Escherichia coli: Derived Murine Interleukin-1β with N-Terminus Partially Nα-Acetylated

Lischwe, Michael A.; Newton, Robert; Huang, Jun-Ru; Yates, Rachel; Breth, Leah A.; Larsen, B.

doi:10.1006/prep.1993.1065

Cited by 6 publications

(3 citation statements)

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“…For example, some E. coli ribosomal proteins have been observed to have N R -acetylated terminal amino acids such as alanine and serine (Terhorst et al, 1973;Wittmann-Liebold and Greuer, 1978;Yaguchi, 1975). A similar phenomenon has also been observed in a number of recombinant proteins expressed in E. coli; they include human interferon-RA (Takao et al, 1987), human interferon-γ (Honda et al, 1989), eglin C (Grutter, et al, 1985), and murine interleukin-1 (Lischwe et al, 1993). Further, as reported by Harbour and co-workers (1992), modification of nonterminal amino acid residues was detected in E. coli-derived recombinant bovine somatotropin; N -acetylation of lysine residues was characterized in that investigation.…”

Section: Resultsmentioning

confidence: 75%

Growth Kinetics of Escherichia coli and Expression of a Recombinant Protein and Its Isoforms under Heat Shock Conditions

et al. 1996

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Preinduction culture conditions were found to have significant impact on the expression and post-translational modification of a recombinant human protein in Escherichia coli under heat shock conditions (30 to 42 degrees C shift). Higher preinduction growth rates (micrograms) favored better cell viability, greater cell mass yields, and increased cloned gene expression during induction. Formation of recombinant protein isoforms (those containing N epsilon-modified lysine residues) exhibited an increasing trend with increasing micrograms. The different extents of post-translational modifications were suspected to be linked to the different concentrations of certain heat shock protein chaperones resulting from different micrograms. In view of the extensive involvement of E. coli heat shock proteins in cellular activities-including the synthesis, processing, modification, and degradation of proteins-at elevated temperatures, it is believed that micrograms dictated the cellular resources available for synthesizing the heat shock proteins required for cell survival, which in turn determined the ability of the cells to respond to the heat shock. With a higher micrograms both the synthesis of host proteins (as indicated by cell growth and survival) and the cloned gene expression were enhanced. The results demonstrate that there exists an intermediate micrograms for optimum production of the unmodified foreign protein in a heat shock environment. More importantly, they also illustrate the feasibility of improving the recombinant protein homogeneity in fermentation, thereby facilitating downstream processing.

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Section: Resultsmentioning

confidence: 75%

Growth Kinetics of Escherichia coli and Expression of a Recombinant Protein and Its Isoforms under Heat Shock Conditions

et al. 1996

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“…Four E. coli-derived recombinant proteins have been found to be partially a-N-acetylated (Grutter et al, 1985;Takao et al, 1987;Honda et al, 1989;Lischwe et al, 1993), which demonstrates that eukaryotic proteins can be a-N-acetylated when expressed in E. coli. Three native ribosomal proteins in E. coli are also a-N-acetylated and are modified by 3 different enzymes (Isono et al, 1978).…”

Section: 914mentioning

confidence: 99%

Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain 6‐N‐acetyllysine

et al. 1994

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Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, e-N-acetyllysine. This amino acid was initially discovered in place of the normal lysine'44 in a modified reversed-phase HPLC (RP-HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP-HPLC purified protein were used to identify this altered residue as e-N-acetyllysine.Ion-exchange chromatography was utilized to prepare low isoelectric point (PI) forms of rpST and rbST, which are enriched in e-N-acetyllysine. Electrospray mass spectrometry demonstrated that the majority of the protein in these low PI fractions contained species 42 Da larger than normal. Immobilized pH gradient electrophoresis (IPG) of the ion-exchange purified low PI proteins was used to isolate several monoacetylated species of rpST and rbST. The location of the acetylated lysine in each IPG-purified protein was determined by tryptic peptide mapping and amino acid sequencing of the altered tryptic peptides. Amino acid analyses of enzymatic digests of rpST and rbST were also used to confirm the presence of e-N-acetyllysine in these recombinant proteins. These data demonstrate that a significant portion of rpST and rbST produced in E. coli contain this unusual amino acid.

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“…The measured mass versus the deduced mass for the species with the acetylated N-terminus was 18,007.4 versus 18,008.0, whereas for the species without the acetyl group it was 17,964.8 versus 17,965.9. Partial N-terminal acetylation of E. coliexpressed proteins of mammalian origin has been confirmed (for references, see Lischwe et al, 1993).…”

Section: Resultsmentioning

confidence: 93%

Production, crystallization, and preliminary X‐ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1‐47) of troponin I

et al. 1997

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Abstract:Troponin is a ternary protein complex consisting of subunits TnC, TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E. coliexpressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, C147 is thought to form a core that is resistant to proteolytic digestion, and the interaction within C147 likely maintains the integrity of the troponin complex. Complex C147 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P3,(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A. The asymmetric unit presumably contains one C147 complex. Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 A resolution, with completeness of 91% and Rmerye = 6.4%. The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous

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Escherichia coli: Derived Murine Interleukin-1β with N-Terminus Partially Nα-Acetylated

Cited by 6 publications

References 0 publications

Growth Kinetics of Escherichia coli and Expression of a Recombinant Protein and Its Isoforms under Heat Shock Conditions

Growth Kinetics of Escherichia coli and Expression of a Recombinant Protein and Its Isoforms under Heat Shock Conditions

Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain 6‐N‐acetyllysine

Production, crystallization, and preliminary X‐ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1‐47) of troponin I

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