Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis

A gene coding for a calmodulin was synthesized and expressed in Escherichia coli. The gene was produced by the enzymatic ligation of 61 chemically synthesized DNA fragments. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis by the replacement of specific gene segments with synthetic double-stranded DNA. An expression vector containing the calmodulin gene was used to transform E. coli. Purification and characterization of calmodulin (VU-1 calmodulin) expressed by these transformants showed that it lacks two posttranslational modifications: an amino-terminal blocking group and N epsilon, N epsilon, N epsilon-trimethyllysine at position 115. The cyclic nucleotide phosphodiesterase activator properties of VU-1, higher plant, and vertebrate calmodulins were not statistically different. However, VU-1 calmodulin was found to activate nicotinamide adenine dinucleotide (NAD) kinase to a maximal level that was at least 3-fold higher than that found with higher plant and vertebrate calmodulins. This higher level of activation is also characteristic of calmodulins from Dictyostelium discoideum and Chlamydomonas reinhardtii [Roberts, D. M., Burgess, W. H., & Watterson, D. M. (1984) Plant Physiol. 75, 796-798; Marshak, D. R., Clarke, M., Roberts, D. M., & Watterson, D. M. (1984) Biochemistry 23, 2891-2899]. The only common feature among Dictyostelium, Chlamydomonas, and VU-1 calmodulins not found in higher plant and vertebrate calmodulins is an unmethylated lysine at position 115. The results indicate that the lack of methylation of lysine-115 may contribute to the maximal level of NAD kinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical synthesis and expression of a cassette adapted ubiquitin gene

A gene encoding the yeast ubiquitin was chemically synthesized and expressed in yeast under regulatory control of the copper metallothionein (CUP1) promoter. The gene was assembled in a one-step ligation reaction from eight oligonucleotide fragments ranging in length from 50 to 64 nucleotides. To facilitate mutagenesis and gene fusion studies, eight unique 6-base-cutting restriction enzyme sites were placed in the reading frame which did not alter the encoded protein sequence or force the utilization of rare codons. In a copper-resistant yeast strain (CUP1r), expression of the gene was induced by copper to approximately 5% of the total yeast proteins, as determined by Coomassie-stained polyacrylamide gels. The protein, purified from yeast, reacted with ubiquitin-specific antibodies and was found to be biologically active in supporting ubiquitin-dependent protein degradation in vitro.     

Chemical synthesis and expression of the human calcitonin gene

A gene coding for the peptide hormone, human calcitonin (hCT), has been constructed and expressed in bacteria using a prokaryotic vector containing the strong T5P25 promoter and a strong ribosome-binding site. The bacterial hCT is different from the natural hCT by having an additional Met residue at the N terminus and a non-amidated C terminus. Despite these differences, the bacterial hCT is biologically active in rat cells. The results are important for future structure-function relationship studies using mutants constructed by genetic engineering.     

Chemical synthesis of a mitochondrial gene designed for expression in the yeast nucleus

A novel DNA sequence coding for subunit 8 of the mitochondrial ATPase of Saccharomyces cerevisiae has been constructed by chemical synthesis. The synthetic gene, termed NAP1, is designed for expression in the yeast nucleus and codes for a 48 amino acid polypeptide identical to that encoded by the mitochondrial aap1 gene of S. cerevisiae. The codons chosen for the NAP1 sequence correspond almost exclusively to those most frequently occurring in highly expressed yeast genes. The NAP1 coding region differs in 31 codons from that of aap1, and is flanked by sequences carrying restriction enzyme sites useful for cloning and for gene expression. A 170 bp double stranded DNA molecule was constructed by assembling 12 oligonucleotides (12 to 45 bases in length) in a single annealing/ligation mixture. This synthetic gene will provide a route for the systematic manipulation, through in vitro mutagenesis, of the structure of a protein normally encoded by mitochondrial DNA.     

Chemical synthesis and expression in E. coli of a human Val8-calcitonin gene by fusion to a synthetic human interferon-gamma gene

A gene coding for human Val8-calcitonin (Val8-hCT) was synthesized by the solid-phase phosphite approach and fused to a synthetic human immune interferon-gamma (IFN-gamma) gene. The IFN gene was previously shown to be expressed at a very high level in E. coli [(1986) Gene, in press] due to the control of a strong synthetic promoter and strong ribosome binding site. The cells harboring the fused gene produced 100-150 micrograms per l of bacterial suspension of immunoreactive calcitonin in the form of hybrid IFN-gamma-Val8-hCT protein consisting of 140 amino acids. The Val8-hCT can be released from this protein by CNBr treatment.     

