High level expression of peptides and proteins using cytochrome b5 as a fusion host.
A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome b5 has been designed. The ability of cytochrome b5 to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., alpha hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase I (ilvM) and II (ilvN), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins from cone snails, and the inactivation gate from the brain voltage gated sodium channel, NaV1.2. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., enterokinase, Factor Xa, and Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained by the fact that the cells take on a bright red color during the course of induction. In all the cases tested so far, the fusion protein accumulates in the soluble fraction to high levels. A novel purification protocol has been designed to purify the fusion proteins using metal affinity chromatography, without the need of a hexahistidine-tag. Mass spectral analysis has shown that the fusion proteins are of full length. CD studies have shown that the solubilized fusion proteins are structured. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. The results presented here demonstrate the versatility of the cytochrome b5 based fusion system for the production of peptides and small proteins (<15 kDa).
Mitra
Ashima
A
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Chakrabarti
Kalyan Sundar
KS
Shahul Hameed
M S
MS
Srinivas
Kalyan V
KV
Senthil Kumar
Ganesan
G
Sarma
Siddhartha P
SP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015786:Cytochromes b5 / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification; D014176:Protein Biosynthesis; D015202:Protein Engineering; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization
D004274:DNA, Recombinant; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5