High-level expression and purification of a recombinant hBD-1 fused to LMM protein in Escherichia coli.
In this work, we present the production of an active 43 aa recombinant human beta-defensin-1 (rhBD-1(43)) in Escherichia coli AD202 cells using specific pLMM1-rhBD-1 expression system. Unique solubility properties of the C-terminal fragment of light meromyosin (LMM) allowed us to overcome foreseeable problems with isolation procedures and toxicity caused by rhBD-1 to the host organism. As a result, the majority of fusion protein (LMM-rhBD-1(43)) was obtained in the soluble state, isolated by a low salt-high salt treatment of total cell protein. The rhBD-1(43) was cleaved from the fusion with Protease 4 and purified on CM Sepharose Fast Flow column with the yield of approximately 1 mg rhBD-1(43) from 6 g of wet weight cells. Purified rhBD-1(43) showed antimicrobial activity against E. coli ML-35p at a concentration of 129 microM. The procedure of rhBD-1 expression and purification we present can provide a reliable and simple method for production of different cationic peptides for biological studies.
Cipáková
Ingrid
I
CPN spol. s r.o., 561 02 Dolní Dobrouc, 401, Czech Republic. icipakova@yahoo.com
Hostinová
Eva
E
Gasperík
Juraj
J
Velebný
Vladimír
V
D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D023083:beta-Defensins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000890:Anti-Infective Agents; C406407:DEFB1 protein, human; D015879:Myosin Subfragments; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins