Certificate of Analysis & Data Sheet

Recombinant superactive human prolactin antagonist 12M, consists of one polypeptide chain containing 199 amino and an additional Ala at N-terminus acids was mono-pegylated and purified by proprietary chromatographic techniques as described by Solomon et al. Endocrinology (in press). Its molecular mass is ~ 39 kDa, however under non-denaturing conditions it behaves as 220 kDa protein due to its increased hydrodynamic volume.

E. coli

Lyophylized white powder

The protein was lyophilized from a concentrated (1mg/ml) solution with 0.02-0.03% NaHCO3.

It is recommended to reconstitute the lyophilized pegylated super active hPRL antagonists in sterile water or 0.4% NaHCO3 adjusted tp pH 8-9, not less than 100µg/ml, which can then be further diluted to other aqueous solutions, preferably in presence of carrier protein.

Lyophilized pegylated del 1-9 G129R hPRLantagonist although stable at room temperature for several weeks, should be stored desiccated below -18C. Upon reconstitution at > 0.1 hPRL mg/ml and up to 3 mg/ml and filter sterilization hPRL can be stored at 4C for several weeks.

Greater than 97.0% as determined by:
(a) Gel filtration analysis (size exclusion chromatography).
(b) Analysis by reducing and non-reducing SDS-PAGE gel.
It containst traces of the non-pegylated antagonists

The sequence of the first six N-terminal amino acids was determined and was found to be Ala-Arg-Ser-Gln-Val-Thr, however the protein was pegylated at its N-terminus.

The purified del 1-9 G129R hPRL consists of > 95% monomers as determined by gel-filtration chromatography.

PLR’s pegylated super active del 1-9 G129R hPRL antagonist is fully biologically active as evidenced by inhibiting hPRL-induced proliferation of Nb2 cells or Baf3 cells stably transfected with hPRL receptors. It also interacts at 1:1 molar ratio with human prolactin receptor extracellular domain as documented by SEC and SPR (Biacore analysis). However its activity is 3-4 fold lower compared to non-pegylaed antagonist.

Less than 0.1 ng/µg (IEU/µg) of del 1-9 G129R hPRL pegylated super active antagonist

Protein content: Protein quantitation was carried out by UV spectroscopy at 280 nm using the absorbency value of 0.96 as the extinction coefficient for a 0.1% (1mg/ml) solution at pH 8.0. This value is calculated by theDNAman computer analysis program of protein sequences.

This material is offered by PLR for laboratory research