Biomedical subjects

Xipeng Liu

Publications and source records attributed to Xipeng Liu.

8 recordsLinked to original sources

PubMed2006

A T7-expression system under temperature control could create temperature-sensitive phenotype of target gene in Escherichia coli.

Temperature-sensitive (TS) mutants of a gene are ones of which the activity or phenotype is very similar to that of wild type only at certain temperature and they provide extremely powerful tool for studying protein function in vivo. Here we report a novel strategy to generate TS phenotype of the interest gene in Escherichia coli based on a temperature-sensitive T7-expression system. A TS T7-RNA polymerase is generated by interrupting it with a TS intein from Saccharomyces cerevisiae vacuolar ATPase subunit (VMA), resulting that the gene flanked by T7-promoter and T7-terminator will be transcribed only at the permissive temperature (18 degrees C), not at the restrictive temperature (37 degrees C). The feasibility to create TS phenotype of this strategy was detected using lacZ as target. Reverse transcriptase polymerase chain reaction (PCR) indicated that at 18 degrees C, transcripts of T7-promoter controlled lacZ were at least 85 times more than those at 37 degrees C. Western blot analysis and enzymatic assay showed that large amounts of active His6-tagged LacZ produced at 18 degrees C but little at 37 degrees C. This strategy appears more promising than other TS creation methods because the target is pre-designed, no modification is introduced, and only simple DNA manipulation is required.

DNA-Directed RNA Polymerases↗

PubMed2006

Chlamydial DNA polymerase I can bypass lesions in vitro.

We found that DNA polymerase I from Chlamydiophila pneumoniae AR39 (CpDNApolI) presents DNA-dependent DNA polymerase activity, but has no detectable 3' exonuclease activity. CpDNApolI-dependent DNA synthesis was performed using DNA templates carrying different lesions. DNAs containing 2'-deoxyuridine (dU), 2'-deoxyinosine (dI) or 2'-deoxy-8-oxo-guanosine (8-oxo-dG) served as templates as effectively as unmodified DNAs for CpDNApolI. Furthermore, the CpDNApolI could bypass natural apurinic/apyrimidinic sites (AP sites), deoxyribose (dR), and synthetic AP site tetrahydrofuran (THF). CpDNApolI could incorporate any dNMPs opposite both of dR and THF with the preference to dAMP-residue. CpDNApolI preferentially extended primer with 3'-dAMP opposite dR during DNA synthesis, however all four primers with various 3'-end nucleosides (dA, dT, dC, and dG) opposite THF could be extended by CpDNApolI. Efficiently bypassing of AP sites by CpDNApolI was hypothetically attributed to lack of 3' exonuclease activity.

Amino Acid Motifs↗

PubMed2005

Chlamydia pneumoniae AP endonuclease IV could cleave AP sites of double- and single-stranded DNA.

Endonuclease IV gene, the only putative AP endonuclease of C. pneumoniae genome, was cloned into pET28a. Recombinant C. pneumoniae endonuclease I V (CpEndoIV) was expressed in E. coli and purified to homogeneity. CpEndoIV has endonuclease activity against apurinic/apyrimidinic sites (AP sites) of double-stranded (ds) oligonucleotides. AP endonuclease activity of CpEndoIV was promoted by divalent metal ions Mg2+ and Zn2+, and inhibited by EDTA. The natural (A, T, C and G) and modified (U, I and 8-oxo-G (GO)) bases opposite AP site had little effect on the cleavage efficiency of AP site of ds oligonucleotides by CpEndoIV. However, the CpEndoIV-dependent cleavage of AP site opposite modified base GO was strongly inhibited by Chlamydia DNA glycosylase MutY. Interestingly, the AP site in single-stranded (ss) oligonucleotides was also the effective substrate of CpEndoIV. Similar to E. coli endonuclease IV, AP endonuclease activity of CpEndoIV was also heat-stable to some extent, with a half time of 5 min at 60 degrees C.

Bacterial Proteins↗

PubMed2005

The mechanism of base excision repair in Chlamydiophila pneumoniae.

Repair of damaged DNA is of great importance in maintaining genome integrity, and there are several pathways for repair of damaged DNA in almost all organisms. Base excision repair (BER) is a main process for repairing DNA carrying slightly damaged bases. Several proteins are required for BER; these include DNA glycosylases, AP endonuclease, DNA polymerase, and DNA ligase. In some bacteria the single-stranded specific exonuclease, RecJ, is also involved in BER. In this research, six Chlamydiophila pneumoniae (C. pneumoniae) genes, encoding uracil DNA glycosylase (CpUDG), endonuclease IV (CpEndoIV), DNA polymerase I (CpDNApolI), endonuclease III (CpEndoIII), single-stranded specific exonuclease RecJ (CpRecJ), and DNA ligase (CpDNALig), were inserted into the expression vector pET28a. All proteins, except for CpDNALig, were successfully expressed in E. coli, and purified proteins were characterized in vitro. C. pneumoniae BER was reconstituted in vitro with CpUDG, CpEndoIV, CpDNApolI and E. coli DNA ligase (EcDNALig). After uracil removal by CpUDG, the AP site could be repaired by two BER pathways that involved in the replacement of either one (short patch BER) or multiple nucleotides (long patch BER) at the lesion site. CpEndoIII promoted short patch BER via its 5'-deoxyribophosphodiesterase (5'-dRPase) activity, while CpRecJ had little effect on short patch BER. The flap structure generated during DNA extension could be removed by the 5'-exonuclease activity of CpDNApolI. Based on these observations, we propose a probable mechanism for BER in C. pneumoniae.

