Purification and on-column refolding of EGFP overexpressed as inclusion bodies in Escherichia coli with expanded bed anion exchange chromatography

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

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Single-step purification of recombinant hepatitis B core antigen Y132A dimer from clarified Escherichia coli feedstock using a packed bed anion exchange chromatography

Process Biochemistry ◽

10.1016/j.procbio.2018.03.003 ◽

2018 ◽

Vol 69 ◽

pp. 208-215 ◽

Cited By ~ 3

Author(s):

Swee Lu Lim ◽

Hon Wei Ng ◽

Made Angga Akwiditya ◽

Chien Wei Ooi ◽

Eng-Seng Chan ◽

...

Keyword(s):

Escherichia Coli ◽

Hepatitis B ◽

Anion Exchange ◽

Packed Bed ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Single Step ◽

Core Antigen ◽

Recombinant Hepatitis ◽

Hepatitis B Core Antigen

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A novel mono-branched lipid phosphate acts as a substrate for dolichyl phosphate mannose synthetase

Biochemical Journal ◽

10.1042/bj2950195 ◽

1993 ◽

Vol 295 (1) ◽

pp. 195-201 ◽

Cited By ~ 14

Author(s):

I B H Wilson ◽

J P Taylor ◽

M C Webberley ◽

N J Turner ◽

S L Flitsch

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Binding Site ◽

Anion Exchange ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Natural Substrate ◽

Dolichyl Phosphate ◽

Lipid Phosphate ◽

Soluble Material

Dolichyl phosphate mannose synthetase (GDP-mannose: dolichyl-phosphate O-beta-D-mannosyltransferase; EC 2.4.1.83) is an enzyme that is involved in glycoconjugate biosynthesis and possesses a putatively conserved dolichol binding site. In order to probe the interaction between the enzyme and the dolichol chain, lipid phosphates varying in length and extent of branching have been tested as substrates in crude microsomal preparations from Saccharomyces cerevisiae. It was found that phytanyl (3,7,11,15-tetramethylhexadecanyl) phosphate was utilized at 60-70% of the efficiency of the natural dolichyl lipid in transfer of [3,4,-3H]mannose from GDP-Man to organic soluble material, whereas addition of S-3-methyloctadecanyl phosphate, which is of similar length to the phytanyl analogue but with only one branch, resulted in approximately 25% of the incorporation of the natural substrate. Incubations with the unbranched tetradecanyl phosphate and with the short, doubly branched R- and S-dihydrocitronellyl (3,7-dimethyloctanyl) phosphates exhibited levels of activity similar to incubations with no exogenous acceptor. These results were qualitatively confirmed with experiments on Escherichia coli harbouring the S. cerevisiae DPM1 gene. The [3H]mannosylated lipid-linked material from microsomal incubations was purified by anion-exchange chromatography. The major saccharide component recovered after hydrolysis was determined to be mannose, but a mannose-containing disaccharide was also present. It is concluded that branching of lipid phosphates is essential for substrates of dolichyl phosphate mannose synthetase and that significant transfer of mannose occurs even if only branching at C-3 is present.

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Biosynthesis Pathway of ADP-l-glycero-β-d-manno-Heptose in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.184.2.363-369.2002 ◽

2002 ◽

Vol 184 (2) ◽

pp. 363-369 ◽

Cited By ~ 111

Author(s):

Bernd Kneidinger ◽

Cristina Marolda ◽

Michael Graninger ◽

Alla Zamyatina ◽

Fiona McArthur ◽

...

Keyword(s):

Escherichia Coli ◽

Anion Exchange ◽

High Performance ◽

Inner Core ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Biosynthesis Pathway ◽

Reaction Products ◽

Novel Gene

ABSTRACT The steps involved in the biosynthesis of the ADP-l-glycero-β-d-manno-heptose (ADP-l-β-d-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-d-β-d-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-d-β-d-heptose. Our results demonstrate that the synthesis of ADP-d-β-d-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional d-β-d-heptose 7-phosphate kinase/d-β-d-heptose 1-phosphate adenylyltransferase), and GmhB (d,d-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-l-β-d-heptose precursor utilized in the assembly of inner core LPS.

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