Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue

Abstract

Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.

Keywords: Mammary Tissue; Milk Lysozyme; Recombinant Protein; Yak.

Figure 1

Alignment of amino acids of yak, cattle, sheep, goat milk lysozymes, and yak stomach lysozymes. The signal peptide is in bold and italics; the active sites are in bold and boxed; cysteine residues are in bold and shaded; Asp-Pro bond is bold. The secondary structural elements are listed under the sequence (7 α-helixes, 3 -sheets and 10 random coils). Accessed GenBank No.: JX946731 in yak, AY684064 in cattle, XP_004022875 in sheep, XP_005680291 in goat milk lysozymes and EU780011 in yak stomach lysozyme.

Figure 2

Polymerase chain reaction product of yak milk lysozyme (YML) gene amplified by specific primer sets. The size of each fragment is marked under their bands. M1, 250bp ladder; M2, DL2000 ladder; M3, DL2000; M4, λ-EcoT14. Lane1, YML1; Lane 2, YML4; Lane 3, YML2; Lane 4, YML3; Lane 5, YML5.

Figure 3

SDS-PAGE analysis of heterologously expressed yak milk lysozyme (YML) in E. coli and P. pastoris. (a) Recombinant yak milk lysozyme1 (rYML1) expressed in E. coli M, protein maker; 1, pET32a after induction for 6 h; 2, pET32a+YML1after induction for 6 h. (b) rYML2 expressed in E. coli M, protein maker; 3, pET32a after induction for 6 h; 4, pET32a+YML2 after induction at 0 h, 5, pET32a+YML2 after induction for 6 h. (c) rYML3 expressed in E. coli M, protein maker; 6, pET32a after induction for 6 h; 7, pET32a+YML3 after induction for 2 h; 8, pET32a+YML3after induction for 6 h. (d) rYML4 expressed in E. coli M, protein maker; 9, pET32a after induction for 6 h; 10, pET32a+YML4 after induction for 2 h; 11, PET32a+YML4 after induction for 6 h. (e) rYML5 expressed in P. pastoris. M, protein Marker; 12, cultured supernatant of the recombinant P. pastoris; 13, purified rYML5. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 4

Antimicrobial activities of the purified recombinant yak milk lysozyme5 (rYML5) against Staphylococcus aureus. The information of plate number is as following: a negative control (0 μg, Plate 1), purified rYML5 (48 μg, Plate 2), purified rYML5 (96 μg, Plate 3).

Figure 5

Expression pattern of the yak milk lysozyme (YML) gene during the lactation cycle. Each value indicates the least squares means of each time point and gene expressed level is calculated as n-fold change relative to −30 d at late pregnancy. Pooled standard error of the mean of YML is 8.26. * Statistically different from −30 d (p<0.01).

Figure 6

The phylogenetic relationship of yak milk lysozymes (YML). (a) The phylogenetic tree of all yak lysozymes. (b) The phylogenetic tree of all yak and cow lysozymes. The bar indicates 5% divergence and there is a triangle behind the name of the YML.