were relatively stable for at least 2 h. Interestingly, the decrease in the amount of the P34 precursor during chase is accompanied by an increase only in the amount of nsP3, which could explain the observed lower amount of nsP4 in infected cells. In cells infected with SFV RNA- mutants ts4 and ts6 maintained at the restrictive temperature, nsP4 but no nsP1, nsP2 or nsP3 accumulated in addition to the ns precursor proteins P1234, P123, P12 and P34. Translation in vitro of mRNAs from a cDNA clone, encoding P1234 with a deletion in the carboxy-terminal proteinase domain of nsP2, did not yield nsP1, nsP2 or nsP3 but only nsP4, indicating that cleavage at the nsP3/4 sites is independent. Evidently, nsP4 is produced by a nascent cleavage of the growing P1234, catalysed by its own proteinase activity; the proteolytic cleavages at the nsP1/2 and nsP2/3 sites are catalysed by the proteinase moiety of nsP2.

Animals↗

The Semliki-Forest-virus-specific nonstructural protein nsP4 is an autoproteinase.

The Semliki-Forest-virus-specific nonstructural proteins are translated as a large polyprotein (2431 amino acid residues), from which the mature polymerase components nsP1, nsP2, and nsP4 are released by proteolytic cleavages. The complete ns polyprotein (P1234) can be cleaved in two alternative ways yielding either P123 (with sequences of nsP1, nsP2 and nsP3) and nsP4 or P12 (nsP1 plus nsP2) and P34 (nsP3 plus nsP4). We studied the possible autoproteolytic role of nsP4 involved in the cleavage between nsP3 and nsP4 in an in vitro transcription-translation system. cDNAs encoding P34 precursor and shorter precursor protein segments covering the nsP3-nsP4 cleavage region, were cloned under the T7 RNA polymerase promoter. The mRNAs synthesized in vitro were capped and translated in rabbit reticulocyte lysates. The translational products were analyzed by SDS/PAGE. The precursor proteins containing nsP4 sequences were cleaved yielding the products with expected sizes, indicating that the cleavage took place at the nsP3-nsP4 junction. By deleting and truncating the cDNA coding for nsP4, the proteolytic activity was mapped within the 102 amino-terminal amino acids of nsP4. The cleavage between nsP3 and nsP4 can be inhibited by pepstatin A and probably takes place in cis, since exogenously added nsP4 was unable to mediate it.

Amino Acid Sequence↗

Nucleotide sequence of the rubella virus capsid protein gene reveals an unusually high G/C content.

The nucleotide sequence of the rubella virus capsid protein (C) gene has been determined from a cDNA clone derived from the 40S genomic RNA. The sequence covers the coding region of the C protein (831 nucleotides), 70 nucleotides of the 5' untranslated region, and the 5' end of the downstream E2 membrane protein gene. The capsid gene is unusually rich in C (41.6%) and G (31.2%) residues (G + C 72.8%), and poor in A (15.4%) and U residues (11.8%). There are regions with long runs of up to 45% C or 35% G residues. The codon usage is non-random, with a strong preference for C and G residues in the third position. Starting from two in-frame AUG codons (seven amino acid residues apart) an open reading frame (ORF) was identified that extended in frame into the ORF coding for the downstream E2 membrane protein gene. Since the amino terminus of the capsid protein is blocked, we could not determine which of the AUGs serve as the initiating codon. To verify that the deduced ORF was correct, we have determined the amino acid sequence of 13 tryptic peptides corresponding to one-third of the C protein. Our data show that the C protein is about 277 residues in length (Mr about 30750). It is very hydrophilic and rich in prolines (14.1%) and arginines (14.4%). Clusters of these amino acids are concentrated in the amino-terminal third of the C protein. No sequence homology to the capsid protein of several alphaviruses was observed. Together with our previous sequence data we have now completed the sequence of the genes coding for the structural proteins C, E2 and E1 of rubella virus.

Amino Acid Sequence↗

Semliki Forest virus-specific non-structural protein nsP3 is a phosphoprotein.

