The IC50 of recombinant HM103p18 as assayed by Cyclin D/CDK4-dependent full read Rb phosphorylation was higher than others have reported for purified p18INK4c protein.33,34 Therefore, some improvements in formulation and/or modifications of HM103p18 by using different MTDs and proteins with and without His tag or NLS sequences might further enhance therapeutic activity of the protein. Finally, we expect improvements to protein refolding and solution-formulation will enhance stability, reduce aggregation, and/or increase fill-finish concentration. In conclusion, this pilot study suggests that HM103p18 could have antitumor therapeutic activity as well as additional therapeutic applications by modulating the proliferation, differentiation and survival of stem cells with specific requirements for CDK4/6.

Materials and Methods Preparation of recombinant p18INK4c fusion proteins. MTD sequences, including MTD103, were identified from a screen of 1,500 signal peptides for sequences with protein transduction activity and were subsequently modified (D. Jo et al., manuscript in preparation). Coding sequences for p18INK4c and EGFP fusion proteins were cloned into pET-28a(+) (Novagen, Darmstadt, Germany) from PCR-amplified DNA segments (Supplementary Table S1). EGFP fusion proteins included a positive control containing the MTS from FGF4 (AAVLLPVLLAAP) and an arbitrary peptide (SANVEPLERL) that served as a negative control. Hp18 consists of an amino terminal 6x histidine tag (MGSSHHHHHHSSLVPRGSH) and NLS (KKKRK) from SV40 large T antigen appended to the human p18INK4c sequence (residues 2�C168).

HMTD103p18, HTatp18, Hphp18, and HAntp18, are identical to Hp18 but contain the hydrophobic MTD103 sequence or protein transduction domains from HIV-I Tat (YGRKKRRQRRR), human Hph-1 (YARVRRRGPRR) and Drosophila Ant (RQIKIWFQNRRMKWKK), respectively (Supplementary Tables S2 and S3). The recombinant proteins were purified from Escherichia coli BL21-CodonPlus (DE3) cells grown to an A600 of 0.6 and induced for 3 hours with 0.6 mmol/l IPTG. Denatured recombinant proteins were purified by Ni2+ affinity chromatography as directed by the supplier (Qiagen, Hilden, Germany). After purification, they were dialyzed against a refolding buffer (0.55 mol/l guanidine HCl, 0.44 mol/l L-arginine, 50 mmol/l Tris�CHCl, 150 mmol/l NaCl, 1 mmol/l EDTA, 100 mmol/l NDSB, 2 mmol/l reduced glutathione, and 0.

2 mmol/l oxidized glutathione) and changed to a physiological buffer such as RPMI 1640 medium. The CKI activity of purified Hp18 and HM103p18 proteins was determined by measuring the inhibition of Rb phosphorylation by CDK6/Cyclin D1 kinase, according to the manufacturer’s instructions (Cell Signaling, Danvers, MA). The structures of MTD- and Entinostat PTD-containing p18INK4c proteins were modeled using the AMBER 10.035,36 package on a KIST supercomputer based on a continuum solvation model.