Single-vector three-frame expression systems for affinity-tagged proteins

An effort is presented to create expression vectors which would allow expression of an inserted gene fragment in three reading frames in a single vector from a single promoter but with three separate ribosome binding sites (RBS). Each expression frame would generate an in-frame fusion with an affinity tag to allow efficient recovery of the produced fusion proteins. In the first generation vector, three identical polyhistidyl tags (His(6)) were used as affinity tags for the three expression frames. In the second generation vector, three different tags, an albumin binding domain derived from streptococcal protein G, an IgG binding Staphylococcus aureus protein A-derived domain (Z) and a His(6) tag, were employed to allow frame-specific affinity recovery. To evaluate the systems, model genes have been inserted in three different frames in both vectors. The first vector was demonstrated to produce fusion proteins in all three frames, whereas for the second, with a much wider spacing between the RBSs and affinity tags, expression could only be demonstrated from the first two translational start sites. For both systems, the first translation start was found to be significantly favored over the others. Nevertheless, we believe that the presented results represent the first successful attempt to create single-vector three-frame expression systems, a concept that could become valuable in future combined cloning-expression vectors.     

Liver (B-type) phosphofructokinase mRNA. Cloning, structure, and expression

Mouse liver mRNA enriched in sequences coding for liver phosphofructokinase by polysome immunoadsorption was used as a template for the synthesis of cDNA. The double-stranded cDNA was inserted into the expression vector lambda gt11 and cloned. Preliminary identification of clones containing cDNA sequences for phosphofructokinase was made by screening the library with anti-rat liver phosphofructokinase serum and horseradish peroxidase-conjugated goat anti-rabbit IgG as second antibody. Subsequently, by selecting antibodies specific to fusion proteins expressed by putative clones and by reacting with Western blots of mouse liver proteins several clones were positively identified as containing liver phosphofructokinase sequences. A cDNA clone corresponding to 2708 nucleotides of liver phosphofructokinase mRNA was further characterized and sequenced. The liver phosphofructokinase mRNA has an open reading frame of 2343 nucleotides followed by a 3'-untranslated region of 303 nucleotides. The G/C-rich (76%) portion of the 5'-untranslated region precedes a characteristic translational start site of CCGCC(AUG). The mRNA coding sequence indicates that the liver phosphofructokinase subunit is composed of 780 amino acid residues and has a Mr of 85,000. Comparison of the deduced amino acid sequence of mouse liver phosphofructokinase with the known rabbit muscle phosphofructokinase shows 68% homology. The N-half of the liver phosphofructokinase has conserved substrate binding sites for ATP and fructose-6-P. The 25 C-terminal residues, which contain the ATP inhibitory site, are the least homologous (20%) but contain a putative phosphorylation site (Arg-Arg-X-X-Ser). The liver phosphofructokinase mRNA is under nutritional and hormonal regulation. The liver phosphofructokinase mRNA level increased 4-fold when previously starved mice were refed a high carbohydrate, fat-free diet. This increase in mRNA level was blocked by 50% by the administration of dibutyryl cAMP. The induction of liver phosphofructokinase mRNA by fasting/refeeding was also diminished in streptozotocin diabetic mice.     

An improved Escherichia coli expression vector for the construction and identification of full-length cDNA clones

An Escherichia coli expression vector designed for the efficient synthesis and identification of a full-length cDNA clone is constructed. The vector allows the synthesis of double-stranded cDNAs downstream from the tandem lac control regions employing the vector-primer and linker procedure of Okayama and Berg [Mol. Cell Biol. 2 (1982) 161-170]. Full-length cDNA clones carrying the 5'-noncoding region in addition to the entire coding and 3'-noncoding regions can be expressed in E. coli cells without fusing their coding region to that of E. coli proteins; these clones are identified by colony immunoassay. The entire cDNA insert can be easily excised from the plasmid, since the multiple cloning sites in the vector are duplicated at both ends of the cDNA insert during its synthesis.     

Multiple polyadenylation sites downstream from the human aFGF gene encoding acidic fibroblast growth factor

We previously reported the isolation of two partial cDNA clones encoding human acidic fibroblast growth factor (aFGF). The nucleotide (nt) sequence throughout the coding region and the deduced amino acid sequence were presented [Jaye et al., Science 233 (1986) 541-545]. In this report, the isolation of additional aFGF cDNA clones and their nt sequence are presented. The human aFGF gene is shown to encode at least four functional polyadenylation sites and multiple regulatory sequences within the 3'-untranslated region. The aFGF open reading frame resides approx. 3100 bp upstream from the most frequently utilized 3' processing and polyadenylation site. Several less abundant cDNA clones provide evidence of polyadenylation at three less distal sites, which are colinear with genomic DNA. Northern-blot analysis reveals three detectable mRNA species, whose sizes and intensities correlate with the length and relative abundance of cDNA clones representing them.     

Molecular cloning of AMP deaminase isoform L. Sequence and bacterial expression of human AMPD2 cDNA.

