A method for the rapid identification of epitopes and other functional Peptide domains.

One of the continuing objectives of molecular biology research is to characterize the functronal domains of proteins. Many proteins contam domains capable of binding specific ligands, such as cofactors, substrates, and domains of other proteins, that form the basis for interactions that drive a large number of biological processes. Protein epitopes are a class of hgand binding domains responsible for eliciting an immune response; in this respect then identification can be of major importance in the development of vaccines and other pharmaceutical compounds.

Expression and Purification of Recombinant Proteins Using the pET System.

The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in Escherichia coli Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and (optionally) translation signals, expression is induced by providing a source of T7 RNA polymerase in the host cell (1-2). T7 RNA polymerase is so selective and active that almost all of the cell's resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein after a few hours of induction. Another important benefit of this system is its ability to maintain target genes transcriptionally silent in the uninduced state. Target genes are initially cloned using hosts that do not contain the T7 RNA polymerase gene, thus eliminating plasmid instability caused by the production of proteins potentially toxic to the host cell. Once established in a nonexpression host, plasmids are then transferred into expression hosts containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control, and expression is induced by the addition of IPTG. Two types of T7 promoter and several hosts that differ in then- stringency of suppressing basal expression levels are available, providing great flexibility and optimizing the expression of a wide variety of target genes. This chapter describes the vectors, hosts, and basic protocols for cloning, expression, and purification of target proteins in the pET System.

Expression of smooth muscle and nonmuscle tropomyosins in Escherichia coli and characterization of bacterially produced tropomyosins.

The cDNA encoding the beta-tropomyosin isoform of chicken smooth muscle (CSM beta) was constructed and expressed in Escherichia coli to produce recombinant, unacetylated beta-tropomyosin (rCSM beta) and a mutant (rCSM beta-7) with a 7-residue deletion at its amino-terminus. Furthermore, the cDNA coding for human fibroblast tropomyosin isoform 3 (hTM3) was also used to produce unacetylated hTM3 (called PEThTM3). All of bacterially-made tropomyosins were high alpha-helical in structure as judged by CD analysis and resistant to heat denaturation. Both the rCSM beta and PEThTM3 exhibited saturable binding to F-actin with apparent binding constants of 1.14 x 10(6) and 2.78 x 10(6) M-1, respectively. The bacterially made, unacetylated smooth muscle tropomyosin (rCSM beta) appeared to have a comparable actin-binding affinity to that of gel-purified CSM beta homodimer (1.25 x 10(6) M-1) but significantly lower than that for native gizzard tropomyosin (CSM-TM) heterodimer (1.28 x 10(7) M-1). The amino-terminal deletion mutant rCSM beta-7 failed to bind to F-actin. Effects of gizzard caldesmon on the actin binding of these bacterially made tropomyosins were also examined. Under the binding condition containing 0.5 mM MgCl2 and 30 mM KCl, caldesmon greatly enhanced the binding of rCSM beta to F-actin. However, under the same condition, there was a slight enhancement in the actin-binding for gel-purified CSM beta or PEThTM3 (1.2-1.6-fold stimulation) and no enhancement for native gizzard tropomyosin. Neither the presence of caldesmon nor native gizzard tropomyosin induced detectable binding of the amino-terminal deletion mutant rCSM beta-7 to F-actin. These results clearly imply the importance of the amino-terminal 7 amino-acid residues of CSM beta in the actin binding and the caldesmon enhancement.

Human fibroblast tropomyosin isoforms: characterization of cDNA clones and analysis of tropomyosin isoform expression in human tissues and in normal and transformed cells.

A tropomyosin-specific oligonucleotide probe (REN29) designed to hybridize to all known human tropomyosin isoforms was used to study tropomyosin mRNA levels in normal and transformed human cells. At least four different sizes of RNAs were detected in normal human fibroblast KD cells by Northern blot analysis. The major bands of 1.1 kb RNA for hTM1 and 3.0 kb RNA for hTM4 were decreased substantially in various transformed cell lines. One of the minor RNA bands (2.0 kb for hTM2 and hTM3) appeared to be absent in a human pancreatic carcinoma cell line. The level of the other minor RNA band (2.5 kb for hTM5) was found to be unchanged or slightly decreased in transformed cells. This differential expression of tropomyosin isoforms at the RNA level was not totally in agreement with the difference in the protein amounts found in normal and transformed cells, suggesting that translational control may also play an important role in the expression of some tropomyosin isoforms. The REN29 probe was further used to screen lambda gt10 and lambda gt11 cDNA libraries, which were constructed from poly(A)+ RNAs of human fibroblast cell lines HuT-14 and WI-38, respectively. In addition to cDNA clones encoding known isoforms, we obtained three classes of new cDNA clones that encode two low M(r) isoforms (hTM5a and hTM5b), and a high M(r) isoform (hTMsm alpha). Sequence comparison revealed that hTM5a and hTM5b are alternatively spliced products derived from the same gene that encodes hTM2 and hTM3. Northern blot analysis and amino acid sequence comparison suggested that the hTMsm alpha represents a smooth muscle tropomyosin which is also expressed in human fibroblasts. The exon specific for, and common to, hTM5a and hTM5b was found to be highly expressed in small intestine. However, there was no detectable expression of this exon in stomach and skeletal muscle. The difference in tissue-specific expression suggests that different isoforms may perform distinct functions in different tissues.

