Expression of recombinant proteins lacking methionine as N-terminal amino acid in plastids: human serum albumin as a case study.
Removal of the N-terminal methionine of a protein could be critical for its function and stability. Post-translational modifications of recombinant proteins expressed in heterologous systems may change amino-terminal regions. We studied the expression of mature proteins lacking methionine as the N-terminal amino acid in tobacco chloroplasts, using human serum albumin (HSA) as an example. Two approaches were explored. First, we fused the Rubisco small subunit transit peptide to HSA. This chimeric protein was correctly processed in the stroma of the chloroplast and rendered the mature HSA. The second approach took advantage of the endogenous N-terminal methionine cleavage by methionine aminopeptidase. Study of this protein processing reveals a systematic cleavage rule depending on the size of the second amino acid. Analysis of several foreign proteins expressed in tobacco chloroplasts showed a cleavage pattern in accordance to that of endogenous proteins. This knowledge should be taken into account when recombinant proteins with N-terminus relevant for its function are expressed in plastids.
Fernández-San Millán
Alicia
A
Instituto de Agrobiotecnología, Universidad Pública de Navarra-CSIC, Campus Arrosadía, 31006 Pamplona, Spain.
Farran
Inmaculada
I
Molina
Andrea
A
Mingo-Castel
Angel M
AM
Veramendi
Jon
J
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D008715:Methionine / Q000235:genetics*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics; D012709:Serum Albumin / Q000096:biosynthesis* / Q000235:genetics; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D012709:Serum Albumin; D008715:Methionine; D012273:Ribulose-Bisphosphate Carboxylase