The superior folding of a RANTES analogue expressed in lactobacilli as compared to mammalian cells reveals a promising system to screen new RANTES mutants

Abstract

Development of effective topical microbicides for the prevention of HIV-1 sexual transmission represents a primary goal for the control of the AIDS pandemic. The viral coreceptor CCR5, used by the vast majority of primary HIV-1 isolates, is considered a primary target molecule. RANTES and its derivatives are the most suitable protein-based compounds to fight HIV-1 via CCR5 targeting. Yet, receptor activation should be avoided to prevent pro-inflammatory effects and possibly provide anti-inflammatory properties. C1C5 RANTES is a chemokine mutant that exhibits high anti-HIV-1 potency coupled with CCR5 antagonism. However, the need for the formation of an N-terminal intramolecular disulfide bridge between non-natural cysteine residues at positions 1 and 5 represents a challenge for the correct folding of this protein in recombinant expression systems, a crucial step towards its development as a microbicide against HIV-1. We report here a rare case of superior folding in a prokaryote as compared to an eukaryotic expression system. Production of C1C5 RANTES was highly impaired in CHO cells, with a dramatic yield reduction compared to that of wild type RANTES and secretion of the molecule as disulfide-linked dimer. Conversely, a human vaginal isolate of Lactobacillus jensenii engineered to secrete C1C5 RANTES provided efficient delivery of the monomeric protein. This and other reports on successful secretion of complex proteins indicate that lactic acid bacteria are an excellent system for the expression of therapeutic proteins, which can be used as a platform for the engineering of conceptually novel RANTES mutants with potent anti-HIV-1 activity.

Fig. 1

Wild type and C1C5 RANTES construct design and protein secretion. (A) Schematic representation of pcDNA3.1+ and p1063 constructs for wt and C1C5 RANTES expression in CHO cells and L. jensenii, respectively. (B) Reducing and non-reducing Western blot analysis of secreted RANTES variants using anti-human RANTES Abs. st, RANTES standard from E. coli; nt, supernatants from non-transfected CHO cells or non-transformed L. jensenii; and wt and C1C5, wt and C1C5 RANTES-containing supernatants, respectively.

Fig. 2

Anti-HIV-1 activity of secreted wt and C1C5 RANTES. Inhibition of HIV-1_BaL infection in PM1 cells was measured by the p24-based assay after 4 days of infection. (A) (circles) E. coli-expressed RANTES standard (ID_50% = 0.8 nM) and (triangles) CHO cell-secreted wt RANTES (ID_50% =0.63 nM). Values are the mean ± SD of two independent experiments performed in triplicate. (B) (white columns) E. coli-expressed RANTES standard; (dotted columns) partially purified wt RANTES from L. jensenii; and (black columns) partially purified C1C5 RANTES from L. jensenii. Values are the mean ± SEM of one experiment performed in triplicate, representative of two experiments yielding similar results.

Fig. 3

Intracellular content of RANTES variants in CHO cells and L. jensenii. Cell lysates were analyzed in Western blot under non-reducing condition and RANTES variants revealed using anti-human RANTES Abs. nt, wt and C1C5 as in Fig. 1.

Fig. 4

Chemical cross-linking of RANTES variants produced by E. coli, CHO cells and L. jensenii. RANTES variants were chemically-cross-linked using DTSSP, resolved by SDS-PAGE under non-reducing or reducing conditions and analyzed by Western blot using anti-human RANTES Abs. wt E. coli, inclusion bodies-refolded wt RANTES from E. coli (standard); wt CHO, CHO cell-secreted wt RANTES; wt L. jensenii, L. jensenii-secreted wt RANTES; and C1C5 L. jensenii, L. jensenii-secreted C1C5 RANTES.

Fig. 5

Purification of L. jensenii-secreted wt and C1C5 RANTES. L. jensenii-secreted wt and C1C5 RANTES were purified to homogeneity using a four-step ionic exchange protocol. After SDS-PAGE in non-reducing conditions, proteins were stained by Coomassie blue or detected by Western blot using anti-human RANTES Abs. st, wt and C1C5 as in previous figures.