In vitro functional characterization of bacterially expressed human fibroblast tropomyosin isoforms and their chimeric mutants.

At least eight tropomyosin isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsm alpha) are expressed from four distinct genes in human fibroblasts. In order to elucidate isoform properties, we have subcloned hTM3 and hTM5 full-length cDNAs, as well as their chimeric cDNAs into the bacterial expression pET8C system. Bacterially expressed tropomyosin isoforms (called PEThTM3, PEThTM5, PEThTM5/3, and PEThTM3/5) were purified and characterized. Under optimal binding conditions, the binding of PEThTM5 isoform to F-actin was stronger than the PEThTM3 isoform. However, analysis of actin-binding by the McGhee and von Hippel equation revealed that PEThTM3 exhibits higher cooperativity in binding than PEThTM5 does. Furthermore, the chimera PEThTM5/3 which possessed the N-terminal fragment of hTM5 fused to the C-terminal fragment of hTM3 had even stronger actin binding ability. The reverse chimera PEThTM3/5 which possessed the N-terminal fragment of hTM3 fused to the C-terminal fragment of hTM5 demonstrated greatly reduced affinity to actin filaments. In addition, both chimeras had different KCl requirements for optimal binding to F-actin than their parental tropomyosins. A bacterially made C-terminal fragment of human fibroblast caldesmon (PETCaD39) and native chicken gizzard caldesmon were both able to enhance the actin-binding of these bacterially expressed tropomyosins. However, PETCaD39's enhancement of binding to F-actin was greater for PEThTM5 than PEThTM3. Under 30 mM KCl and 4 mM MgCl2, the low M(r) isoform PEThTM4 appeared to be able to amplify the actin-activated HMM ATPase activity by 4.7 fold, while the high M(r) isoform PEThTM3 stimulated the activity only 1.5 fold. The higher enhancement of ATPase activity by PEThTM5 than by PEThTM3 suggested that the low M(r) isoform hTM5 may be more involved in modulating nonmuscle cell motility than hTM3. These results further suggested that different isoforms of tropomyosin might have finite differences in their specific functions (e.g., cytoskeletal vs. motile) inside the cell.

Calponin and tropomyosin interactions.

The interaction between chicken gizzard calponin and tropomyosin was examined using viscosity, light scattering, electron microscopy and affinity chromatography. At neutral pH, 10 mM NaCl and in the absence of Mg2+, calponin induced tropomyosin filaments to form paracrystals thus decreasing the viscosity while increasing dramatically the light scattering of the tropomyosin solution. Electron micrographs of the uranyl acetate stained calponin-tropomyosin complex showed the presence of spindle shaped paracrystals with regular striation patterns and repeating units of about 400 A. Under similar conditions, smooth muscle caldesmon also induced tropomyosin to form paracrystals. To localize the calponin-binding site on tropomyosin, binding of fragments of tropomyosin, generated by chemical and mutational means, to a calponin-affinity column was studied. The COOH-terminal tropomyosin fragment Cn1B(142-281) and the NH2-terminal fragment CSM-beta(1/8/12-227) bound to a calponin-affinity column with an affinity similar to that of intact tropomyosin; while the NH2-terminal fragment, Cn1A(11-127), did not bind, indicating that the calponin-binding site(s) resides within residues 142-227 of tropomyosin. To determine the involvement in calponin binding of the area around Cys-190 of tropomyosin, fragments with cleavage sites near or at Cys-190 were used. Thus, while fragments Cy2(190-284) and CSM-beta(1/8/12-200) bound weakly to the calponin-affinity column, fragment Cy1(1-189) did not. These results demonstrate that calponin binds to tropomyosin between residues 142 and 227, and that the integrity of the region around Cys-190 of tropomyosin is important for strong interaction between the two proteins.

Characterization of cDNA clones encoding a human fibroblast caldesmon isoform and analysis of caldesmon expression in normal and transformed cells.