Human AMPD2 cDNA clones have been isolated from T-lymphoblast and placental lambda gt11 libraries utilizing a previously cloned rat partial AMPD2 cDNA as the probe. Alignment analysis of all cDNA clones indicates the presence of intervening sequences in several placental isolates. This has been confirmed by sequencing human AMPD2 genomic clones. Intervening sequences can be removed from the cDNA clones by restriction with endonucleases at unique sites within the proposed open reading frame. This results in a 3292-base pair cDNA proposed to contain the entire AMPD2 open reading frame, which would encode a 760-amino acid polypeptide with a predicted subunit molecular mass of 88.1 kDa. Nucleotide and predicted amino acid comparisons with the 264 base pairs of proposed coding sequences in the rat AMPD2 cDNA demonstrate 91% similarity and identity, respectively. A comparison of the predicted human AMPD1 and AMPD2 polypeptides demonstrates homology in their C-terminal domains. Included in this region is the conserved motif, SLSTDDP, proposed to be part of the catalytic site of all AMP deaminases. In contrast, the predicted N-terminal domains of the human AMPD1 and AMPD2 polypeptides are unique. When placed in a prokaryotic expression vector, the human AMPD2 cDNA expresses AMP deaminase activity which can be precipitated with polyclonal antisera specific for isoform L.     

Isolation and characterization of bovine and mouse terminal deoxynucleotidyltransferase cDNAs expressible in mammalian cells.

We have isolated nearly full-length cDNA clones of terminal deoxynucleotidyltransferase (TdT) from calf thymus and mouse lymphoma cDNA libraries. The libraries were constructed using the pcD vector system which permits the expression of cDNA inserts in mammalian cells. The bovine TdT cDNA clone contains an open reading frame coding for 520 amino acids, Mr 59,678. The mouse TdT cDNA clone contains an open reading frame of 1,587 bp, whose translated cDNA encodes a 60,004 dalton protein. The mouse TdT cDNA clone contains 60 bp in the 3' end region of the coding sequence not found in the bovine TdT cDNA sequence, otherwise, the clones share about 80% homology. A possible nuclear-localization-sequence (Pro-Arg-Lys-Lys-Arg-Pro-Arg) was conserved in the N-terminal region in the mouse and bovine cDNA clones. Bovine and mouse cDNAs transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 60,000 which was detected immunologically using polyclonal rabbit antibody against bovine TdT. Bovine TdT expressed in COS7 cells by nearly full-length cDNA clone was localized in the nucleus and the translational product of pOK103 lacking the nuclear-localization-sequence was localized in the cytoplasm.     

Molecular cloning, sequencing, and expression of mouse ferrochelatase

The cDNA encoding mouse ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) was isolated from a mouse erythroleukemia (MEL) cell cDNA library in lambda gt11 expression vector, by immunoscreening with a polyclonal antibody. Two full-length clones containing cDNA inserts of 2.2 and 2.90 kilobases were obtained. These clones have the same entire enzyme coding region, but alternative putative polyadenylation sites in the 3'-noncoding regions. From the deduced primary structure, a putative leader sequence of 53 amino acid residues resulted in a precursor protein of 420 amino acid residues (Mr 47,130) and a mature protein of 367 residues (Mr 41,692). The cDNA allows for the expression of active ferrochelatase by transfected culture cells. RNA blot analysis showed two species of ferrochelatase mRNA consistent with findings of two polyadenylation sites. Both the mRNAs increased by treatment of the MEL cells with dimethyl sulfoxide. The band pattern of the RNA of the mouse liver was the same as that of the MEL cells. Based on these results, we deduce that ferrochelatase in erythroid and hepatic cells can be only of one type.     

Construction of a multiframe vector to express coding sequences in Escherichia coli

Cloning of foreign DNA fragments for coding sequence analysis in Escherichia coli usually involves sets of three vectors. To simplify this, we constructed an expression vector named pMFV7 containing three ATG codons in different frames downstream of a Shine-Dalgarno sequence, assuming that the ribosome can use any of the three start codons in an alternative manner. Translation beginning at either of the start codons would drive the expression of any coding fragment cloned downstream. To test the feasibility of this proposal, we cloned DNA fragments of the lacZ gene in each of the possible reading frames downstream from pMFV7 start codons. Sequence analysis of the N-terminus regions around the fusion sites indicates that ribosomes indeed initiate translation at each of the three initiation codons. In one case, levels of beta-galactosidase activity depended largely on the N-terminus of the translation products. We conclude that pMFV7 may be useful for expressing coding sequences regardless of their reading frame.     

Molecular cloning and sequence analysis of full-length cDNA coding for mouse contrapsin

Four overlapping cDNA clones encoding contrapsin were isolated from a mouse liver cDNA library constructed in the expression vector, lambda gt11. M13 vector sequence analysis revealed that contrapsin cDNA contained an open reading frame of 1,254 bases encoding 418 amino acids. The N-terminal amino acid sequence of the isolated contrapsin matched residues 30 to 48 of the sequence deduced on nucleotide analysis. One clone, which had the longest 3' untranslated region, contained two sets of tandem polyadenylation signals, AATACA and AATAAA, which were located 497 bases apart, while the remaining three clones terminated at the first signal. The entire reading frame sequence of contrapsin cDNA showed 64% homology with that of human alpha-1-antichymotrypsin.     

Cloning, nucleotide sequence and expression of thioltransferase (glutaredoxin) cDNA from Schizosaccharomyces pombe

Thioltransferase (TTase), also known as glutaredoxin (Grx), is an enzyme that catalyzes the reduction of a variety of disulfide compounds, including protein disulfides, in the presence of reduced glutathione. TTase acts as a cofactor for various enzymes such as ribonucleotide reductase. We previously purified a TTase from Schizosaccharomyces pombe and its molecular size was determined. In the present study, a cDNA coding TTase was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization, which was constructed in a plasmid vector pGAD GH, and its corresponding insert was confirmed by Southern hybridization. The nucleotide sequence of the 375 bp long cDNA clone reveals an open reading frame, which encodes a protein of 101 amino acids. The coding region of the original clone was transferred after the lac promoter of pUC13 vector for expression in E. coli, and simultaneously, a suitable Shine-Dalgarno (SD) sequence was added in front of the coding region by PCR. The two primers used for PCR also separately contained BamHI and HindIII restriction sites. The E. coli strain (A434) harboring the pUC13 derivative pKU10 showed a 17.3-fold increase in TTase activity compared to the strain with only the vector plasmid.     

Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning

We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.     

Identification of novel homologues of three low molecular weight subunits of the mitochondrial bc1 complex

Large-scale random cDNA sequencing projects have been started for several organisms and are a valuable tool for the analysis of quantitative and qualitative aspects of gene expression. However, the reliability of the obtained data is limited as most of the clones are only partially analysed on one strand. As a consequence the sequence entries derived from random cDNA sequencing projects usually comprise incomplete open reading frames. They nevertheless define complete and reliable coding sequences, if two prerequisites are fullfilled: (i) the clones encode very small proteins, and (ii) the clones have a high frequency in the cDNA-banks. The present study describes the use of cDNA databases for the identification of homologues of three low-molecular-weight subunits of the mitochondrial bc₁ complex, termed the QCR6, QCR9 and QCR10 proteins. These polypeptides are only characterized for a small number of organisms, have a scarcely defined function and exhibit a low degree of structural conservation if compared between different species. Several clones were identified for each polypeptide by searches with TBLASTN using the known sequences as probes. Most of the database entries contain complete open reading frames and sequencing queries could be excluded due to the abundancy of the clones. Multiple sequence alignments are presented for all three polypeptides and consensus sequences are given which may provide a basis for the investigation of the proteins by site-directed mutagenesis.     

Preparation of DNA libraries of Plasmodium falciparum for searching calmodulin binding protein genes,GOGAT and Pfmyo A

A cDNA library of Plasmodium falciparum (Colombian strain FCB2) asexual stage was constructed in the lambda ZipLox vector. The lambda ZipLox library and a lambda ZAPII (Dd2 strain) were screened for genes coding for proteins that bind with or are related to calmodulin (CaM). Screening was accomplished with Hot start PCR assays and hybridization with radiolabeled probes. Actin I, CaM, glutamate synthase (GOGAT) and the three myosin clones--Pfmyo A, Pfmyo B and Pfmyo C--were identified. The clones coding for actin I, CaM and GOGAT were retrieved from the lambda ZipLox library, and the GOGAT and Pfmyo A clones from the lambda ZAP II library. The GOGAT clone contained an insert of 2,413 base pairs corresponding to 24.8% of the reported sequence. The Pfmyo A insert was 2,457 base pairs long, and represented the complete mRNA coding for this gene. Finally, the first report of a complete cDNA clone containing the P. falciparum myosin A is presented.     

Human hepatic triglyceride lipase: cDNA cloning, amino acid sequence and expression in a cultured cell line

By immunoscreening of a human cDNA expression library and hybridization of colonies, four partially overlapping cDNA clones of human hepatic triglyceride lipase (HTGL) mRNA were isolated. The clones included the complete coding sequence, the 3'- and at least part of the 5'-untranslated region. The length of the composite HTGL cDNA segment (1.7 kb) was consistent with the size of the mRNA identified in an established human hepatoma cell line. DNA-sequence analysis of cDNAs of partially unspliced mRNAs, and of cloned genomic DNA indicated that the HTGL coding sequence comprises at least six exons. As predicted from the cDNA, the unprocessed HTGL protein has a molecular weight of 56, three potential glycosylation sites, and a signal peptide of 23 amino acids. Sequence comparison with cDNA of other lipases, including rat hepatic lipase, revealed 30%-75% protein-sequence homology. The data establish that HTGL is a secretory protein produced in the hepatocyte, and that its synthesis can be continued in permanent cell lines of hepatoma origin. Our studies also showed that HTGL is another member of a lipase gene family which has interfacial binding sites and possibly other functional domains in common.