Antisera were raised in rabbits against fusion proteins consisting of beta-galactosidase and partial amino acid sequences of Semliki Forest virus (SFV)-specific non-structural proteins nsP1, nsP2, nsP3 and nsP4. The antisera were specific since each of them precipitated only one labelled protein of a size expected for nsP1, nsP2, nsP3 or nsP4 from lysates of [35S]methionine-labelled SFV-infected BHK-21 cells. The specific antisera also precipitated p220 (with sequences of nsP1, nsP2 and nsP3), p155 (nsP1 and nsP2) and p135 (nsP3 and nsP4) which have been previously shown to be cleavage products of the polyprotein precursor of the non-structural proteins. nsP1, nsP4 and most of nsP3, together with the virus-specific RNA polymerase activity, were in the mitochondrial pellet (P15) fraction of infected BHK-21 cells whereas nsP2 was evenly distributed between P15 and the supernatant fraction (S15). Only antisera directed against nsP3 sequences precipitated a labelled protein from cells incubated with [32P]orthophosphate during SFV infection. Treatment of the immunoprecipitate with calf alkaline intestinal phosphatase reduced the amount of labelled nsP3 considerably. Immunoprecipitated 32P-labelled nsP3, isolated by SDS-PAGE, was subjected to acid hydrolysis. Both phosphoserine and phosphothreonine but not phosphotyrosine could be identified in the hydrolysate. Approximately twice as much [32P]serine as [32P]threonine was detected in nsP3. P15 and S15 fractions were prepared from [35S]methionine- and 32P-labelled SFV-infected cells and the 35S/32P ratio of nsP3 was determined after immunoprecipitation and SDS-PAGE. The nsP3 in S15 was less heavily phosphorylated (about 50%) than P15-associated nsP3. Anti-nsP3 serum revealed large cytoplasmic vesicles in SFV-infected cells in indirect immunofluorescence microscopy.

Antibody Specificity↗

Evolution of poliovirus during an outbreak: sequential type 3 poliovirus isolates from several persons show shifts of neutralization determinants.

An outbreak of poliomyelitis in Finland resulted in the widespread circulation of wild-type 3 poliovirus strains that had antigenic properties distinct from the strains used to produce the attenuated and inactivated vaccines. Considerable variation was observed in the ability of broadly reacting monoclonal antibodies directed against type 3 poliovirus to neutralize the 54 strains examined. Sequential isolates from several persons showed an antigenic drift with these monoclonal antibodies and selected human sera. In addition, some faecal specimens were found to contain more than one antigenic variant. Primer extension sequencing of genomic RNAs of three plaque-purified antigenic variants isolated from one patient showed base substitutions in the region coding for the major antigenic site of poliovirus type 3. The resulting difference in the amino acid sequence in the virion protein VP1 could explain the differences observed in the neutralization of these strains by the monoclonal antibodies. Whether the observed changes in the antigenic characteristics of the sequential isolates represent true antigenic drift under immunological pressure or whether the emergence of the new variants is based on other modes of selection during replication is not known.

Antibodies, Monoclonal↗

Nucleotide sequence of the genes coding for the membrane glycoproteins E1 and E2 of rubella virus.

The complete nucleotide sequence of the genes coding for the two membrane glycoproteins E1 and E2 of rubella virus has been determined from cloned cDNA derived from the 40S genomic RNA. A sequence of 2451 nucleotides extending from the poly(A) tract towards the 5' end is presented. Within one continuous open reading frame E2 is located amino-terminally followed by E1 and a 58 residue long untranslated 3' region preceding the poly(A) tract. The coding regions of E2 and E1 are unusually G/C rich, 71.4% and 66.4% respectively. At the carboxy-terminal end of the coding region of E1, there is an inverted complementary nucleotide sequence, which could form a 13 base pair hairpin structure. Mature E2 and E1 are both preceded by a stretch of uncharged mainly non-polar amino acids, 21 and 20 residues in length, respectively. These could serve as signal peptides that mediate the membrane translocation of the proteins. At the carboxy termini of both proteins there are stretches of hydrophobic amino acids, 19 and 27 residues in length, which probably represent the transmembrane anchors of the proteins. The size of mature E1 is 481 amino acids (mol. wt. 51,502), whereas the exact size of mature E2 could not be established as its carboxy-terminal end could not be located in the sequence. A maximum size of 262 amino acids (mol. wt. 28,277) is, however, suggested. Between the E2 and E1 genes, there is a stretch of seven amino acids, five of which are arginines, which may serve as cleavage sites for a trypsin-like protease. E1 contains three and E2 four potential sites for asparagine-linked glycosylation. Both proteins are cysteine-rich (5%). Comparison of the rubella virus amino acid sequence to those of several alphaviruses indicated no sequence homology.

Amino Acid Sequence↗

Replication of the genome of alphaviruses.

