ABSTRACT
Transglutaminase (TG) posttranslational modification of antibody permits more precisely conjugating. Based on the amino acid sequence of an anti-CD24 antibody (cG7), this article is aimed to generate a deglycosylated cG7 mutant (cG7Q). Firstly, we introduced additional glutamines at position 297 (N297Q) by site-directed mutagenesis, and then transfected the recombinant plasmids into CHO-s cells via electroporation method and screened by Dot blot assay. Subsequently, cG7Q was expressed and purified through Protein A affinity chromatography, further identified by SDS-PAGE electrophoresis and Western blot. Its affinity was detected with surface plasmon resonance and flow cytometry assay, and ADCC effect was determined by lactate dehydrogenase (LDH) release. Eventually, a cG7 mutant, cG7Q was successfully expressed with sequence-specific conjugation sites for further study.
ABSTRACT
In order to investigate the role of His180 residue, located in the non-conserved region of the σ70 subunit of Escherichia coli RNA polymerase, two mutant variants of the protein with substitutions for either alanine or glutamic acid were constructed and purified using the IMPACT system. The ability of mutant σ70 subunits to interact with core RNA polymerase was investigated using native gel-electrophoresis. The properties of the corresponding reconstituted holoenzymes, as provided by gel shift analysis of their complexes with single- and double-stranded promoter-like DNA and by in vitro transcription experiments, allowed one to deduce that His180 influences several steps of transcription initiation, including core binding, promoter DNA recognition and open complex formation.
ABSTRACT
A serine residue Ser463, required for proper function of E. coli -glutamyltranspeptidase (EcGGT) was identified by site-directed mutagenesis on the basis of sequence alignment of human, pig, rat, and three bacterial enzymes. Thr-, Asp-, and Lys-substituted variants were overexpressed in E. coli M15 cells and the recombinant proteins were purified to near homogeneity by nickel-chelate chromatography. With the exception of S463T, the other two variants completely lost GGT activity, implying the importance of this residue in EcGGT. Moreover, substitution of Ser463 with either Lys or Asp impaired the capability of autocatalytic processing of the precursor into - and -subunit. Computer modeling showed that the critical bonding distance of Gln390 C-Thr391 OG1 was significantly increased in S463D and S463K, indicating that these distance changes might be responsible for the lack of enzyme maturation. Measurements of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues in S463D and S463K, while circular dichroism (CD) spectra were nearly identical for wild-type and all mutant enzymes. The temperature-dependent signal in the far-UV region for S463T was consistent with that of wild-type enzyme, but S463D and S463K showed a different sensitivity towards temperature-induced denaturation. These results implied that a significant conformational change occurred as a result of Asp- and Lys-substitution.
Subject(s)
Amino Acid Sequence , Aspartic Acid/chemistry , Catalysis , Circular Dichroism , Escherichia coli/enzymology , Glutamine/chemistry , Lysine/chemistry , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Serine/chemistry , Spectrometry, Fluorescence/methods , Threonine/chemistry , Tryptophan/chemistry , gamma-Glutamyltransferase/chemistry , gamma-Glutamyltransferase/geneticsABSTRACT
In this paper,by means of PCR amplification,site-specific mutagenesis,DNA recombination in vitro and optimization of translation initiation,we constructed a recombinant plasmid pBV-IL-6 that overproduces biologically active human interleukin-6(rhIL-6)in E.coli.The expressed rhIL-6 lacks the first 25 amino acids of mature hIL-6 and accounts for 71% of the total bacteria proteins.The reasons why a deleted rather than mature IL-6 was chosen for expression in E.coli are as follows:1)The specific bio-activities of the deleted hIL-6 are the same as,even more than that of the mature form according to the published data;2)The deleted part of IL-6 coding region is rich in C-G andeasy to form secondary structure in mRNA,which is not good for expression;3)The re-designed region for initiation site of translation encodes a hydrophilic amino-end which favours highly translation initiation and expression of hIL-6 in E.coli.