Expression of novel lipocalin-like milk protein gene is developmentally-regulated during lactation in the tammar wallaby, Macropus eugenii.

We have identified a novel whey protein (late lactation protein B; LLPB) that is first secreted in the milk of the tammar wallaby around day 200 of lactation. The LLPB cDNA clone of 843 base pairs encodes a mature protein of 156 amino acids. LLPB shares 65 and 48% nucleotide and deduced amino acid identity, respectively, with a previously identified late lactation protein A (LLPA). Both these proteins share significant amino acid sequence homology with the lipocalin protein family. Expression of the LLPB gene is induced between days 200 and 240 of lactation, in contrast to expression of the LLPA gene, which is induced at around 145 days of lactation. Maximal expression of both genes in mammary explants from tammars at 213 days of lactation required a combination of prolactin, insulin and hydrocortisone. Transcripts of LLPA, LLPB and beta-lactoglobulin (TBLG) were localized to the same cells by in situ hybridization. A substantial level of alveolar maturation is required for expression of the LLP genes, unlike TBLG, which is expressed in immature alveoli. We hypothesize that the temporal expression of the LLPB and LLPA genes may be regulated both by endocrine stimuli and factors intrinsic to the mammary gland.

Dealing with intractable protein cores: protein sequencing of the Mcg IgG and the Yvo IgM heavy chain variable domains.

The VH domains of two human monoclonal antibodies, designated Mcg IgG1(lambda) and Yvo IgM(kappa), were particularly intractable to standard protein sequencing protocols. Peptides liberated from the VH domains of these proteins, using standard enzymatic or chemical cleavages, invariably precipitated during the procedures. Boiling in SDS containing buffers dissolved precipitates and the peptides were separated using SDS-PAGE. Fully overlapped VH sequences were obtained with a series of 'in-gel' cleavages, followed by passive/differential transfers of peptides onto PVDF membranes. Both the in-gel cleavages and passive transfers could be applied to 'wet' or 'dry' gels so that gels could be archived and used at a later date to obtain additional sequence information from a fragment of interest. Repetitive yields of even the most insoluble peptides were such that the sequences of various peptides from relatively complex mixtures of peptides could be assigned with confidence. Despite the overall success of the sequencing, we occasionally referred to electron density maps, calculated for crystals of the Fab of Yvo IgM, to resolve particular sequences and confirm ambiguous amino acid assignments. Methods we describe in this report should be generally useful for obtaining sequences of proteins with intractable cores and may find many applications in the 'post genomic era'.