The genome of Semliki Forest virus (SFV) is 11,442 nucleotides with a 5' cap-structure and a 3' poly(A) tail of about 100 residues. The genome of the closely relate Sindbis virus (SIN) is slightly longer (11,703 nucleotides). The parental RNA is first translated from the 5' two thirds to yield; nsP1, nsP2, nsP3 and nsP4, which are cleaved from a polyprotein of 2431 amino acids (SFV). The parental genome is copied to a full-length minus strand with poly(U) at the 5' end. The minus strand is used as template for the synthesis of 42 S RNA in membrane-bound replicative-intermediate (RI) structures. In addition to 42 S RNA, a 3'-coterminal subgenomic 26 S mRNA, coding for the structural proteins, is synthesized by internal initiation at the minus strand. Capping and methylation of both plus-strand RNAs occur concomitantly with their synthesis. Analysis of Sindbis virus temperature-sensitive RNA-negative mutants have shown that one complementation group (B) is specifically associated with the synthesis of minus strands. Another, group F, is involved in the polymerization step of both minus- and plus-strand 42 S RNA, and of the 26 S mRNA. The synthesis of minus strands is normally dependent on protein synthesis. There is a shut off of the minus-strand RNA synthesis at about 3 h post-infection. This is apparently regulated by a virus-specific protein, represented by the complementation group A. The same protein is involved in the regulation of the initiation of 26 S RNA together with a component represented by group G mutants. Comparative analysis of SFV and SIN RNAs and DI RNAs of both viruses suggests that perhaps only 19 nucleotides from the 3' end and about 150 nucleotides from the 5' end are needed for replication of the alphavirus RNAs. In some SIN DI RNAs the proposed secondary structure at the 5' end is replaced by a cellular tRNA(ASP) suggesting that the secondary structure rather than nucleotide sequence is sufficient for the recognition by the viral polymerase. Even when the primary structure of the four non-structural proteins of both SFV and SIN is known, the correlation of the genetic data with the individual proteins has not yet been possible.

Alphavirus↗

Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus.

The nucleotide sequence coding for the nonstructural proteins of Semliki Forest virus has been determined from cDNA clones. The total length of this region is 7381 nucleotides, it contains an open reading frame starting at position 86 and ending at an UAA stop codon at position 7379-7381. This open reading frame codes for a 2431 amino acids long polyprotein, from which the individual nonstructural proteins are formed by proteolytic processing steps, so that nsPl is 537, nsP2 798, nsP3 482 and nsP4 614 amino acids. In the closely related Sindbis and Middelburg viruses there is an opal stop codon (UGA) between the genes for nsP3 and nsP4. Interestingly, no stop codon is found in frame in this region of the Semliki Forest virus 42S RNA. In other aspects the amino acid sequence homology between Sindbis, Middelburg and Semliki Forest virus nonstructural proteins is highly significant.

Amino Acid Sequence↗

Amino acid sequence of alpha-amylase from Bacillus amyloliquefaciens deduced from the nucleotide sequence of the cloned gene.

We have isolated by molecular cloning the gene coding for the alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1.) from Bacillus amyloliquefaciens and determined its complete nucleotide sequence. The gene cloned in the plasmid pUB110 using Bacillus subtilis as a host, was contained in a 2.3-kilobase insert. Starting from an ATG initiator codon, an open reading frame comprising a total of 514 amino acids (1542 base pairs) was found within the cloned DNA fragment. The gene region encoding the COOH terminus of alpha-amylase was located by direct COOH-terminal analysis of the purified exoenzyme. The NH2-terminal portion of the gene encodes a 31 amino acid-long signal peptide (Palva, L., Pettersson, R. F., Kalkkinen, N., Lehtovaara, P., Sarvas, M., Söderlund, H., Takkinen, K., and Kääriäinen, L. (1981) Gene 15, 43-51). Since the signal peptide is correctly cleaved in the new host, as shown here by direct NH2-terminal sequence analysis, the exoamylase consists of 483 amino acid residues, corresponding to a molecular weight of 54,778. The reading frame used to deduce the amino acid sequence was found to be correct by comparison with partial amino acid sequence data published previously (Detera, S. D., and Friedberg, F. (1979) Int. J. Peptide Protein Res. 14, 364-372; Chung, H., and Friedberg, F. (1980) Biochem. J. 185, 387-395). Several differences between the sequence presented here and the partial ones published previously, however, were found. The nucleotide sequences both 5' and 3' to the alpha-amylase gene revealed palindromic structures including a stretch of six T-residues, suggesting transcription termination signals on both sides of the gene. Thus, it appears that alpha-amylase is translated from a monocistronic mRNA.

Amino Acid Sequence↗