Chemical synthesis and expression of a gene coding for hirudin, the thrombin-specific inhibitor from the leech Hirudo medicinalis

A DNA containing the coding sequence for the proteinase inhibitor protein hirudin from the leech Hirudo medicinalis has been obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides. The 226 bp synthetic gene carries signals for the translation initiation and termination. Fragment synthesis was performed by the Khorana ligation method as well as by the fill-in method. Efficiencies of these two methods are compared. The synthetic gene was expressed in E. coli as a fusion protein with beta-galactosidase under the control of the lac-promoter as well as a non-hybrid protein under the control of the lambda PL-promoter. The non-hybrid expression product was shown to have similar biological properties as the authentic protein isolated from the leech.     

Identification, sequence determination, and expression of the flavodoxin gene from Desulfovibrio salexigens

Restriction fragments of genomic DNA from Desulfovibrio salexigens (ATCC 14822) containing the structural gene coding for the flavodoxin protein were identified using the entire coding region of the gene for the Desulfovibrio vulgaris (Hildenborough) flavodoxin as a probe (Krey, G.D., Vanin, E.F., and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443). A 1.4-kb PstI-HindIII fragment was ultimately identified which contains an open reading frame coding for a polypeptide of 146 amino acid residues that was highly homologous to the D. vulgaris flavodoxin, sharing a sequence identity of 55%. When compared to the X-ray crystal structure of the D. vulgaris protein, the homologous regions were largely confined to those portions of the protein which are in the immediate vicinity of the flavin mononucleotide cofactor binding site. Tryptophan-60 and tyrosine-98, which reside on either side of the isoalloxazine ring of the cofactor, are conserved, as are the sequences of the polypeptide loop that interacts with the phosphate moiety of the flavin. Acidic residues forming the interface of model electron-transfer complexes with certain cytochrome c proteins are retained. The flavodoxin holoprotein is over-expressed in E. coli from the cloned gene using its endogenous promoter.     

Chemical synthesis, molecular cloning and expression of gene coding for a Bowman-Birk-type proteinase inhibitor

A gene coding for a Bowman-Birk-type proteinase inhibitor was synthesized chemically, cloned and expressed in Escherichia coli as a fusion protein with a beta-galactosidase fragment. The corresponding mutant inhibitor, carrying a P1 = Arg16 instead of Lys and an Ile27 instead of Met was obtained after cyanogen bromide cleavage, refolding and affinity chromatography on trypsin-Sepharose. Dissociation constants of complexes with trypsin of this mutant and wild-type Bowman-Birk inhibitor are identical within experimental error. This is explained by differential patterns of hydrogen bonds between side-chains of Arg or Lys in proteinase inhibitors and the primary specificity pocket of trypsin.     

Cloning and characterization of the flavodoxin gene from Desulfovibrio desulfuricans

The gene coding for the flavodoxin protein from Desulfovibrio desulfuricans [Essex 6] (ATCC 29577) has been cloned and sequenced. The gene was identified on Southern blots of HindIII-digested genomic DNA by hybridization to the coding region for the flavodoxin from Desulfovibrio vulgaris [Hildenborough] (Krey, G.D., Vanin, E.F. and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443). Ultimately, a 1.8 kb TaqI fragment was cloned which contains an open reading frame of 447 nucleotides coding for an acidic protein of 148 amino acids and calculated molecular weight of 15,726. The derived amino acid sequence of this protein is 47% identical to the flavodoxin from D. vulgaris. Regions of the polypeptide which form the flavin mononucleotide binding site are largely homologous; however, some perhaps significant differences are noted. The aromatic amino acid residues that flank the flavin isoalloxazine ring in the D. vulgaris structure, i.e., tryptophan-60 and tyrosine-98, are conserved in this flavodoxin.     

Chemical synthesis,cloning and expression of human preproinsulin gene

A 355 base pair DNA sequence coding for human preproinsulin has been assembled by joining 55 synthetic deoxyoligonucleotide fragments prepared by the modified phosphotriester methodology. Proinsulin was expressed underlac promoter control and truncatedΒ-galactosidase 590 amino acid long sequence. The fusedΒ-galactosidase proinsulin protein was produced in amount to 30 % of the totalEscherichia coli proteins. It was also expressed in M13 bacteriophage and yeast system.     

Cloning, nucleotide sequence, and expression of the flavodoxin gene from Desulfovibrio vulgaris (Hildenborough)

