Defensin (DF) is a newer cytolytic antimicrobial peptide having molecular weight of 4 kDa having antimicrobial activity against wide range of gram positive and gram negative bacteria. The present work describes method development of recombinant Defensin protein using internal standard Bovine serum albumin (BSA). A simple, precise and accurate reverse phase high performance liquid chromatography method has been developed and validated for quantification of Defensin protein using BSA as an internal standard using Enable Q C18 column (250mm x 4.6 mm I.D, 5μm particle size 300ºA) as column, water: acetonitrile (60:40 v/v) with Trifluoroacetic acid 0.1% as mobile phase, flow rate of 1ml/min and detection was carried out at 280 nm. The retention time of BSA and Defensin was 2.342 and 5.279 min respectively. The linearity range was found to be 10-50 μg/ml, co-relation coefficient was found to be 0.999. The limits of detection and quantitation of the method were 1.010 and 3.062 μg/ml respectively. So, the developed method was accurate, precise and robust.
Key words: Recombinant Defensin, BSA, RP-HPLC, validation.
Human Defensins (1-5) are small, cationically charged, cysteine-rich endogenous antibiotic peptides with antimicrobial and cytotoxic properties that contain 29-35 amino acid residues, including six invariant disulphide linked cysteines moiety having a molecular weight of 4-5 kDa. The novel trend in drug development is the design and development of biopharmaceuticals, thus, the pharmaceutical industry has focused on bio molecules method development and validation (8-9). HPLC is a widely used technique for proteins and peptides because of its ease of usage, higher selectivity and faster analysis. In this study one of the antimicrobial peptide Defensin i.e. antimicrobial and cytolytic Cysteine rich peptide was expressed in the E.Coli cell. develop a suitable method for this newly cloned Defensin protein. Literature survey does not reveal any method for estimation of Defensin using internal standard BSA(10-15). So, the present paper describes simple, sensitive, rapid, accurate and robust method for estimation of recombinant Defensin protein.
The HPLC apparatus was a Shimadzu chromatographic system equipped with an injection valve; Shimadzu LC 20AT UV dual absorbance detector (SPD 20A) was used. A reversed-phase C18 column Q (Enable 250mm x 4.6mm, 5 µm, 300A°). Peak area integration was performed using LC solution software.
Recombinant Defensin which was extracted from E.Coli is used for analysis of drug and it is confirmed by IR spectroscopy and sodium dodecyl Polyacrylamide gel electrophoresis. HPLC grade Acetonitrile (ACN) was purchased from Fischer scientific Ltd. Trifluoroacetic acid (TFA) AR grade was from SD Fine Chem., Mumbai, India. Triple distilled water was used for analysis. Calibrated glasswares were used throughout the work.
The mobile phase used in this work was ACN: water: TFA (0.1%) 40:60 (v/v) which was...