Bacterial Proteins↗

PubMed2005

Recombinant human alphaA-crystallin can protect the enzymatic activity of CpUDG against thermal inactivation.

alpha-Crystallin is one of the major protein components in mammalian lens fiber cells. It is composed of alphaA and alphaB subunits that have structural homology to the family of mammalian small heat shock proteins. Horwitz firstly characterized native alpha-crystallin as a molecular chaperone in vitro based on its ability to prevent heat-induced aggregation of lens proteins and enzymes. Andley et al. cloned and expressed human alphaA-crystallin in Escherichia coli and confirmed its chaperone activity by suppression of thermal aggregation and singlet oxygen-induced opacification. Although alphaA-crystallin acts as a chaperone protein, there is no report showing on its ability to protect enzymes against thermal inactivation. Here, we present data showing that alphaA-crystallin can prevent thermal inactivation of CpUDG that catalyzes uracil removal from DNAs.

Base Sequence↗

PubMed2005

Signal peptide does not inhibit binding of biotin to streptavidin.

Three recombinant polypeptides of streptavidin: the full-length streptavidin with a signal peptide (rsavS), full-length streptavidin (rsavF) and core streptavidin (rsavC), were expressed in E. coli strain BL21 (DE3) and purified by Ni-NTA chromatography. Although all three recombinant streptavidins had biotin-binding activity, the stability and solubility of rsavC tetraunits were much better than those of rsavS and rsavF, indicating that signal peptide and/or extra amino acid residues in rsavS and rsavF have negative effects on streptavidin. Meanwhile, the signal peptide and extra amino acid residues in rsavS and rsavF made it difficult for polypeptides to fold into functional proteins. After refolding of denaturing-purified proteins in vitro, both the specific activities and biotin binding sites of renatured streptavidins were 1.4-times as that of proteins obtained by native Ni-NTA purification. Because the denaturing-purified rsavC is easy of refolding into functional protein, the better strategy for production of active rsavC is to isolate the protein from IPTG-induced E. coli extracts by denaturing Ni-NTA affinity chromatography followed by refolding of purified polypeptide in vitro.

Binding Sites↗

PubMed2005

Expression of both Chlamydia pneumoniae RNase HIIs in Escherichia coli.

Both genes encoding the RNase HIIs from Chlamydia pneumoniae AR 39 (discriminated as CpRNase HIIa and CpRNase HIIb in this report) were cloned and efficiently expressed in Escherichia coli. These genes amplified from Chlamydial genomes with PCR were digested with restriction endonucleases and then cloned into plasmid pET-28a predigested with the same enzymes. DNA sequencing confirmed that the constructs were correct in translation frame and coding sequence. Recombinant RNase HIIs were over-expressed by 0.5 mM IPTG induction. CpRNase HIIa existed mainly as inclusion bodies while CpRNase HIIb mainly as soluble fractions in E. coli. The soluble proteins were 20% of total expressed CpRNase HIIa and 65% of total expressed CpRNase HIIb, respectively. Native purification and denaturing Ni-NTA purification were performed to recover the recombinant CpRNase HIIs from induced bacteria. 3.36 mg CpRNase HIIa and 18 mg CpRNase HIIb were, respectively, obtained from 1 g wet bacteria with native Ni-NTA purification. Denaturing Ni-NTA purification recovered 14.48 mg CpRNase HIIa and 10.4 mg CpRNase HIIb from 1 g wet bacteria, respectively. Although the proteins recovered by denaturing Ni-NTA purification were inactive, re-folding by dialysis against decreased concentrations of urea could generate CpRNase HIIa and CpRNase HIIb as active as those recovered by native Ni-NTA purification. These efforts offered basis for further study on the structure-function relationships and their biological importance of Chlamydial RNase HIIs.

Base Sequence↗

PubMed2004

Cloning, expression, and characterization of uracil-DNA glycosylase of Chlamydia pneumoniae in Escherichia coli.

A uracil-DNA glycosylase gene was cloned from Chlamydia pneumoniae AR39 and expressed in E. coli strains BL21 (DE3) and BL21 (DE3) pLysS. After purification by Ni-NTA His x Bind Resin and DEAE Sepharose Fast Flow column chromatography, recombinant CpUDG with a specific activity of 1,000,000 U/mg was obtained. The enzymatic activity of the purified CpUDG protein was further characterized using oligodeoxyribonucleotides carrying uracil bases as substrates. The base opposite to uracil in double strand DNAs affected uracil removal efficiencies in the order: U/- > U/T > U/C > U/G > U/A. Free uracil and abasic sites (AP site) could inhibit the reaction. The optimal temperature and pH for uracil removal by CpUDG were 37 degrees C and pH 8.0, respectively. Site-directed mutagenesis studies indicated that amino acids D77, H200, and A205 were important for the catalytic activity of CpUDG. Together, these data suggest that CpUDG is a member of the UDG family-I protein. This is the first report on cloning, expression, and characterization of Chlamydia uracil-DNA glycosylase.

Amino Acid Sequence↗