Overlapping cDNA clones encoding a low M gamma human nonmuscle caldesmon isoform (HUM 1-CaD) span the entire coding region (538 amino acids) as well as 111 base pairs (bp) of 5'-noncoding and 1249 bp of 3'-noncoding region. Northern blot probes derived from either the coding or 3'-noncoding region hybridized to a 4.3-kilobase mRNA in nonmuscle cells and a 5.2-kilobase mRNA in stomach tissue. Primer extension results indicated that the 5'-noncoding region of the HUM 1-CaD mRNA is approximately 700 bp in length and also suggested that 1-CaD mRNAs with common 5'-noncoding regions are expressed in both liver and fibroblast cells. Comparisons of the human, rat, and chicken 1-CaD amino acids sequences demonstrated that although each isoform has unique characteristics, extensive regions of conservation exist. Amino acids 27-53 and 97-127 are 100% identical in these isoforms while amino acids 297-531 of HUM 1-CaD are 94 and 85% identical to the rat and chicken 1-CaDs, respectively. In addition, the levels of HUM 1-CaD mRNA and protein appeared to be decreased by 2-4 fold in the transformed derivatives of KD and WI38 cell lines as judged by Northern and Western blot analysis. The results suggest that the decrease of 1-CaD protein in these transformed cells is a direct result of decreased 1-CaD mRNA synthesis and/or increased mRNA turnover.

Epitope mapping of monoclonal antibodies against caldesmon and their effects on the binding of caldesmon to Ca++/calmodulin and to actin or actin-tropomyosin filaments.

The effects of monoclonal anti-caldesmon antibodies, C2, C9, C18, C21, and C23, on the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin were examined in an in vitro reconstitution system. In addition, the antibody epitopes were mapped by Western blot analysis of NTCB (2-nitro-5-thiocyanobenzoic acid) and CNBr (cyanogen bromide) fragments of caldesmon. Both C9 and C18 recognize an amino terminal fragment composed of amino acid residues 19 to 153. The C23 epitope lies within a fragment ranging from residues 230 to 386. Included in this region is a 13-residue repeat sequence. Interestingly this repetitive sequence shares sequence similarity with a sequence found in nuclear lamin A, a protein which is also recognized by C23 antibody. Therefore, it is likely that the C23 epitope corresponds to this 13-residue repeat sequence. A carboxyl-terminal 10K fragment contains the epitopes for antibodies C2 and C21. Among these antibodies, only C21 drastically inhibits the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin. When the molar ratio of monoclonal antibody C21 to caldesmon reached 1.0, a maximal inhibition (90%) on the binding of caldesmon to F-actin filaments was observed. However, it required double amounts of C21 antibody to exhibit a maximal inhibition of 70% on the binding of caldesmon to F-actin-tropomyosin filaments. These results suggest that the presence of tropomyosin in F-actin enhances caldesmon's binding. Furthermore, C21 antibody also effectively inhibits the caldesmon binding to Ca++/calmodulin. The kinetics of C21 inhibition on caldesmon's binding to Ca++/calmodulin is very similar to the inhibition obtained by preincubation of caldesmon with free Ca++/calmodulin. This result suggests that there is only one Ca++/calmodulin binding domain on caldesmon and this domain appears to be very close to the C21 epitope. Apparently, the Ca++/calmodulin-binding domain and the actin-binding domain are very close to each other and may interfere with each other. In an accompanying paper, we have further demonstrated that microinjection of C21 antibody into living chicken embryo fibroblasts inhibit intracellular granule movement, suggesting an in vivo interference with the functional domains [Hegmann et al., 1991: Cell Motil. Cytoskeleton 20:109-120].

Caldesmon-binding sites on tropomyosin.

The interaction of chicken gizzard caldesmon with fragments of tropomyosin, generated by chemical, enzymatic, and mutational means, was studied to determine the caldesmon-binding site(s) on tropomyosin. Binding was examined by fluorescence spectroscopy and affinity chromatography. Removal of residues 1-141 and 228-284, respectively, from the NH2 and COOH ends of tropomyosin did not affect its binding to caldesmon significantly, indicating that the major, caldesmon-binding region lies between residues 142-227. The Escherichia coli produced chicken gizzard beta-tropomyosin mutant, CSM-beta (1/8/12-227), bound caldesmon about 2-fold stronger than a similar mutant of residues 8-200. This further focused the primary caldesmon-binding site to residues 201-227. Cleavage of tropomyosin at CYS-190 weakened markedly the binding of the two resulting fragments, residues 1-189 and 190-284, to caldesmon suggesting the requirement for the integrity of the caldesmon-binding region between residues 142227 of tropomyosin for strong interaction with caldesmon. Based on data from this study and others, we have proposed models for the interaction of tropomyosin with caldesmon in vitro, as well as the possible arrangement of the smooth muscle thin filament proteins in vivo.