High Level Expression and Purification of Atl, the Major Autolytic Protein of Staphylococcus aureus

Table 1

Oligonucleotide primers used in this study.

Primer	Sequence	Amplicon^1	Plasmid^2	Protein ID	Predicted MW/PI^3	Number of amino acids absent^4
Atl-FW	GGATCCATGGCGAAAAAATTCAATTAC	3790	pRSETA-atl	Atl	141258/9.59	0
Atl-FW-1	GGATCCGCTGGTTATAGTTTAGTTGATG	3382	pRSETA-atl1	Atl-1	126872/9.57	136
Atl-FW-2	GGATCCCCACAAGTAAACTCTTCAAT	3136	pRSETA-atl2	Atl-2	118310/9.59	218
Atl-FW-3	GGATCCCATGCATTTGTTGATGGGGATCG	2920	pRSETA-atl3	Atl-3	110038/9.62	290
Atl-FW-4	GGATCCGGTGGTACTGACCATGCCGATCC	2659	pRSETA-atl4	Atl-4	100288/9.73	377
Atl-FW-5	GGATCCGTATACGACAAAACTGGTAAAG	2401	pRSETA-atl5	Atl-5	91053/9.75	463
Atl-FW-6	GGATCCGTGTCTGGCTCTGGAAACCAAACA	2143	pRSETA-atl6	Atl-6	81641/9.75	549
Atl-FW-7	GGATCCAACGGTGTTGCTCAAATTAATGC	1942	pRSETA-atl7	Atl-7	74557/9.71	616
Atl-FW-8	GGATCCTCACCTGTAAATGTAATGCAAAC	1729	pRSETA-atl8	Atl-8	66778/9.71	687
Atl-RV	GAATTCATGTTGCTTATTTATATTG

All PCR were carried out using the indicated forward primers and a common reverse primer. The forward primers included a BamHI site (underlined) and the reverse primer included an EcoRI site (underlined) to facilitate the subcloning of the amplified fragment. ¹The size of the resulting amplicon is indicated in base pairs. ²The construct where the corresponding amplicon was cloned in vector pRSETA at BamHI and EcoRI sites. ³The molecular weight (MW) and the PI of the recombinant proteins likely to be expressed in E. coli  BL21 with the corresponding plasmid constructs. The MW/PI was calculated using the website http://web.expasy.org/compute_pi/. ⁴The numbers indicate the lack of Atl N-terminal amino acids. All recombinant Atl proteins include an extra 34 amino acids at the N-terminus that arises from the vector pRSETA.