The gene coding for the flavodoxin protein from Desulfovibrio vulgaris (Hildenborough) has been identified, cloned, and sequenced. DNA fragments containing the flavodoxin gene were identified by hybridization of a mixed synthetic heptadecanucleotide probe to Southern blots of SalI-digested genomic DNA. The nucleotide sequences of the probe were derived from the published protein primary structure (Dubourdieu, M., LeGall, J., and Fox, J. L. (1973) Biochem. Biophys. Res. Commun. 52, 1418-1425). The same oligonucleotide probe was used to screen libraries (in pUC19) containing size-selected SalI fragments. One recombinant, carrying a 1.6-kilobase (kb) insert which strongly hybridizes to the probe, was found to contain a nucleotide sequence which codes for the first 104 residues of the amino-terminal portion of the flavodoxin protein sequence but lacked the remainder of the gene. Therefore, a PstI restriction fragment from this clone was used as a probe to isolate the entire gene from a partial Sau3AI library in Charon 35. Of the plaques which continued to hybridize strongly to this probe through repeated screenings, one recombinant, containing a 16-kb insert, was further characterized. The entire flavodoxin gene was localized within a 1.4-kb XhoI-SacI fragment of this clone. The complete nucleotide sequence of the structural gene for the flavodoxin protein from Desulfovibrio vulgaris and flanking sequences which may include promoter and regulatory sequences are reported here. The cloned flavodoxin gene was placed behind the hybrid tac promoter for overexpression of the protein in Escherichia coli. Modification to the 5'-end of the gene, including substitutions at the second codon, were required to obtain high levels of expression. The expressed recombinant flavodoxin protein is isolated from E. coli cells as the holoprotein with physical and spectral properties similar to the protein isolated from D. vulgaris. To our knowledge, this is the first example of the expression of a foreign flavodoxin gene in E. coli using recombinant DNA methods.     

Chemical synthesis of a gene for human stefin A and its expression in E. coli

A DNA containing the coding sequence for the human cysteine proteinase inhibitor stefin A was obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides, using the Khorana ligation method. The 306-bp synthetic gene carries signals for the initiation and termination of its translation. The gene was expressed in E. coli using a cytoplasmic expression vector and stefin A was secreted under the control of the E. coli alkaline phosphatase signal sequence, respectively. The secreted hybrid protein was shown to exhibit biological properties similar to the native protein isolated from human plasma.     

Chemical synthesis and cloning of a gene for human beta-urogastrone

A DNA duplex coding for the 53 amino acids of human beta-urogastrone has been synthesised. Computer assisted design of the gene included restriction endonuclease sites for plasmid insertion, a termination codon and two triplets coding for lysine at the 5'-end of the structural gene. The synthesis involved preparation of 23 oligodeoxyribonucleotides by phosphotriester procedures coupled to rapid HPLC techniques. The gene was constructed in two halves by enzymatic ligation of the oligonucleotides and cloned into a specially constructed chimeric plasmid vector. Escherichia coli K12 MRC8 was transformed by the plasmid and clones containing the full gene sequence were isolated and characterised.     

Chemical synthesis of a gene for somatomedin C.

A synthetic gene for somatomedin C, a human growth factor, has been assembled by a single ligation of 23 oligodeoxyribonucleotides, which were chemically synthesized by an improved solid phase phosphotriester method.     

Cloning and expression of a Clostridium kluyveri gene responsible for diaphorase activity

A small enzyme showing diaphorase activity was purified from culture supernatant of Clostridium kluyveri and its N-terminal amino acid sequence was determined. This sequence identified a gene (diaA) encoding a protein (DiaA) of 229 amino acids with a predicted molecular weight of 24,981 in the genomic DNA sequence database of C. kluyveri constructed by the Research Institute of Innovative Technology for the Earth. The predicted protein was composed of a flavin reductase-like domain and a rubredoxin-like domain from its N-terminus. The diaA gene was cloned into an expression vector and expressed in an Escherichia coli recombinant. Recombinant enzyme rDiaA showed NADH/NADPH diaphorase activity with 2,6-dichlorophenolindophenol and nitro blue tetrazolium. The enzyme was most active at pH 8.0 at 40 degrees C. The UV-visible absorption spectrum and thin layer chromatography (TLC) analyses indicated that one rDiaA molecule contained a tightly bound FMN molecule as a prosthetic group. An iron molecule was also detected in an enzyme molecule.     

Development and characterization of a xylose-inducible gene expression system for Clostridium perfringens

A xylose-inducible gene expression vector for Clostridium perfringens was developed. Plasmid pXCH contains a chromosomal region from Clostridium difficile (xylR-P(xy)(lB)): xylR, encoding the xylose repressor, xylO, the xyl operator sequence, and P(xylB), the divergent promoter upstream of xylBA encoding xylulo kinase and xylose isomerase. pXCH allows tightly regulated expression of the chloramphenicol acetyltransferase reporter and the α-toxin genes in response to the inducer concentration. Thus, pXCH could constitute a new valuable genetic tool for study of C. perfringens.     

Chemical synthesis and expression of copper metallothionein gene of Neurospora crassa

The gene coding for the Neurospora crassa copper metallothionein (MT) was synthesized and inserted in the lacZ' gene of pUC18 plasmid to give the same translational reading frame as the latter gene. The MT-beta-galactosidase fused gene was expressed in Escherichia coli to produce a fused protein in which the amino and carboxy termini of MT are linked to the beta-galactosidase through methionine residues. An MT derivative containing an extra homoserine residue at the carboxy terminus was prepared by cyanogen bromide cleavage of the fused protein followed by a reverse-phase HPLC separation. The spectral features of the MT derivative and its copper complex were similar to those of the corresponding native MTs.