Molecular cloning and expression in yeast of caprine prochymosin.
We cloned and characterized a preprochymosin cDNA from the abomasum of milk-fed kid goats. This cDNA contained an open reading frame that predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively. Comparison of the caprine preprochymosin sequence with the corresponding sequences of lamb and calf revealed 99 and 94% identity at the amino acid level. The cDNA fragment encoding the mature portion of caprine prochymosin was fused in frame both to the killer toxin signal sequence and to the alpha-factor signal sequence-FLAG in two different yeast expression vectors. The recombinant plasmids were transformed into Kluyveromyces lactis and Saccharomyces cerevisiae cells, respectively. Culture supernatants of both yeast transformants showed milk-clotting activity after activation at acid pH. The FLAG-prochymosin fusion was purified from S. cerevisiae culture supernatants by affinity chromatography. Proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed kappa-casein.
Vega-Hernández
Maria C
MC
Molecular Biology Laboratory, Research Unit, Nuestra Señora de Candelaria University Hospital, 38010, Santa Cruz de Tenerife, Spain.
Gómez-Coello
Alicia
A
Villar
Jesús
J
Claverie-Martín
Félix
F
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry; D012085:Chymosin / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D006041:Goats; D006868:Hydrolysis; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics*; D008892:Milk / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D002364:Caseins; D004792:Enzyme Precursors; D011994:Recombinant Proteins; C032443:prorennin; D012085:Chymosin